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UNIVERSIDAD POLITÉCNICA DE MADRID
ESCUELA TÉCNICA SUPERIOR
DE INGENIEROS AGRÓNOMOS
ESTUDIOS TERMODINÁMICOS DE LOS
PROCESOS DE VITRIFICACIÓN EN LA
CRIOCONSERVACIÓN DE GERMOPLASMA
VEGETAL
- Tesis doctoral -
ALINE SCHNEIDER TEIXEIRA
Licenciada en Biología
2013
DEPARTAMENTO DE BIOLOGÍA VEGETAL
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS
ESTUDIOS TERMODINÁMICOS DE LOS PROCESOS DE
VITRIFICACIÓN EN LA CRIOCONSERVACIÓN DE
GERMOPLASMA VEGETAL
THERMODYNAMIC STUDIES OF VITRIFICATION
PROCESSES IN PLANT GERMPLASM
CRYOPRESERVATION
- Tesis doctoral –
ALINE SCHNEIDER TEIXEIRA
Licenciada en Biología
Directores:
María Elena González Benito (Doctora Ingeniero Agrónomo)
Antonio Diego Molina García (Doctor en Ciencias Químicas)
2013
La doctora María Elena González Benito, Catedrática de Universidad del Departamento de
Biología Vegetal (Universidad Politécnica de Madrid), y el doctor Antonio Diego Molina
García, Científico Titular del ICTAN-CSIC.
CERTIFICAN QUE la presente Tesis Doctoral titulada “Estudios termodinámicos de los
procesos de vitrificación en la crioconservación de germoplasma vegetal/ Thermodynamic
studies of vitrification processes in plant germplasm cryopreservation”, ha sido realizada bajo
su dirección por la licenciada en Biología Dña. Aline Schneider Teixeira.
Fdo María Elena González Benito Fdo. Antonio Diego Molina García
Caminante, no hay camino
Caminante son tus huellas El camino nada más;
caminante no hay camino se hace camino al andar. Al andar se hace camino y al volver la vista atrás
se ve la senda que nunca se ha de volver a pisar.
Caminante, no hay camino sino estelas sobre el mar.
¿Para qué llamar caminos A los surcos del azar...?
Antonio Machado
AGRADECIMIENTOS
Gracias a la Universidad Politécnica de Madrid y al Programa de Doctorado en
Biotecnología y Recursos Genéticos de Plantas y Microorganismos Asociados, y al CSIC por
la beca JAE-Pre y al proyecto �“CRYODYMINT�” (AGL2010-21989-C02-02) del Ministerio
de Ciencia e Innovación del Gobierno de España.
Gracias a los directores de esta tesis, el Dr. Antonio Diego Molina García, del Instituto de
Ciencia y Tecnología de los Alimentos y Nutrición (ICTAN-CSIC), y la Dra. María Elena
González Benito, del Dpto. de Biología Vegetal de la Universidad Politécnica de Madrid
(UPM), por su paciencia, visión crítica y espíritu didáctico. Mi más sincero agradecimiento
por toda su ayuda y por las oportunidades que me han brindado en el ámbito de la
investigación.
El doctorado ha supuesto una gran aventura para mí en muchos aspectos como sumergirme
en el fascinante mundo de la CRIOCONSERVACIÓN, donde además de aprender, conocí y
compartí con gente fantástica a la que no puedo dejar de agradecer.
Una de ellas es el �“Profesor Antonio�”, por los muchos momentos compartidos en el
instituto, por las largas tardes de mate y charlas. Siempre disponible para ordenar una idea en
un dibujo. Y a su esposa Lola, quien siempre encuentra tiempo para hacer se presente, por la
amistad y por varios momentos alegres.
Gracias al personal del ICTAN, de la USTA: Rubén Domínguez, Inmaculada Álvarez y
Estela Vega, por la cobertura técnica en los análisis de las muestras. A los técnicos de
informática: Luis Canet y Roberto Arcoya por la disponibilidad y amabilidad y al personal de
la administración Nuria Serrenes, Víctor Martín y Buenaventura Rodríguez. A Óscar García
Bodelón por las charlas y los cafés. A las investigadoras del CIB y del ICTAN, Dras. Begoña
de Ancos, María Escribano, Maite Serra, Pilar Rupérez, Sara Pérez, Sylvia Rodríguez, Miguel
Ángel Peñalva y José Luis García por la agradable compañía a la hora de comer. Al personal
de seguridad, por estar siempre atentos durante mi permanencia en el despacho por las
noches.
Mi amiga, Mirari Arancibia compañera de instituto, de investigación, de piso, de viajes,
paseos y de muchos findes de películas. Gracias por ser �“mis dos manos más�”.
Mi técnico querido Fernando Pinto, que trabaja en el Instituto de Ciencias Agrarias del
CSIC, en el Servicio de Microscopía Electrónica, que siempre aceptó los desafíos de mis
muestras, siempre dispuesto a ayudarme. Gracias por la agradable convivencia.
Mi más sincero agradecimiento a todos los profesores y a los técnicos que trabajan en el
laboratorio de cultivo in vitro de la E.U.I.T. Agrícola de la UPM, Carlos Ruiz y Marta
Huertas.
Mientras estudiaba en la Universidad Politécnica de Madrid, he tenido el privilegio de
contar con las enseñanzas y la amistad de varios compañeros con los que he compartido tan
buenos momentos. Entre ellos, quiero destacar a Carolina Kremer y Natacha Coelho (por ser
un soplo de aire fresco en el laboratorio); Shanez Zai, Narcizo Mesa, Cesar Tapia y James
Quiroz por el ánimo fuera de la Escuela; Julia Quintana, Juan Fernández y Manuel Rodelo mi
gran familia española; Mónica García la ultima incorporación a la familia pero no menos
querida y Alina Gheorghe que de compañera de clase pasó a compañera de instituto y amiga.
Amigos que fueron fundamentales en el trayecto, Fábia Andrade, Bartolomeu de Souza,
Danniely Campos Ferreira, Augusto Campos Ferreira, Paula Buarque, Melyssa Negri, Nadia
Silva, Bruna Catarina Fonseca gracias: por apoyarme en todo lo que me propuse y por hacer
me ver que lo imposible solo tarda un poco más.
Amigos a que no les importo la distancia y que estuvieron siempre presentes: Roberta
Casanova, Roberta Gomes, Fabiana Rotta, Fernando Lang, Catalina Guzmán, Pinar Singur,
Sophie Lalanne, Pachi Marino, Popi Coppola, Lucia Klein, Joaquin Hasperué, João Vieira,
Anai Loreiro, Simone Barrionuevo, Maria do Carmo Ruaro Peralba, Ady García, Osmar
Conte, Regilene Souza, Renata Azevedo, Giovani Brandão Mafra de Carvalho, Catarina
Fardilha, Susi Poersch, Margarete Cegolini, Cristiane Dalla Vecchia, Giselle Baccan, David
Duran, Ademir Coser, Erika Cavalcante y Rodrigo Morais de Souza.
Gracias al equipo del CIDCA de La Plata, en Argentina, dónde hice mi primera estancia:
mis amigos, las Dras. Miriam Martino, Lorena Deladino, Alba Navarro, Estela Bruno, Natalia
Graiver y Noemí Zaritzky y el M.Sc. Alex Fernando, por su apoyo, optimismo e incentivo.
Al equipo del �“USDA-ARS�”, de Fort Collins, en EEUU donde fue recibida por los Drs.
Christina Walters y Daniel Ballesteros, por los M.Sc. Jennifer Crane, Fernanda Pierruzi y
Patrick Reeves y por Lisa Hill y John Waddell, por su amabilidad y confianza, por creer en mí
y apoyarme en la realización del proyecto. Me llevé la sonrisa de cada uno de ellos, como
señal del buen ambiente y amistad.
Al equipo del �“Crop Research Institute�”, de Praga, en la Republica Checa, donde fui
recibida por los Drs. Milos Faltus, Jiri Záme ník, Renata Kotková, Jane Záme ník y Alois
Bilav ík, por la oportunidad, por la experiencia inolvidable y por las charlas de la cultura
checa, y a la Dra. Helena Stavelikova que me permitió hacer un recorrido de bicicleta por los
campos de cultivo del instituto VÚRV-Olomouc.
Gracias al personal del �“National Centre for Macromolecular Hydrodynamics, School of
Biosciences, University of Nottingham�”, de Gran-Bretaña, Gary Adams and Fahad Almutairi
por recibir mis muestras y amablemente ayudar en la construcción de ese trabajo.
Finalmente, agradezco de todo corazón a mi familia, a la que amo y extraño muchísimo:
mis hermanos (Denise y Fábio), mi cuñada (Valquiria), mi adorable ahijado (Otavio), mis
tíos, primos; pero fundamentalmente a mis padres (Glaci y Diamarante), por su gran amor y
por hacerme sentir que están tan cerca, gracias a todos porque sin sus constantes muestras de
apoyo y cariño nada de esto hubiera sido posible.
INDICE/INDEX
INDICE/INDEX
Página/Page
1.1. INTRODUCCIÓN/INTRODUCTION............................................................................ 1
1. 1.Crioconservación ........................................................................................................... 1
1.2. Técnicas de crioconservación basadas en la vitrificación ............................................ 2
1.2.1. Técnicas de vitrificación (en sentido estricto)...................................................... 3
1.2.2. Técnicas de vitrificación-�“droplet�” ...................................................................... 4
1.2.3. Técnicas de encapsulación-deshidratación........................................................... 4
1.2.4. Técnicas de encapsulación-vitrificación .............................................................. 5
1.3. Aspectos físicos de la crioconservación ....................................................................... 5
1.3.1. Formación de cristales de hielo ............................................................................ 5
1.3.2. La transición vítrea ............................................................................................... 7
1.3.3. La velocidad de cambio de temperatura en crioconservación............................ 10
1.3.4. Soluciones crioprotectoras.................................................................................. 11
1.3.5. Encapsulación en alginato .................................................................................. 12
1.3.6. Quitosano............................................................................................................ 13
1.4. La menta como modelo en el estudio de la crioconservación .................................... 14
2 OBJETIVOS/AIMS............................................................................................................. 16
3. MEASUREMENT OF COOLING AND WARMING RATES IN VITRIFICATION-
BASED PLANT CRYOPRESERVATION PROTOCOLS................................................ 17
3.1. Introduction ................................................................................................................ 17
3.2. Materials and Methods ............................................................................................... 19
3.2.1. Plant materials pre-culture and shoot tips extraction.......................................... 19
iv
INDICE/INDEX
3.2.2. Dehydration of shoot tips in the vitrification method ........................................ 19
3.2.3. Vitrification......................................................................................................... 20
3.2.4. Droplet-vitrification............................................................................................ 20
3.2.5. Plant recovery and viability................................................................................ 20
3.2.6. Temperature measurement and cooling and warming rates ............................... 21
3.2.7. Thermocouples ................................................................................................... 21
3.2.8. Experimental set-up and temperature change rate measurement design ............ 22
3.3. Results and Discussion............................................................................................... 23
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS .............. 33
4.1. Introduction ................................................................................................................ 33
4.2. Materials and Methods ............................................................................................... 36
4.2.1. Plant material pre-culture and shoot tips extraction ........................................... 36
4.2.2. Incubation and dehydration of shoot tips ........................................................... 36
4.2.3. Plant recovery and viability................................................................................ 37
4.2.4. Low temperature scanning electron microscopy ................................................ 38
4.2.5. Dry matter and water content determination ...................................................... 39
4.2.6. Differential scanning calorimetry ....................................................................... 39
4.2.7. Sucrose content assessment in shoot tips............................................................ 41
4.3. Results and Discussion............................................................................................... 42
4.3.1. Detection of glassy state and ice by cryo-SEM.................................................. 42
4.3.2. Determination of pre-culture effecte by cryo-SEM............................................ 43
4.3.3. Observed plant recovery and viability................................................................ 43
4.3.4. Analysis of the droplet-vitrification protocol steps by cryo-SEM...................... 45
v
INDICE/INDEX
4.3.5. Changes in the dry mass and water content during the droplet-vitrification
protocol......................................................................................................................... 45
4.3.6. Measurement of ice heat of fusion using DSC ................................................... 47
4.3.7. Effects of quench-cooling on heat of fusion of ice............................................. 50
4.3.8. Detection of glass transition by DSC.................................................................. 51
4.3.9. Changes in sucrose content during the droplet-vitrification protocol ................ 51
4.4. General discussion...................................................................................................... 53
5. CRYOPRESERVATION OF MINT SHOOT TIPS BY ENCAPSULATION-
DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-
SEM AND DSC................................................................................................................ 57
5.1. Introduction ................................................................................................................ 57
5.2. Materials and Methods ............................................................................................... 59
5.2.1. Plant material and shoot tips extraction.............................................................. 59
5.2.2. Shoot tips cryopreservation procedure ............................................................... 59
5.2.3. Water content determination............................................................................... 60
5.2.4. Differential scanning calorimetry ....................................................................... 61
5.2.5. Temperature measurement and cooling and warming rates ............................... 62
5.2.6. Low temperature scanning electron microscopy ................................................ 63
5.3. Results and Discussion............................................................................................... 64
5.3.1. Water content reduction during the drying period.............................................. 64
5.3.2. Calorimetric studies on water status ................................................................... 66
5.3.3. Cooling and warming rate determination ........................................................... 68
5.3.4. Low temperature scanning electron microscopy ................................................ 71
vi
5.3.5. Cryopreserved shoot tips survival and re-growth............................................... 74
INDICE/INDEX
5.4. General discussion...................................................................................................... 74
6. GLASS TRANSITION AND ANNEALING BEHAVIOUR OF PLAN
VITRIFICATION SOLUTIONS ................................................................................... 77
6.1. Introduction ................................................................................................................ 77
6.2. Materials and Methods ............................................................................................... 79
6.2.1. Standard compounds .......................................................................................... 79
6.2.2. Solutions ............................................................................................................. 79
6.2.3. Plant material...................................................................................................... 80
6.2.4. Differential scanning calorimetry conditions ..................................................... 80
6.2.5. Glass transition characterization for PVS........................................................... 81
6.2.6. Cooling rate test .................................................................................................. 81
6.2.7. Warming rate test................................................................................................ 81
6.2.8. Storage test ......................................................................................................... 82
6.2.9. Annealing test ..................................................................................................... 82
6.2.10. Statistical analysis ............................................................................................ 83
6.3. Results and Discussion............................................................................................... 83
6.3.1. Vitrification behaviour of different vitrification solutions ................................. 83
6.3.2. Effect of cooling rate on vitrification behaviour ................................................ 85
6.3.3. Effect of warming rate on vitrification behaviour .............................................. 89
6.3.4. Crystallinity of garlic shoot tips ......................................................................... 89
6.3.5. Effect of the storage time on the vitrification behaviour of PVS ....................... 91
6.3.6. Annealing of PVS1 and PVS3............................................................................ 93
7. THE APPLICATION OF CHITOSAN FILM ON ALGINATE BEADS FOR
CRYOPRESERVATION USES .................................................................................... 95
vii
INDICE/INDEX
7.1. Introduction ................................................................................................................ 95
7.2. Materials and Methods ............................................................................................... 99
7.2.1. Chitosan types and hydrodynamic characterization ........................................... 99
7.2.2. Alginate beads .................................................................................................. 100
7.2.3. Chitosan solution and alginate bead treatment ................................................. 100
7.2.4. water content determination and air dehydration studies ................................. 100
7.2.5. Differential scanning calorimetry and frozen and unfrozen water fractions .... 101
7.2.6. Low temperature scanning electron microscopy observations......................... 101
7.2.7. Environmental scanning electron microscopy.................................................. 102
7.3. Results and Discussion............................................................................................. 102
7.3.1. Chitosan hydrodynamic characterization ......................................................... 102
7.3.2. Water content determination and air dehydration ............................................ 105
7.3.3. Calorimetric studies on water status................................................................. 106
7.3.4. Scanning electron microscopy of alginate beads with and without chitosan ... 107
8. DISCUSIÓN GENERAL/GENERAL DISCUSSION................................................... 110
9. CONCLUSIONES/CONCLUSIONS.............................................................................. 122
10. REFERENCIAS/REFERENCES ................................................................................. 124
11. ANEXOS/ANNEXES ..................................................................................................... 154
Annex I. COMPOSITION OF VITRIFICATION SOLUTIONS ................................... 153
Annex II. PUBLICATIONS............................................................................................ 155
viii
INDICE/INDEX
LISTA DE FIGURAS/LIST OF FIGURES
Página/Page
Figura 1.1. Ilustración sobre la transición vítrea en comparación con una fusión/cristalización de una sustancia pura no polimérica. Se denota el estado de equilibrio mediante un trazo continuo y el de no equilibrio por trazos discontinuos. (a) Volumen específico, b) viscosidad aparente, (c) capacidad calorífica (primeras derivadas), representando la dirección hacia arriba una transición endotérmica. (Figura tomada de Walstra, 2003).................................................9
Figura 1.2. Ilustración sobre la transición vítrea de una solución de vitrificación (PVS3) en comparación con una fusión/cristalización de ápices de ajo. Se denota el estado de equilibrio mediante un trazo continuo y el de no equilibrio por trazos discontinuos .................................9
Figura 1.3. Ilustración sobre la transición vítrea en comparación con la fusión en un termograma de DSC (A) y el acercamiento de las temperaturas, por aumento de TG y disminución de Tf (B) ...............................................................................................................10
Figure 3.1. Experimental set up for thermal change rate measurement for (a) vitrification, or (b), droplet-vitrification protocols ...........................................................................................22
Figure 3.2. Cooling (a) and warming (b) evolution determined in samples treated after the vitrification protocol. Data are the average of at least three independent determinations. Control data were obtained with the thermocouple tip inserted in an empty cryovial. The lines shown represent the individual data points measured .............................................................24
Figure 3.3 a and b. Measurements of cooling rate obtained following the droplet-vitrification protocol: (a) AFS inside cryovial and (b) naked AFS. Data are the average of at least three repeats ......................................................................................................................................25
Figure 3.4 a and b. Measurements of warming rates obtained following the droplet protocols. Data are the average of at least three repeats............................................................................26
Figure 3.5. Mint shoot tip survival and re-growth percentages after different cryopreservation techniques and a 4-week recovery period. Means of survival or re-growth with the same letter are not significantly different according to the Duncan�’s Multiple Range Test at alpha = 0.05. Bars: standard error .................................................................................................................27
Figure 4.1. Scheme of the steps of the droplet cryopreservation protocol employed, see text for more details on media and solutions employed .................................................................37
Figure 4.2. Cartoon showing the three mint tips on the microscopy holder before insertion in the microscope (a) and after equatorial freeze fracture (b) ... ..................................................38
Figure 4.3. Cryo-SEM micrographs of shoot tips cooled in LN in the microscope cryo-unit (see Methods). Control (A); stage a, cold treatment (B); stage b, preculture (C and D); stage c, loading (E and F), and stage d, dehydration (G and H). The bar corresponds to 10 µm (A, B, D, F, H) or 50 µm (C, E, G) ...............................................................................................44
ix
INDICE/INDEX
Figure 4.4. Typical DSC thermograms corresponding to re-warming processes of shoot tips in different stages of the droplet cryopreservation protocol: cold treatment (stage a), preculture (stage b), loading (stage c) and dehydration (stage d). Each experiment was performed with five shoot tips. The insert shows an expanded thermogram section which allows the glass transition, obtained with 30 tips, to be appreciated. Scanning rate was 10ºC min-1. (See Materials and Methods and Figure 4.1. for more details). The ordinates scale of the thermograms baseline is arbitrar ........................................................................................48
Figure 4.5. Typical DSC thermograms corresponding to re-warming processes of the cryopreservation solutions employed in different starges of the droplet cryopreservation protocol: preculture (stage b): liquid MS medium containing 0.3M sucrose, loading (stage c): loading solution and dehydration (stage d): PVS2. The insert shows an expanded thermogram section which allows the glass transition to be appreciated. Scanning rate was 10ºC min-1. (See Materials and Methods and Figure 14 for more details). The ordinates scale of the thermograms baseline is arbitrary ............................................................................................49
Figure 4.6. DSC scans showing warming at 10ºC min-1 for shoot tips treated at the preculture stage (b) of the droplet cryopreservation protocol. Cooling rate was 10ºC min-1 or quenching (direct immersion of the DSC pan in LN) ...............................................................................50
Figura 5.1. Scheme of the steps of the encapsulation-dehydration protocol employed, see text for more details on media and solutions used ..........................................................................60
Figura 5.2. Experimental set up for thermal change rate measurement for encapsulation-dehydration ..............................................................................................................................63
Figura 5.3. Cartoon showing the three mint tips-containing beads on the microscopy holder before insertion in the microscope (a) and after equatorial freeze fracture (b) .......................64
Figura 5.4. Typical DSC thermograms corresponding to re-warming processes of shoot tips with beads of the encapsulation-dehydration protocol. Beads were desiccated under the air flow of a laminar-flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h and T6 = 6 h. Data are the mean of at least three repeats. Each experiment was performed with three beads. The inserts show an expanded thermogram section which allows seeing the glass transition. Scanning rate was 10ºC m-1. The ordinates scale of the thermograms baseline is arbitrary ........................................................................66
Figure 5.5. Ratio Wf(dm) Wu(dm)-1 for mint shoot tips (control and preculture) and shoot tip-
containing beads (for drying times 0 to 6 hours). Bars: standard error ...................................67
Figure 5.6. Temperature evolution, during cooling by immersion in LN, of cryovials with 10 alginate beads containing mint shoot tip and treated after the encapsulation-dehydration protocol. Beads were desiccated under the air flow of a laminar flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = cryovial without beads. Data are the mean of at least three repeats .................70
Figure 5.7. Temperature evolution, during warming in a 45ºC water bath for 1 min and then in 25ºC for 1 min of cryovials with 10 alginate beads containing mint shoot tip and treated after the encapsulation-dehydration protocol. Beads were desiccated under the air flow of a laminar flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3
x
INDICE/INDEX
h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = cryovial without beads. Data are the mean of at least three repeats ....................................................................................................................70
Figura 5.8. Cryo-SEM micrographs of alginate beads. View of beads external surface at drying time 0 (A) and 6 hours (B), and internal cryofracture surface, at drying time 0 (C) and 6 hours (D). The bar corresponds to 10 µm .............................................................................71
Figura 5.9. Cryo-SEM micrographs of mint shoot tips in different stages of the encapsulation-dehydration protocol. Drying times (hours): A (0), B (1), C (2), D (3), E (4), F (5), G (6). H, tip growing for one week, after warming. The bar corresponds to 10 µm ........73
Figura 5.10. Mint shoot tip survival and re-growth percentages after dehydration for different times following the encapsulation-dehydration protocol. Observations were made after a 4-week recovery period. Beads were desiccated under the air flow of a laminar flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = before cooling in LN. Data are the mean of at least three repeats. Means of survival or re-growth with the same letter are not significantly different according to the Duncan�’s Multiple Range Test at alpha = 0.05. Bars: standard error ...............................75
Figura 6.1. Scheme of the steps of the annealing protocol used; see text for more details on solutions employed ..... .............................................................................................................83
Figure 6.2. PVS3 thermograms showing the glass transition, performed at a warming rate of 10ºC min-1 and after cooling at different rates. Quenching: direct immersion of the DSC pan in LN; cryovial: pan included in a cryovial and then in LN. White arrows mark the glass transition inflection point ........................................................................................................86
Figure 6.3. Glass transition temperature (a) and glass transition heat capacity changes (b) for the different PVS and garlic shoot tips (tips in the last step of the droplet-vitrification method), obtained in warming (10ºC min-1) DSC experiments, after being cooled at different rates. * Rate not tested for garlic samples. Bars: standard error .............................................88
Figure 6.4. Glass transition temperature (a and b) and heat capacity (c and d) obtained in DSC during warming at different rates (5, 10 or 20°C min-1). DSC pans had been cooled either at 10°C min-1 in the calorimeter or by quenching in LN. Bars: standard error ..... ........90
Figura 6.5. Typical scan of shoot tips of garlic after treatment with PVS3 for 2 h. Both cooling and warming rates were 10ºC min-1. The black arrows mark to the glass transition inflection point in cooling and warming scans, while gray arrows signal the freezing and melting events...........................................................................................................................91
Figure 6.6. Typical thermogram of PVS1 on the step E-F of Figure 6.1. The annealing areas were comprised between the curve and the extended baseline ...............................................93
Figura 6.7. Annealing area versus temperature (D in Figure 6.1) of the solutions PVS1( ) and PVS3(�•) .............................................................................................................................94
Figure 7.1. Schematic representation of the chemical structures of the chitin and chitosan (adapted from Goy et al., 2009) ..............................................................................................96
Figure 7.2. Plots for extrapolation of the sedimentation coefficient of the different chitosan types to infinite dilution: a = MMW, b = LMW and c = C chitosan .....................................103
xi
INDICE/INDEX
Figure 7.3. Evolution of the water content over total sample mass, Wc(s), with air flow drying time, for alginate beads covered with chitosan or not, prepared with 2 and 3% sodium alginate concentration .........................................................................................................................105
Figure 7.4. Cyro-SEM micrographs showing the external AB (right) and ACB (left) bead surface after quench cooling to liquid nitrogen temperature. The bar corresponds to 5 µm .....................................................................................................................................................107
Figure 7.5. Environmental SEM micrographs showing bead surfaces after different air-flow dehydration times: a) AB and c) ACB (0 hours -not dehydrated beads); b) AB, d) and e) ACB (5 hours dehydration). The bar corresponds to 50 m for a) and e) and to 100 m for b), c) and d) .....................................................................................................................................108
xii
INDICE/INDEX
LISTA DE TABLAS/LIST OF TABLES
Página/Page
Table 3.1. Cooling and re-warming rate measurements obtained following the vitrification protocols ..................................................................................................................................30
Table 3.2. Cooling and re-warming rate measurements obtained following the droplet-vitrification protocols ..............................................................................................................31
Table 4.1. Compositional and thermal parameters (mean standard deviation) of the thawing events obtained from DSC thermograms for mint shoot tips specimens at different stages (a, b, c and d) of the droplet cryopreservation protocol ...............................................................46
Table 4.2. Solute content (mean standard deviation) of mint shoot tips at the different stages of the droplet cryopreservation protocol.... ..............................................................................53
Table 5.1. Compositional and thermal parameters of the thawing events obtained from gravimetry and DSC thermograms for mint shoot tips specimens at different stages of the encapsulation-dehydration cryopreservation protocol .............................................................64
Table 5.2. Cooling and warming rate measurements obtained following the encapsulation-dehydration protocol ................................................................................................................68
Table 6.1. Composition of vitrification solutions ...................................................................80
Table 6.2. Glass transition parameters of different plant vitrification solutions (cooling and warming rates were both 10°C min-1) .....................................................................................84
Table 6.3. Crystallization parameters (mean standard deviation) of garlic shoot tips in the last stage of the droplet-vitrification protocol (in PVS3). Onset of the melting endotherm and crystallinity as crystallization percentage, both measured in the warming scan. Cooling scans were performed at 10ºC min-1 and warming scans at different rates .......................................91
Table 6.4. Cryopreservation solutions glass transition temperature (mean standard deviation) versus storage time .................................................................................................92
Table 6.5. Cryopreservation solutions heat capacity change (mean standard deviation) versus storage time ..................................................................................................................92
Table 7.1. Analytical ultracentrifugation-derived and chitosan size parameters (mean ± standard deviation) ................................................................................................................103
Table 7.2. Water and dry matter content (mean ± standard deviation) for alginate beads (AB) and chitosan-covered alginate beads (ACB). See Materials and Methods for parameter description .............................................................................................................................105
Table 7.3. Calorimetric parameters (mean ± standard deviation) obtained for 3% alginate beads (AB) and chitosan-covered alginate beads (ACB). See Materials and Methods for parameter description ............................................................................................................106
xiii
RESUMEN/ABSTRACT
RESUMEN
En la actualidad, las técnicas de crioconservación poseen una importancia creciente para el
almacenamiento a largo plazo de germoplasma vegetal. En las dos últimas décadas, estos
métodos experimentaron un gran desarrollo y se han elaborado protocolos adecuados a
diferentes sistemas vegetales, utilizando diversas estrategias como la vitrificación, la
encapsulación-desecación con cuentas de alginato y el método de “droplet”-vitrificación. La
presente tesis doctoral tiene como objetivo aumentar el conocimiento sobre los procesos
implicados en los distintos pasos de un protocolo de crioconservación, en relación con el estado
del agua presente en los tejidos y sus cambios, abordado mediante diversas técnicas biofísicas,
principalmente calorimetría diferencial de barrido (DSC) y microscopía electrónica de barrido a
baja temperatura (crio-SEM). En un primer estudio sobre estos métodos de crioconservación, se
describen las fases de enfriamiento hasta la temperatura del nitrógeno líquido y de
calentamiento hasta temperatura ambiente, al final del periodo de almacenamiento, que son
críticas para la supervivencia del material crioconservado. Tanto enfriamiento como
calentamiento deben ser realizados lo más rápidamente posible pues, aunque los bajos
contenidos en agua logrados en etapas previas de los protocolos reducen significativamente las
probabilidades de formación de hielo, éstas no son del todo nulas. En ese contexto, se analiza
también la influencia de las velocidades de enfriamiento y calentamiento de las soluciones de
crioconservación de plantas en sus parámetros termofísicos referente a la vitrificación, en
relación su composición y concentración de compuestos. Estas soluciones son empleadas en la
mayor parte de los protocolos actualmente utilizados para la crioconservación de material
vegetal. Además, se estudia la influencia de otros factores que pueden determinar la estabilidad
del material vitrificado, tales como en envejecimiento del vidrio. Se ha llevado a cabo una
investigación experimental en el empleo del crio-SEM como una herramienta para visualizar el
estado vítreo de las células y tejidos sometidos a los procesos de crioconservación. Se ha
comparado con la más conocida técnica de calorimetría diferencial de barrido, obteniéndose
resultados muy concordantes y complementarios. Se exploró también por estas técnicas el
efecto sobre tejidos vegetales de la adaptación a bajas temperaturas y de la deshidratación
inducida por los diferentes tratamientos utilizados en los protocolos. Este estudio permite
observar la evolución biofísica de los sistemas en el proceso de crioconservación. Por último,
se estudió la aplicación de películas de quitosano en las cuentas de alginato utilizadas en el
protocolo de encapsulación. No se observaron cambios significativos en su comportamiento
RESUMEN/ABSTRACT
frente a la deshidratación, en sus parámetros calorimétricos y en la superficie de las cuentas. Su
aplicación puede conferir propiedades adicionales prometedoras.
RESUMEN/ABSTRACT
ABSTRACT
Currently, cryopreservation techniques have a growing importance for long term plant
germplasm storage. These methods have undergone great progress during the last two decades,
and adequate protocols for different plant systems have been developed, making use of diverse
strategies, such as vitrification, encapsulation-dehydration with alginate beads and the droplet-
vitrification method. This PhD thesis has the goal of increasing the knowledge on the processes
underlying the different steps of cryopreservation protocols, in relation with the state of water
on tissues and its changes, approached through diverse biophysical techniques, especially
differential scanning calorimetry (DSC) and low-temperature scanning electron microscopy
(cryo-SEM). The processes of cooling to liquid nitrogen temperature and warming to room
temperature, at the end of the storage period, critical for the survival of the cryopreserved
material, are described in a first study on these cryopreservation methods. Both cooling and
warming must be carried out as quickly as possible because, although the low water content
achieved during previous protocol steps significantly reduces ice formation probability, it does
not completely disappear. Within this context, the influence of plant vitrification solutions
cooling and warming rate on their vitrification related thermophysical parameters is also
analyzed, in relation to its composition and component concentration. These solutions are used
in most of the currently employed plant material cryopreservation protocols. Additionally, the
influence of other factors determining the stability of vitrified material is studied, such as glass
aging. An experimental research work has been carried out on the use of cryo-SEM as a tool for
visualizing the glassy state in cells and tissues, submitted to cryopreservation processes. It has
been compared with the better known differential scanning calorimetry technique, and results
in good agreement and complementary have been obtained. The effect on plant tissues of
adaptation to low temperature and of the dehydration induced by the different treatments used
in the protocols was explored also by these techniques. This study allows observation of the
system biophysical evolution in the cryopreservation process. Lastly, the potential use of an
additional chitosan film over the alginate beads used in encapsulation protocols was examined.
No significant changes could be observed in its dehydration and calorimetric behavior, as well
as in its surface aspect; its application for conferring additional properties to gel beads is
promising.
INDICE/INDEX
ACRÓNIMOS Y ABREVIATURAS
AB: �“alginate beads�”, cuentas de alginato
ABC: �“chitosan-treated alginate beads�”, cuentas de alginato recubiertas de quitosano
AFS: tira de papel alumínio
Aw: peso molecular del agua
aw: actividad del agua
Cpi: capacidad calorífica a presión constante del hielo
Cpw: capacidad calorífica a presión constante del agua
cryo-SEM: microscopía electrónica de barrido de baja temperatura
DA: grado de acetilación
dm: contenido de materia seca
DMSO: dimetil sulfóxido
DSC: calorimetría diferencial de barrido
HPLC: cromatografía de líquidos de alto rendimiento
IBPGR: �“International Board for Plant Genetic Resources�”
IC: cromatografía iónica de alto rendimiento
LMW chitosan: tipo comercial de quitosano, de bajo peso molecular
LN: liquid nitrogen (nitrógeno líquido)
M: peso molecular
MMW chitosan: tipo comercial de quitosano, de peso molecular medio
MS: medio Murashige y Skoog (1962)
NaCleq: concentración molar de cloruro sódico equivalente
NL: nitrógeno líquido
PAD: detección amperométrica por pulsos
PVS: soluciones de vitrificación de plantas
xiv
INDICE/INDEX
PVS1: �“Plant vitrification solution nº 1�”, solución de vitrificación de plantas nº 1 (Uragami et al., 1989)
PVS2: �“Plant vitrification solution nº 2�”, solución de vitrificación de plantas nº 2 (Sakai et al., 1990)
PVS2 mod: solución de vitrificación de plantas nº 2, modificada
PVS3: �“Plant vitrification solution nº 3�”, solución de vitrificación de plantas nº 3 (Nishizawa et al., 1993).
PVS3 mod: solución de vitrificación de plantas nº 3, modificada.
So20,W: coeficiente de sedimentación a dilución infinita, en agua y a 20ºC
Suc: contenido de sacarosa
Suc(dm): concentración de sacarosa respecto a la masa seca de la muestra.
Suceq: concentración molar de sacarosa equivalente
Suc(s): concentración de sacarosa respecto a la masa total de muestra
Tf: temperatura o punto de congelación (o fusión) de equilibrio
Tf*: temperatura de fusión de equilibrio del agua pura
Tf(onset): temperatura de inicio (onset) de la fusión de hielo detectada mediante DSC
Tf(peak): temperatura del pico de la fusión de hielo detectada mediante DSC
TG: Temperatura de transición vítrea
Wc: contenido de agua
Wc(dm): contenido de agua con respecto a la masa seca de muestra
Wc(s): contenido de agua con respecto a la masa total de muestra
Wf: contenido de agua congelada
Wf(dm): contenido de agua congelada respecto a la masa seca de muestra
Wf(s): contenido de agua congelada respecto a la masa total de muestra
Wf(w): contenido de agua congelada respecto al contenido total de agua
Wu: contenido de agua no congelada
Wu(dm): contenido de agua no congelada respecto a la masa seca de muestra
Wu(s): contenido de agua no congelada respecto a la masa total de muestra
xv
INDICE/INDEX
Wu(w): contenido de agua no congelada respecto al contenido total de agua
Hf: calor latente de cambio de fase o entalpía de fusión
Hf(dm): entalpía de cambio de fase respecto a la masa de materia seca
Hf(s): entalpía de cambio de fase respecto a la masa de muestra total
Hf(w)*: entalpía específica de fusión del agua pura
Hf(w): entalpía de cambio de fase respecto al contenido de agua
T: gradiente térmico
Tf: depresión de la temperatura de fusión
s: factor pre-exponencial para el cálculo del peso molecular a partir del coeficiente de sedimentación
: osmolalidad
: Densidad
xvi
1. INTRODUCCIÓN/INTRODUCTION
1. INTRODUCCIÓN
1.1. Crioconservación
Las técnicas de crioconservación, ampliamente empleadas en la actualidad para la
conservación de muy diversos tipos de materiales biológicos, tienen su origen en
investigaciones iniciales llevadas a cabo con células y tejidos animales. Gracias al
descubrimiento de las cualidades del glicerol y del dimetilsulfóxido (DMSO; Mounib et al.,
1968; Graybill & Horton, 1969) capaces de disminuir los efectos negativos que podría
ocasionar la inmersión de tejido vivo en nitrógeno líquido, se obtuvieron los primeros éxitos
significativos en la crioconservación y recuperación de espermatozoides y células sanguíneas.
Más adelante, la técnica se aplicó a estructuras vegetales, tales como semillas y tejidos
preparados para soportar situaciones de estrés (baja temperatura), a consecuencia de presentar
un bajo contenido hídrico y acumular substancias de reserva y compuestos con capacidad
crioprotectora, como aminoácidos (glicina, betaína y prolina) y azúcares, principalmente,
manitol (Reed, 1988). De entre los distintos sistemas organizados, semillas y embriones se
consideran los más adecuados, siempre que sea posible, para la conservación de la diversidad
de los recursos fitogenéticos (Henshaw et al., 1980). Mediante el empleo de las técnicas
desarrolladas específicamente para diversos tejidos y especies, los materiales crioconservados
pueden recuperar satisfactoriamente su funcionalidad tras el almacenamiento (Benson et al.,
1996; Sakai & Engelmann, 2007).
La mayoría de las células vegetales contienen elevadas cantidades de agua por lo que son
extremamente sensibles a las temperaturas por debajo de 0ºC. Para impedir la formación de
cristales de hielo, evitando daños a las membranas y a otros elementos celulares, se recurre a
inducir procesos de deshidratación y aumento de la concentración intracelular de solutos y
adicionar sustancias de actividad crioprotectora. Además, los cambios de temperatura
(enfriamiento y calentamiento) tienen un papel determinante en la formación de hielo y en su
localización y efecto fisiológico. Estas dos etapas de los protocolos de crioconservación,
deshidratación y cambios térmicos, deben ser optimizadas.
La formación de hielo intracelular se considera generalmente letal, si bien el mecanismo
del daño por congelación (freeze injury) resulta controvertido en la literatura (Benson, 2008).
1
1. INTRODUCCIÓN/INTRODUCTION
El sitio primario de la lesión por congelación en sistemas biológicos parece ser la membrana
celular (Steponkus, 1992). Estudios morfológicos apoyan esa idea demostrando la estrecha
relación entre los cambios en la ultraestructura de la membrana plasmática y las tensiones de
enfriamiento y calentamiento (Singh, 1979; Fujikawa, 1980, 1981; Pearce, 1988). La
formación de hielo extracelular, que puede tener lugar incluso tras una exposición prolongada
de las células a soluciones concentradas o su deshidratación, si el enfriamiento es
especialmente lento, también puede provocar daños a la membrana y otros componentes
celulares (Mazur, 1970).
Según el �“International Board for Plant Genetic Resources�”, IBPGR (1982), la
crioconservación es una tecnología indicada para el mantenimiento de especies con
propagación vegetativa, plantas con semillas recalcitrantes, o especies amenazadas de
extinción. Es considerado un método eficiente, práctico y de bajo coste para la preservación
germoplasma vegetal, y adicionalmente, posee la capacidad de mantener el material viable
por un tiempo considerado indefinido (Touchell & Dixon, 1994).
1.2. Técnicas de crioconservación basadas en la vitrificación
La conservación de material biológico a baja temperatura aprovecha la reducción de
velocidades de difusión y reacciones asociadas al descenso térmico para evitar cambios y
deterioro del material, pero al descender por debajo de la temperatura de equilibrio de
congelación, la formación de cristales de hielo suele constituir un importante riesgo para la
integridad celular. Las técnicas de crioconservación se basan en la estabilización del sistema,
por la práctica anulación de los procesos metabólicos que tiene lugar por debajo de la
transición vítrea. Así, los sistemas vitrificados, mediante diferentes metodologías, serían
básicamente estables frente al tiempo, pues los procesos basados en movilidad molecular no
podrían tener lugar por debajo de esta transición. Estos procesos incluyen la formación de
hielo misma, que requiere de una masiva reorganización de la estructura del agua líquida, y
por tanto, estaría completamente impedida en este estado. Una vez alcanzada la temperatura
final, de nitrógeno líquido, la formación de hielo no es posible. Sin embargo, existen dos
momentos fundamentales a considerar para evitar daños en el material que se desea
crioconservar: el riesgo de formación de cristales de hielo al inicio (enfriamiento) y al final
(calentamiento) del proceso.
2
1. INTRODUCCIÓN/INTRODUCTION
Las técnicas de crioconservación pueden dividirse, según la velocidad de enfriamiento
empleada, en métodos clásicos, donde se logra una deshidratación inducida por el descenso
controlado de la temperatura (mediada por la formación de hielo extracelular), y métodos más
novedosos, donde se produce una vitrificación global de las soluciones contenidas en los
tejidos, es decir, tanto de la soluciones intra- como extracelulares (Benson, 2008).
El método clásico se basa en el descenso lento de la temperatura. En este caso, el agua del
medio intracelular es extraída hacia el medio extracelular, donde se forman preferentemente
los cristales de hielo, por tener una concentración de solutos menor, por lo que las soluciones
del citoplasma se concentran, facilitando su entrada en el estado vítreo. El resultado es una
región extracelular con hielo y crecientes cantidades de agua, y un citoplasma cada vez más
concentrado y viscoso, libre de hielo y sin daños celulares.
Se suele operar en dos etapas. Tras una etapa de enfriamiento lento (por ejemplo, a una
velocidad de 0,5-2,0ºC min-1, hasta -40ºC), el espécimen se sumerge en nitrógeno líquido
(LN) (-195,9ºC), lo que constituye un enfriamiento mucho más rápido. Se pueden usar
congeladores programables que realizan estas etapas a velocidad de enfriamiento controlada
(González-Benito et al., 2004).
Los nuevos métodos se basan en la vitrificación de todas las soluciones del sistema
mediante un descenso rápido de la temperatura obtenido por la inmersión directa del material
en nitrógeno líquido. Las soluciones intra- y extracelulares, cuyo contenido en agua y solutos
ha sido previamente modulado, pasan directamente al estado vítreo y el enfriamiento ocurre
sin formación alguna de hielo ni daño para las células. Estas técnicas se dividen en:
vitrificación en sentido estricto, vitrificación-droplet, encapsulación-deshidratación,
encapsulación-vitrificación.
1.2.1. Técnicas de vitrificación (en sentido estricto)
Como se ha indicado, la vitrificación conlleva la práctica solidificación del líquido, sin
cristalización de hielo, sino mediante una elevación extrema de la viscosidad durante el
enfriamiento. Para alcanzarlo, los sistemas biológicos deben ser sometidos a diversos
procesos previos. Los tejidos son primeramente cultivados en medios con altas
concentraciones de sacarosa u otros agentes osmóticos, con la finalidad de reducir su
contenido en agua e inducir la síntesis de sustancias de defensas naturales frente a estrés.
3
1. INTRODUCCIÓN/INTRODUCTION
Posteriormente se transfieren a una solución glicerol-sacarosa, llamada solución de carga
(Towill, 1990; Sakai et al., 2000). A continuación se reduce el contenido en agua de las
soluciones intra y extracelulares, exponiendo los tejidos a soluciones crioprotectoras
altamente concentradas (solución de vitrificación); posteriormente el espécimen (en el interior
de un criovial) se sumerge rápidamente en nitrógeno líquido. Tras el calentamiento (rápido),
la solución de vitrificación se retira del criovial y se añade una solución de, generalmente,
1,2M sacarosa y se sustituye una vez con solución fresca. Por último el material vegetal se
transfiere al medio de cultivo.
La reducción del contenido acuoso y la disminución de la movilidad molecular
conseguidos mediante estos tratamientos permiten que, mediante un proceso de enfriamiento
lo suficientemente rápido (inmersión directa en nitrógeno líquido) las soluciones vitrifiquen.
1.2.2. Técnicas de vitrificación-droplet
La técnica de vitrificación-droplet puede considerarse una modificación de la anterior, si
bien deriva de la técnica de congelación de gotas desarrollada por Kartha et al. (1982). En
este protocolo, los sistemas vegetales son tratados con una solución de vitrificación. Gotas de
esta solución conteniendo el material a crioconservar son dispuestas sobre pequeñas tiras de
papel de aluminio y el conjunto es rápidamente sumergido en nitrógeno líquido. En el
posterior proceso de recuperación, el calentamiento se realiza sumergiendo el soporte de papel
de aluminio en medio líquido, a temperatura ambiente.
La ventaja principal de ésta técnica es la posibilidad de alcanzar una velocidad muy alta de
enfriamiento y calentamiento, debido al volumen reducido de la solución de vitrificación
donde están sumergidas las estructuras vegetales y a la escasa masa del conjunto.
1.2.3. Técnicas de encapsulación-deshidratación
La encapsulación-deshidratación, desarrollada por Fabre & Dereuddre (1990), consiste en
la inclusión de estructuras vegetales en cuentas de alginato, seguida de su cultivo en
soluciones de sacarosa altamente concentradas y, posteriormente, de una etapa de
deshidratación física, que elimina parte del agua congelable a 0ºC, e inmersión directa en NL.
4
1. INTRODUCCIÓN/INTRODUCTION
La deshidratación física se lleva a cabo empleando gel de sílice o el flujo de aire de una
campana de flujo laminar (Paulet et al., 1993).
La técnica de vitrificación permite el procesado de los sistemas biológicos para su
conservación en protocolos relativamente rápidos. Bajo condiciones óptimas, produce
mayores niveles de recuperación y reduce enormemente el tiempo necesario para la
deshidratación de muestras (Sakai et al., 1990). Sin embargo, los principales puntos débiles de
esta técnica son la dificultad de tratar un gran número de especímenes a la vez y la duración
de los pasos del protocolo, muy reducida y necesariamente exacta, lo que dificulta la
manipulación de pequeños explantes.
Por el contrario, los protocolos de encapsulación-deshidratación suelen ser más
prolongados comparados con los de vitrificación. Sin embargo, los explantes encapsulados
son más fáciles de manipular, gracias al tamaño relativamente grande del recubrimiento de
alginato. Desventajas de ésta técnica son, en general, su menor porcentaje de supervivencia y
de recuperación, y el mayor grado de deshidratación que ocasiona en las estructuras vegetales
(Matsumoto & Sakai, 1995) comparada con la técnica de vitrificación.
1.2.4. Técnicas de encapsulación-vitrificación
La encapsulación-vitrificación consiste en una combinación de las ventajas de la técnica de
encapsulación-deshidratación y vitrificación. Se reunieron en un mismo protocolo la facilidad
de manipulación de los explantes encapsulados y el tratamiento de las muestras mediante
sustancias crioprotectoras (rapidez de ejecución), seguido por la deshidratación con
soluciones de vitrificación, previamente a la etapa de enfriamiento (Sakai & Engelmann,
2007). En el posterior proceso de recuperación, el calentamiento se realiza sumergiendo el
criovial en un baño de agua a temperatura controlada.
1.3. Aspectos físicos de la crioconservación
1.3.1. Formación de cristales de hielo
La formación de cristales de hielo, es decir, la cristalización del agua, se produce en tres
etapas, denominadas nucleación, propagación y maduración. Así, cada cristal parte de una
5
1. INTRODUCCIÓN/INTRODUCTION
estructura microscópica inicial o núcleo, que se forma por acción de los movimientos
Brownianos, al azar, de las moléculas de agua. El cristal se desarrolla creciendo por adición
de más moléculas de agua al núcleo original. A tiempos más prolongados, los cristales ya
formados se reorganizan y maduran, creciendo los mayores a costa de los más pequeños. Por
lo tanto, la formación de hielo es un fenómeno que depende fuertemente de la movilidad
molecular y el tiempo, y está directamente relacionado con la temperatura y el contenido de
agua (Roos & Karel, 1991). Otro parámetro importante para la formación de cristales de hielo
es la viscosidad de la solución, muy directamente relacionada con la movilidad a escala
molecular. Mayores concentraciones de solutos y menores contenidos de agua suelen estar
asociados a altas viscosidades y movilidades reducidas, que ralentizan tanto la nucleación del
hielo como el crecimiento posterior de sus cristales.
La nucleación de hielo ocurre siempre por debajo de la llamada temperatura de equilibrio
de congelación, o de fusión (Tf, también del inglés freezing), que caracteriza al cambio de fase
termodinámico entre el agua líquida y sólida. Sin embargo, debido a la necesidad de
formación de núcleos estables, en la práctica, la formación de hielo no suele ocurrir justo por
debajo de Tf, sino frecuentemente bastantes grados por debajo. La probabilidad de formación
de hielo está controlada de manera compleja por la temperatura, la movilidad molecular (a su
vez dependiente de la temperatura) el tamaño del sistema y el tiempo, siendo un fenómeno
básicamente estocástico, controlado cinética y no termodinámicamente (Walstra, 2002). La
temperatura de congelación depende directamente del contenido en solutos, siendo el
descenso de Tf proporcional al de la concentración (Levine & Slade, 1992).
La extensión de los daños causados por el crecimiento de cristales de hielo depende de su
localización y del tamaño del cristal. Las perturbaciones observadas en las células están
frecuentemente relacionadas con el estado de las membranas celulares, que pueden sufrir
diversos daños funcionales, incluyendo la pérdida de contenido celular, evidenciado durante
el proceso de calentamiento en la recuperación. A pesar de la imagen intuitiva del cristal de
hielo rompiendo la membrana, por presión mecánica, en su crecimiento, los daños derivados
de la formación intracelular de hielo suelen estar asociados a la deshidratación de membranas
y sistemas enzimáticos, que por otra parte, son también responsables del daño observado en
otros contextos similares, tales como en alimentos congelados e incluso en plantas cultivadas
sometidas a bajas temperaturas pero por encima del punto de congelación (Levitt, 1980;
Steponkus & Webb, 1992; Thomashow, 1998; Salinas, 2002).
6
1. INTRODUCCIÓN/INTRODUCTION
Así, para conseguir que la formación de hielo sea más improbable durante un enfriamiento
rápido, se deben inducir cambios en las propiedades físico-químicas celulares, reduciendo el
punto de congelación del sistema y la probabilidad de formación de hielo, por medio de la
deshidratación, aumento de la microviscosidad citoplasmática y reducción la movilidad
molecular (Blanshard & Franks, 1987; Angell 2000; Wesley-Smith et al., 2001), lo que
favorece la entrada en estado vítreo.
1.3.2. La transición vítrea
Los líquidos, a temperaturas suficientemente bajas, pierden movilidad molecular,
traslacional y rotacional, y se convierten en sólidos amorfos, sin orden cristalino, duros y
frágiles, siendo denominados vidrios (Sperling, 1986). Con el aumento de la temperatura, la
estructura del material pierde rigidez, permitiendo así que exista alguna movilidad molecular.
Este proceso puede ser descrito como una transición entre el estado vítreo y el estado de goma
o líquido, y se produce a una temperatura conocida como temperatura de transición vítrea (TG,
del inglés glass transition; Levine & Slade, 1992; Angell, 2000).
A pesar de que la movilidad está muy reducida en el estado vítreo, alguna lenta
reorganización puede tener lugar en el ámbito del agua, los gases disueltos y algunas
pequeñas moléculas (Ubbink & Krüger, 2006). La transición entre el estado vítreo y el líquido
no debe ser considerada como una transición de fase termodinámica propiamente, ya que las
temperaturas de transición vítrea no son precisamente constantes y el proceso tiene lugar, en
realidad, en un intervalo de temperatura (Walstra, 2002). Los solutos y el contenido en agua
tienen gran influencia sobre el valor de TG de los sistemas acuosos. Distintas sustancias
poseen efectos especialmente intensos sobre esta transición. Estudios realizados con azúcares
muestran que existe una relación entre el peso molecular de los mismos y su correspondiente
temperatura de transición vítrea (Roos & Karel, 1991; Levine & Slade, 1992).
Los cambios más importantes que ocurren en la transición vítrea están relacionados con el
volumen específico, la viscosidad aparente y la capacidad calorífica. Un ejemplo de estas tres
propiedades, tanto en relación con la temperatura de transición vítrea, como con la
temperatura de fusión, se puede observar en la Figura 1.1. La variación del volumen
específico (el inverso de la densidad, ) frente a la temperatura tiene lugar de manera
diferente según sea la velocidad del proceso. Si el líquido se enfría de manera suficientemente
7
1. INTRODUCCIÓN/INTRODUCTION
lenta, al llegar a la temperatura de cambio de fase tiene lugar el fenómeno de cristalización y
se observa una disminución abrupta de 1/ . Si el sistema sigue siendo enfriado, la reducción
del volumen específico continúa, pero a una velocidad mucho más lenta. Sin embargo, si el
líquido se enfría muy rápidamente, se evita la cristalización y el volumen específico continúa
disminuyendo al mismo ritmo que antes, hasta alcanzar la TG. En este punto, el volumen
específico disminuye con la misma dependencia con la temperatura que en el sólido cristalino
(Walstra, 2002).
La transición vítrea afecta las propiedades mecánicas del material como consecuencia del
aumento de la movilidad molecular, es decir, el coeficiente de difusión de las moléculas. Al
aumentar la temperatura por encima de TG, la viscosidad disminuye drásticamente (Roos,
1995). La entalpía del sistema (Figuras 1.1 y 1.2) se comporta como una derivada de primer
orden. El pico estrecho que se produce en Tf corresponde a la fusión de la estructura
cristalina, mientras que el pequeño salto que se observa corresponde a la TG. Cuando se
representa la segunda derivada, se observa un máximo que corresponde a la temperatura de
transición vítrea, por lo que se considera que se puede hablar de una transición de segundo
orden (Walstra, 2002). En las temperaturas superiores a TG, se observa un aumento en la
movilidad molecular y una disminución de la viscosidad que posibilitan la cristalización del
agua, si se dispone de tiempo suficiente para la realización del proceso (Roos & Karel, 1991).
Cuando la temperatura está por debajo de la de transición vítrea, el sistema se puede
considerar estable, porque no es posible la formación de hielo, ya que en estas condiciones la
movilidad molecular está muy reducida. Con frecuencia, la dificultad está en el control de la
TG, ya que es un parámetro muy sensible a la proporción de agua y otras moléculas pequeñas,
y pequeñas cantidades de agua puede causar grandes variaciones en TG (Roos & Karel, 1991;
Roos, 1995; Zamecnik et al., 2007).
Por lo tanto, en el intervalo de temperatura entre los parámetros mencionados, Tf y TG, es
donde puede haber riesgo de formación de hielo, tanto durante el enfriamiento como en el
calentamiento (Figura 1.3A). Así, es de interés el recudir este intervalo (disminuyendo el
valor de Tf y aumentando el de TG) (Figura 1.3B). Además, como se trata de
comportamientos cinéticos, el sistema debe permanecer el menor tiempo posible en este
intervalo, para minimizar la probabilidad de formación de hielo.
8
1. INTRODUCCIÓN/INTRODUCTION
TG Tf
Figura 1.1. Ilustración sobre la transición vítrea en comparación con una fusión/cristalización de una sustancia pura no polimérica. Se denota el estado de equilibrio mediante un trazo continuo y el de no equilibrio por trazos discontinuos. (a) Volumen específico, b) viscosidad aparente, (c) capacidad calorífica (primeras derivadas), representando la dirección hacia arriba una transición endotérmica (Figura tomada de Walstra, 2002).
9
1. INTRODUCCIÓN/INTRODUCTION
Figura 1.2. Ilustración sobre la transición vítrea de una solución de vitrificación (PVS3) en comparación con una fusión/cristalización de ápices de ajo. Se denota el estado de equilibrio mediante un trazo continuo y el de no equilibrio por trazos discontinuos.
Tf TG
Tf TG
Figura 1.3. Ilustración sobre la transición vítrea en comparación con la fusión en un termograma de DSC (A) y el acercamiento de las temperaturas, por aumento de TG y disminución de Tf (B).
1.3.3. La velocidad de cambio de temperatura en crioconservación
Las velocidades de enfriamiento y calentamiento están determinadas por la masa térmica
del material (su capacidad calorífica total) y las propiedades de transferencia de calor del
sistema (Bald, 1987). La dificultad de la inducción de cambios de temperatura (enfriamiento o
calentamiento) muy rápidos, es mayor cuanto mayor es la masa del sistema, especialmente si
el contenido en agua (de alta capacidad calorífica específica) es elevado, ya que es necesario
transferir una mayor cantidad de calor (Franks, 1986). Esta transferencia de calor debe
alcanzar al centro de la muestra y para eso debe, frecuentemente, atravesar capas de
materiales malos conductores térmicos, como son las paredes de contenedores plástico y
crioviales (Song et al., 2010).
Para diseñar procedimientos de crioconservación adecuados, en los cuales la rapidez de las
etapas de enfriamiento y calentamiento es esencial, resulta necesario controlar los diversos
10
1. INTRODUCCIÓN/INTRODUCTION
factores concurrentes: tamaño de la masa de muestra (y demás componentes del sistema),
contenido en agua y transferencia de calor. El resultado es una interacción compleja entre las
propiedades físicas citadas de los diversos componentes del sistema (muestra, contenedores,
soportes, soluciones), que tiene como resultado directo las velocidades de variación de
temperatura aquí determinadas y, una vez comprendido dentro de un proceso de
crioconservación real, la obtención de mayores o menores porcentajes de viabilidad
(Pennycooke & Towill, 2000).
1.3.4. Soluciones crioprotectoras
Blanshard & Franks (1987) llevaron a cabo numerosos estudios sobre el control de la
cristalización del agua: inhibición y/o control de la nucleación, control del crecimiento de los
cristales de hielo y empleo del estado vítreo. Se ha comprobado que la adición de polímeros y
azúcares puede retrasar la cristalización (Iglesias & Chirife, 1978). Este efecto se ha
observado también con adición de fructosa (Roos & Karel, 1991) y relacionado con la
variación de la viscosidad y el correspondiente efecto en la movilidad en la estructura
molecular (Roos, 1995).
A partir de estas observaciones se han desarrollado tratamientos alternativos con
soluciones crioprotectoras, como agentes osmóticos (ej.: manitol), que causan deshidratación
reduciendo el contenido de agua intracelular, y crioprotectores (ej.: DMSO), que penetran en
las células para estabilizar proteínas y membranas (Engelmann & Takagi, 2000; Benson,
2008). Sin embargo, el modo de acción de cada componente de la solución crioprotectora es
específico, siendo modulado por las condiciones de incubación, y por otra parte, su presencia
puede interferir en el desarrollo de estrategias de defensa de cada sistema biológico frente a la
congelación.
Los protocolos seguidos en los distintos métodos de crioconservación se basan en
combinaciones de diversas sustancias, desarrolladas básicamente mediante metodología
empírica (prueba y error). El tiempo de exposición y condiciones deben ser, por tanto,
optimizadas para obtener la protección suficiente contra la formación de hielo sin dañar las
células por estrés osmótico o toxicidad química de las soluciones de vitrificación (Volk &
Walters, 2006).
11
1. INTRODUCCIÓN/INTRODUCTION
Diferentes soluciones de vitrificación han sido desarrolladas por varios equipos de
investigación en todo el mundo. Sin embargo, las soluciones más utilizadas son las soluciones
en las que uno de los componentes es el glicerol, como por ejemplo PVS1, PVS2 y PVS3
(Plant vitrification solutions 1, 2 y 3). La solución PVS1 contiene 19% p/v de glicerol, 13%
p/v de etilenglicol, 13% p/v de propilenglicol, 6% p/v de DMSO en medio líquido MS y 0,5
M de sorbitol (Uragami et al., 1989). La PVS2 contiene 30% p/v de glicerol, 15% p/v de
etilenglicol, 15% p/v de DMSO y 0,4 M de sacarosa. La PVS3 se prepara con 40% p/v de
glicerol y de sacarosa en medio de cultivo básico (Sakai & Engelmann, 2007).
Soluciones acuosas del polímero sintético de alcohol polivinílico, su copolímero de bajo
peso molecular y el polímero poliglicerol están siendo utilizadas como soluciones de
vitrificación alternativas, pues reducen la actividad de nucleación y el crecimiento de los
cristales de hielo (Kami et al., 2008).
1.3.5. Encapsulación en alginato
El empleo de cuentas de gel formadas por el polímero alginato cálcico se ha generalizado,
como medio auxiliar en los protocolos de crioconservación de germoplasma vegetal, ya que
confiere una protección frente a los tratamientos de deshidratación empleados.
Las cuentas de gel suelen ser generadas en dos etapas. Primeramente los especímenes pre-
tratados son introducidos en una solución de alginato sódico (soluble en agua) que contiene
nutrientes y azúcares (por ejemplo, medio MS líquido + 0,35 M sacarosa + 3% alginato).
Después, los especímenes son absorbidos con la ayuda de una pipeta y dispensados, en gotas
de la solución de alginato, en el seno de otra solución con el medio de encapsulación o
polimerización (una sal soluble de calcio). Las gotas de solución de alginato con el material
biológico incluido forman, entonces, esferas de consistencia creciente, a medida que la
difusión del calcio externo permite la formación del gel de alginato cálcico. Tras un periodo
de gelificación adecuado (minutos) las cuentas pueden ser extraídas de la solución y tratadas,
ya sea mediante soluciones concentradas o deshidratadas al aire, porque el gel es permeable a
los solutos y al agua.
El alginato es un polisacárido extraído de tres especies de algas pardas (Laminaria
hyperborea, Ascophyllum nodosum y Macrocystis pyrifera), en las cuales representa más del
40% del peso seco. Se encuentra como sal de varios cationes comunes en el mar como Mg2+,
12
1. INTRODUCCIÓN/INTRODUCTION
Sr2+, Ba2+ y Na+. Químicamente es un polisacárido lineal compuesto por bloques alternados
de residuos de ácido -L-gulurónico (G) y ß-D-manurónico (M) unidos (Draget, 2000;
George & Abraham, 2006; Coviello et al., 2007). Las distintas regiones presentan geometrías
diferentes, en hélice y plana. Cuando dos regiones de bloques gulurónicos se alinean lado a
lado, forman un hueco en forma de diamante el cual es ideal para alojar iones divalentes
unidos cooperativamente. Las propiedades físicas de los alginatos están determinadas por la
composición y extensión de las secuencias de ácido manurónico, gulurónico y de la estructura
alternada de ambos, así como por su peso molecular (George & Abraham, 2006).
La propiedad más importante de los alginatos es la de formar geles con cationes divalentes
como el Ca2+. La afinidad de los alginatos por los metales alcalino térreos aumenta en el
orden Mg < Ca < Sr < Ba; la selectividad por éstos aumenta marcadamente con el mayor
número de residuos -L-gulurónicos en las cadenas (Draget, 2000; George & Abraham,
2006). La gelificación y el entrecruzamiento del polímero se logran por el intercambio de los
iones sodio de los ácidos gulurónicos con los cationes divalentes y el apilamiento de estos
grupos gulurónicos para formar la estructura.
Cuando la solución de alginato de sodio entra en contacto con la solución gelificante que
contiene iones calcio, ocurre la gelificación instantánea en la interfase, con el tiempo los iones
calcio difunden en la solución e interaccionan con la solución de alginato. La máxima dureza
del gel se registra en la superficie, ya que la concentración de alginato es mayor en ésta y
disminuye hacia el centro del gel (King, 1983; Draget, 2000).
1.3.6. Quitosano
Las cuentas de alginato cálcico son empleadas en otros contextos como protectores o
vehículos para nutrientes u otros compuestos. Sus propiedades de permeabilidad a gases, agua
y solutos, así como su resistencia mecánica han sido moduladas mediante la adición de otros
polisacáridos (Anbinder et al., 2011). Tal es el caso del quitosano, que añade a sus
características de interés, la de ser un efectivo agente antimicrobiano de amplio espectro
(Eaton et al., 2008).
El quitosano se obtiene directamente a partir de la quitina, por desacetilación. Es el
principal componente estructural del exo-esqueleto de los crustáceos y también se encuentra
en moluscos, insectos y hongos. La forma más común de obtención es el -quitosano, a partir
13
1. INTRODUCCIÓN/INTRODUCTION
de la quitina de los desechos de caparazón de cangrejos y camarones, representando alrededor
del 70% de sus componentes orgánicos. En su obtención, los caparazones molidos son
desproteinizados y desmineralizados por tratamiento sucesivos con bases y ácidos, y a
continuación se extrae la quitina la cual es desacetilada a quitosano por hidrólisis alcalina a
alta temperatura (George & Abraham, 2006).
La quitina es un co-polímero muy ordenado de 2-acetoamido-2-deoxi- -D-glucosa (como
componente principal) y 2-amino-2-deoxi- -D-glucosa. A diferencia de otros polisacáridos
abundantes, como la celulosa, la quitina contiene nitrógeno (Terbojevich & Muzzarelli, 2000).
El quitosano presenta propiedades biológicas favorables para el uso en la biotecnología
como nula toxicidad, biocompatibilidad, biodegradabilidad (Sinha & Kumria, 2001) y
actividad antimicrobiana contra una amplia cantidad de hongos filamentosos, levaduras y
bacterias (No et al., 2002; Eaton et al., 2008). Sin embargo, el estudio de encapsulados
obtenidos a partir de esto polímero en el campo de la crioconservación es un campo aún por
explorar.
1.4 La menta como modelo en el estudio de la crioconservación
La familia Lamiaceae (Labiatae) está formada por aproximadamente 200 géneros y 3200
especies de distribución cosmopolita, pero son especialmente abundantes en la región del
Mediterráneo y Centro-Este asiático. Más de la mitad de las especies pertenecen a ocho
géneros: Salvia (500), Hyptis (350), Scutellaria (200), Coleus (200), Plectranthus (200),
Stachys (200), Nepeta (150) y Teucrium (100). Otros géneros de la familia con un elevado
número de especies son Lavandula, Marrubium, Mentha y Thymus (Cronquist, 1981). La
familia se caracteriza por la presencia de flavonoides y terpenoides (Cole, 1992), metabolitos
secundarios con importantes aplicaciones en medicina y como condimento alimentario.
El género Mentha L. tiene amplia distribución en todos los continentes, a excepción de
Sudamérica y la Antártida. Sus centros de diversidad son Europa, Australia y Asia Central. En
Europa hay una gran diversidad de especies, encontrándose unas 22 entre especies e híbridos
(Flora Europaea, versión on line, Chambers, 1992).
La menta es una planta de interés económico, principalmente en la industria farmacéutica y
cosmética (Gupta, 1991; Munsi, 1992), y es cultivada en los Estados Unidos, Italia, Francia,
14
1. INTRODUCCIÓN/INTRODUCTION
Hungría y otros países (Fahn, 1979; Lawrence, 1985). Por la gran cantidad de aceites
esenciales que contienen, entre ellos mentol, mentona y mentofurano, con acción
antimicrobiana, antiespasmódica, antiulcerosa y antiviral, forma parte de los productos
estratégicos industriales en Biotecnología. Estos compuestos se encuentran en mayor cantidad
en las hojas y son ampliamente investigados tanto desde el punto de vista agronómico como
químico (Schilcher, 1988; Martins, 1998). La infusión de sus hojas puede ser utilizada para
facilitar la digestión, eliminación de gases del aparato digestivo (Lorenzi & Matos, 2002;
Simões & Spitzer, 2007) y en medicina popular (Piccaglia et al., 1993).
En el trabajo aquí desarrollado, se utilizó Mentha ×piperita Linn., siendo un híbrido estéril
entre Mentha aquatica L. y Mentha spicata L. (Arvy & Gallouin, 2006). Esta es una planta
herbácea, perenne, aromática, de aproximadamente 30 cm de altura, con ramas de color verde
oscuro a morado, tallo ramificado, flores lilas a azuladas y las hoja opuestas pecioladas, oval
y borde cerrillado (Matos, 1998).
La elección de esta planta como modelo para el estudio de los procesos de
crioconservación responde a su frecuente uso comercial con multiplicación vegetativa,
presencia en diversos métodos y protocolos publicados, así como a su buen comportamiento
frente a la crioconservación (Bhat et al, 2001; Wang & Reed, 2003; Islam et al., 2005; Volk
& Walters, 2006; Volk et al., 2006; Sakai & Engelmann, 2007; Senula et al., 2007; Uchendu
& Reed, 2008).
15
2. OBJETIVOS/AIMS
2. OBJETIVOS
La crioconservación de germoplasma vegetal es una alternativa eficiente a la conservación
clásica (semillas o colecciones en campo), que adicionalmente permite conservar a largo
plazo material vegetal utilizado u obtenido mediante biotecnología. En la actualidad, resulta
necesario poner a punto los protocolos adecuados para cada especie, e incluso, genotipo,
mediante ensayo y error. Entender los procesos subyacentes a los diversos pasos de un
protocolo de crioconservación facilitará el desarrollo de nuevos protocolos. En el presente
trabajo se estudiaron tres técnicas diferentes de crioconservación: vitrificación, vitrification-
droplet, y encapsulación-deshidratación. El objetivo principal de este proyecto de tesis es
aumentar el conocimiento sobre los procesos implicados en los distintos pasos de un
protocolo de crioconservación, en relación con el estado del agua presente en los tejidos y sus
cambios, abordado mediante diversas técnicas biofísicas, principalmente calorimetría
diferencial de barrido y la microscopía electrónica de barrido a baja temperatura, estudiando
la dinámica de formación de hielo y el estado vítreo. La especie objeto de estudio en la mayor
parte de los capítulos ha sido la menta (Mentha ×piperita M186). En dicha especie existían ya
puestos a punto protocolos de crioconservación basados en todas las técnicas mencionadas
anteriormente (Senula et al., 2007; Uchendu & Reed, 2008; Volk & Walters, 2006).
Los objetivos concretos de la tesis fueron:
1) Estudio comparativo de las velocidades de enfriamiento y calentamiento en las diferentes
técnicas de crioconservación.
2) Estudio de formación de cristales de hielo y los procesos de vitrificación en las distintas
etapas del protocolo de vitrificación-droplet, mediante crio-SEM y DSC en menta.
3) Estudio de los distintos estados de deshidratación en el protocolo de encapsulación-
deshidratación, mediante crio-SEM y DSC, en menta.
4) El efecto de la velocidad de enfriamiento en la temperatura de transición vítrea y capacidad
calorífica en diversas soluciones de vitrificación de plantas.
5) Modulación del papel del encapsulado de alginato mediante la adición de otros
polisacáridos.
16
3. MEASUREMENT OF COOLING AND WARMING RATES
3. MEASUREMENT OF COOLING AND WARMING RATES
IN VITRIFICATION-BASED PLANT CRYOPRESERVATION
PROTOCOLS
3.1. Introduction
Cryopreservation of living tissues for its functional recovery is a widely employed
procedure, for the multi-purpose preservation of medical, veterinary and crop and wild plant
species materials. The basis of cryopreservation is the drastic reduction of most chemical and
physical activities in the cells. While temperature reduction over the physiological fluids
freezing point can only slow these processes, freezing confers a higher degree of activity
stoppage, due to the associated reduction of water activity. Unfortunately, intracellular ice
formation has been found to be lethal for most tissues and specimens. Satisfactorily stable
storage conditions are found for most cases when storage is carried out at temperatures well
below the freezing point: in the glassy state. When viscosity of liquids increases, molecular
mobility is reduced. This reduction can be so extreme that most processes are virtually
detained (Mazur, 1984).
The change between liquid and vitreous state is a second order phase change, not
determined by an enthalpy increment as first class changes, but by a step in some of the
physical properties of the system, such as the heat capacity, specific volume and apparent
viscosity (Walstra, 2002). Glassy state is kinetically controlled, so it can be considered to be
metastable, though the very slow mobility guarantees the actual stoppage of most processes.
Moreover, ice formation, a process requiring the assembly of an initial nucleus of water
molecules driven by Brownian movement, is completely impaired in glasses (Slade & Levine,
1995).
The molecular mobility reduction required for entering the glassy state is achieved by
either a temperature drop or by an increase of the solute concentration. The glassy state
conditions can be achieved by dehydration (which causes the effective solute concentration to
rise) and it can be employed for preservation of systems surviving extreme dehydration, such
as most seeds (orthodox), even at room temperature (Sacandé et al., 2000). Most systems
would be damaged by such dehydration degree, and, therefore, a reduction of temperature is
17
3. MEASUREMENT OF COOLING AND WARMING RATES
also required, to reach the glass transition temperature TG, which is lower the higher the
solvent (water) content is. However, cooling below the equilibrium freezing temperature Tf,
gives rise to a problem: the wide temperature gap between Tf and TG must be crossed and ice
can be formed in this region.
Ice crystal formation takes place always under the equilibrium freezing temperature, but its
initial nucleation step is stochastic in nature, depending on the random rearrangement of water
molecules to achieve the initial nucleus conformation able to spontaneously grow. That
implies that there is an increasing probability of ice being formed the longer the permanence
between Tf and TG. For this reason thermal change (cooling and warming) rates are
considered critical for the success of cryopreservation (Blanshard & Franks, 1987; Angell,
2000; Wesley-Smith et al., 2001).
Cryopreservation is customarily applied to animal, plant and microbial specimens. Plant
tissues are often preserved after vitrification protocols (Towill, 1990; Mazur et al., 2007;
Nadarajan et al., 2008), which imply a previous set of steps designed to increase solute
content and reduced water in cells, as well as to promote the plant natural defences towards
stress. Cooling by plunging into liquid nitrogen (LN) completes the procedure. The recovery
process is nearly symmetric, and first re-warming is carried out by immersion in a warm
water bath, followed by a rehydration and solute unloading stage before specimen culture. A
method frequently used in the cryopreservation of plant germplasm is that named directly as
vitrification (Sakai & Engelmann, 2007). Another usual procedure is droplet-vitrification, and
the main difference with the former is the mass (of specimen plus cryoprotectant solution and
container) to be cooled or warmed (Sakai & Engelmann, 2007; Senula et al., 2007).
The need for a fast temperature change in the vitrification and de-vitrification processes is
deemed as being of crucial importance, and is closely associated to the success of the
procedure (Wesley-Smith et al., 2001). In spite of this, measurement of actual cooling and
warming rates in these protocols has rarely being recorded and reported. Actually, most
temperature rate changes reported for cryopreservation processes correspond to animal or
human origin samples, where the applied protocols are often of different nature. The few
reports of cooling/warming rates in plant cryopreservation lack precision and their
methodology is scarcely described (Pennycooke & Towill, 2000; Towill & Bonnart, 2003).
The cause for this lack of measurements is, most likely, related to the difficulty of performing
reliable fast thermal measurements. In the present work, the rates of cooling and warming in
18
3. MEASUREMENT OF COOLING AND WARMING RATES
cryopreservation experiments, performed with mint shoot tips and following the vitrification
and droplet-vitrification protocols, were determined using a fast temperature determination
system. The viability of apices after cryopreservation was also studied, to ascertain the
validity of the protocol followed.
3.2. Materials and Methods
3.2.1. Plant material pre-culture and shoot tip extraction
Shoot tips were extracted from in vitro shoots of Mentha ×piperita (genotype �“MEN 186�”
obtained from the IPK Genebank, Gatersleben, Germany). In vitro plants were monthly
subcultured on medium MS (Murashige & Skoog, 1962) with 3% sucrose, and incubated at
constant temperature (25°C) with a photoperiod of 16 h, and an irradiance of 50 µmol m-2 s-1
from fluorescent tubes. One-node segments were obtained from these shoots, transferred to
fresh medium and incubated at alternating temperatures of 25°C (day) and -1°C (dark),
always with 16 h photo- and thermoperiod, 50 µmol m-2 s-1 irradiance, provided by
fluorescent tubes (Senula et al., 2007). After 3-weeks of culture under these conditions, shoot
tips (1-2 mm) were excised from axillary buds.
3.2.2. Dehydration of shoot tips in the vitrification method
All excised shoot tips were pre-cultured for 24 h on filter paper in liquid MS medium
containing 0.3 M sucrose, at 25ºC. Thereafter, the explants were transferred to a Petri dish
with 2 mL of loading solution (2 M glycerol + 0.4 M sucrose) over filter paper, for 20 min, at
room temperature. Finally, they were osmotically dehydrated in 2 mL PVS2 (plant
vitrification solution 2: 30% w/v glycerol, 15% w/v ethylene glycol, 15% w/v DMSO and 0.4
M sucrose in half-strength MS liquid medium; Sakai et al, 1990) in a Petri dish, on filter
paper, for 30 min at 0ºC. All constituents of PVS2 were autoclaved except DMSO, which was
filter-sterilised. All solutions stated here or thereafter were prepared in liquid medium MS.
19
3. MEASUREMENT OF COOLING AND WARMING RATES
3.2.3. Vitrification
Modifications of the protocols for mint from Volk & Walters (2006) and Uchendu & Reed
(2008) were used. After dehydration in PVS2, ten shoot tips were placed in a 1.0 mL cryovial
containing 0.5 mL or 1.0 mL PVS2. Then, this container was submerged into liquid nitrogen
(LN) and, after a short period in LN conditions (approx. 1 h), the cryovial was removed and
rewarmed in 40°C water for 2 min, stirring gently. Then shoot tips were washed with 1.2 M
sucrose for 20 min at room temperature and subsequently cultured.
3.2.4. Droplet-vitrification
Modifications of the protocol developed by Senula et al. (2007) for mint were studied.
After dehydration in PVS2, shoot apices were transferred into droplets of 2 µL PVS2 placed
on aluminium foil strips (AFS; 5 droplets per strip, each containing a shoot tip). The strips
with the adhered droplets were immersed into LN, either directly (naked) or placed inside
cryovials. After a short period in LN conditions (approx. 1 h), cryovials/AFS were retrieved
and warmed. Cryovials were plunged into a water bath at 40°C for 3 to 5 s; the AFS were then
removed from the cryovial and the apices were incubated in a 1.2 M sucrose solution for 20
min at room temperature. When naked aluminium strips had been immersed in LN, they were
directly plunged into the 1.2 M sucrose solution at room temperature and shoot tips incubated
for 20 min.
3.2.5. Plant recovery and viability
Shoot tips exposed to the vitrification or droplet-vitrification techniques were cultured on
re-growth medium (semi-solid MS medium supplemented with 0.5 mg/L BAP and 3%
sucrose), incubated in the dark for 24 h and thereafter under 16 h photoperiod, at 25ºC. For
control samples (-LN), all steps were carried out except immersion in LN and rewarming
Survival and re-growth were calculated as percentages over the total number of shoot tips
used 4 weeks after culture. Survival was defined including all forms of visible viability
(evidence of green structures or callus). Re-growth was defined as the formation of small
plantlets. The number of replicates was 3 or 6 for the vitrification and droplet-vitrification
20
3. MEASUREMENT OF COOLING AND WARMING RATES
protocols, respectively. Data were subjected to analysis of variance and Duncan�’s Multiple
Range Test (Alpha = 0.05) was used for comparison of means.
3.2.6. Temperature measurement and cooling and warming rates
The specimen temperature changes were acquired using a fast data acquisition
multichannel temperature measuring system, MW100-UNV-H04 (Yokogawa, Tokio, Japan).
Several reading speeds were tested and finally a reading speed of 20 data per second
(measuring interval 50 s) was selected as optimal. Liquid nitrogen and ice were used for
calibration points (-196 and 0ºC, respectively), and calibration was performed frequently
between measurements. Cooling and warming rates were defined as the ratio of a
predetermined temperature interval T1-T2, over the time elapsed between the reported
measurement and of T1-T2.
The determination of cooling and warming rates was carried out, at least in triplicate,
during the cooling and warming phases of the described protocols.
3.2.7. Thermocouples
Several types of thermocouples and wire gauges were tested and finally, T-type
thermocouples (copper-constantan) were selected for its good performance in the temperature
region studied (45 to -150ºC). Thin (diameter 0.5 mm) but steel-shielded and mineral-isolated
thermocouples (TC Medida y Control, S.A., Madrid, Spain) were chosen, in order to
minimize external electromagnetic influences on measurements. They were placed either on
the surface of AFS (commercial household aluminium foil, strips of approx. 20 x 5 mm, 5
mg) or in the centre of cryovials, introduced through a hole in the lid, depending on the
cryopreservation technique studied.
Controls were set for empty cryovials and thermocouples without specimens. Additional
controls were set for evaluating the effect of heat losses through the thermocouple wire,
comparing results with those obtained with additional thermocouple tips inserted in the
sample. No significant differences were found that could justify a heat loss correction.
Thermocouples were frequently calibrated as a part of the measurement set-up, as described
above, and especially after any thermocouple exchange or reconnection.
21
3. MEASUREMENT OF COOLING AND WARMING RATES
3.2.8. Experimental set-up and temperature change rate measurement design
A scheme of the experimental set up used for temperature change rate is shown in Figure
3.1. The aim of the experiment was to reproduce the conditions used in customary
cryopreservation, which involves a certain degree of variability due to not completely
controlled factors and the operator action.
Figure 3.1. Experimental set up for thermal change rate measurement for (a) vitrification, or (b), droplet-vitrification protocols.
The sample holders (either a criovial or an AFS) were handled by means of the attached
thermocouple wire, by means of isolated tongs, in order to reduce the heat exchange sources.
For cooling rate determination cryovials/AFS were tempered at 1-4ºC before immersion in
LN, and data collection started at 0ºC, and continued till a plateau near -196ºC was reached.
For warming, data collection started when the specimens were immersed and thermally
equilibrated in LN and continued till room temperature equilibrium. Rates were calculated
after adopting an ice risk temperature window, by dividing the thermal difference between the
window extremes by their time difference.
Temperature change rates were determined on specimens being submitted to the
vitrification protocol, using a cryovial containing 0.5 or 1 mL of PVS2 solution (containing
10 mint shoot tips), as well as an empty cryovial for blank comparison. Rates were also
measured on specimens treated with the droplet-vitrification protocol, introducing variations
in several parameters. Two experimental conditions were tested (with different masses and
specimen distributions): an AFS with 5 droplets of 2 L (containing each one a mint shoot
tip), naked or inside a cryovial. Two controls were added: an AFS with no shoot tip sample,
inside or not a cryovial.
22
3. MEASUREMENT OF COOLING AND WARMING RATES
3.3. Results and Discussion
Figure 3.2 shows cooling and warming experiments corresponding to the vitrification
protocol. Figure 3.2a shows the temporal evolution during cooling of the sample, consisting
of 10 shoot tips in a 1.0 mL cryovial containing 0.5 mL or 1.0 mL PVS2. Figure 3.2b shows
data for the same samples during warming. A control performed with an empty cryovial is
also shown in both cases. Given the relatively slow rates for this case, the data points obtained
at 50 µs interval form a continuous line. In spite of the visible dependence of rate with the
thermal difference with the surrounding fluid, data cannot be properly fitted to a function, as
many smaller scale phenomena have a variable influence on both cooling and warming
processes. This includes the changes in heat capacity and conductivity of the containers, fluids
and sample components during this wide temperature span, the possible phase changes
(including to and from glassy state) in all components, involving, when not a heat effect, an
additional heat capacity step change, the heat interchanges derived from mechanical forces
when the volume of components are altered, etc. Also the complex convention of the external
fluid (and Leidenfrost effect in LN), would generate differences in the instantaneous heat
exchange rates (Leindenfrost, 1966; Song et al., 2010).
The cryoprotecting solution content seems to be a major determinant of the rate, both for
cooling and warming: the empty cryovial, with less total heat capacity than those with
solution, shows faster rates. Those of 0.5 and 1.0 mL samples do present a very similar
behaviour, probably compensating the higher heat capacity of the larger volume sample and
its higher heat conductivity, due to the higher heat exchange surface. The trends observed are
quite symmetric for cooling and warming, both for samples and control measurements.
Control observations carried out using several thermocouple wires indicated that any effect
on thermal change rates was negligible, allowing discarding reductions of the measured rate
due to the wire contribution to thermal mass or to heat fluxes through it (data not shown).
Figures 3.3 and 3.4 show cooling and warming experiments corresponding to the droplet-
vitrification protocol. Figure 3.3 a and b show temperature temporal evolution determined
during cooling by immersion in LN, for different masses and sample distributions (naked AFS
or inside a cryovial and with different number of droplets).
23
3. MEASUREMENT OF COOLING AND WARMING RATES
Figure 3.2. Cooling (a) and warming (b) evolution determined in samples treated after the vitrification protocol. Data are the average of at least three independent determinations. Control data were obtained with the thermocouple tip inserted in an empty cryovial. The lines shown represent the individual data points measured.
Figure 3.4 a and b show temperatures obtained during the warming process as described
in the droplet-vitrification protocol, for the same samples as in Figure 3.3. Data obtained
were fairly reproducible and, in spite of the additional error introduced by specimen
variability and operator manipulation, their scattering was much smaller than the differences
observed among different experimental conditions. Comparison of these results showed a
diverse behaviour in both cooling and warming rates for the different conditions. The
presence of PVS2 cryoprotecting solution drops in the droplet technique induces a reduction
of both cooling and warming rates, when compared with the same methodology but without
the drops.
In those systems based on an aluminium foil strip, the small additional mass of this strip
(aprox. 3-4 mg) can be compensated by the increased heat diffusion granted by the larger
interchange surface, and it can be observed that both cooling and heating rates of these strips
24
3. MEASUREMENT OF COOLING AND WARMING RATES
(with the measuring thermocouple attached) were larger than those determined with the
thermocouple alone.
Figure 3.3 a and b. Measurements of cooling rate obtained following the droplet-vitrification protocol: (a) AFS inside cryovial and (b) naked AFS. Data are the average of at least three repeats.
The rates in the warming process for this protocol show larger differences from those of
cooling than in the case of the vitrification protocol, due to the particularities of the warming
process. The introduction of the AFS + 5 x 2 µL PVS2 drops sample in the sucrose solution at
room temperature has the effect of a reduction of the warming rate, probably due to different
interface contact and heat exchange behaviour than that resulting of the immersion in LN.
Because of this slower rate, the warming process of this sample has been shown in a different
sub-figure than the cooling, faster, one.
In Figure 3.4 b), a slope change in the warming process can be appreciated for the two
samples considered, at 0.3-0.4 s. This was related to the physical transfer from nitrogen to air
25
3. MEASUREMENT OF COOLING AND WARMING RATES
and then to the warming medium. It could not be appreciated in the data sets of Figure 3.4 a)
because it was obscured by the larger duration of the warming process. The end of the 3-5 s
warming step in the water bath (and exit to room temperature air), for the two samples treated
in that way and shown in Figure 3.4 a), per contra, does not give rise to any warming rate
irregularity. This may be a result of both these samples being included in a cryovial, whose
isolation could mask fast thermal effects. As expected, when comparing the results from both
vitrification and droplet-vitrification protocols, it can be seen that the inclusion of specimens
in a cryovial considerably reduces both cooling and warming rates.
Figure 3.4 a and b. Measurements of warming rates obtained following the droplet protocols. Data are the average of at least three repeats.
The data dispersion in the thermal change rates observed comprise both the effect of real
experimental errors and those derived from the possible small variations of the
cryopreservation procedure. The experimental methodology followed was intended to
reproduce the usual operations in cryopreservation techniques, rather than to minimize the
causes of variability, so that the sources of variation associated to manual manipulations when
transferring samples, the holding with tongs and the degree of stirring have not been
26
3. MEASUREMENT OF COOLING AND WARMING RATES
standardized. In the same way, an extreme important factor, as it is the position of the active
thermocouple tip within the experimental set-up, especially inside cryovials, has not been
fixed. A controlled position in the cryovial centre would have produced more homogeneous
data, but it would not have been representative of the real temperature values in positions
closer to its wall. All these factors add to an increase in the dispersion of the obtained data.
Figure 3.5 shows how control specimens, not immersed in liquid nitrogen (LN), present a
100% survival, proving that the dehydration and chemical treatments applied (including
exposure to PVS2) have no toxic effect that could damage apices. Survival and regrowth
values were fairly similar for all cases. The highest regeneration percentage was observed
(96%) when the droplet-vitrification procedure was employed with direct immersion in LN, a
value decreasing with the inclusion in a cryovial. The vitrification protocol, yielded similar
regeneration values for the treatments with 0.5 or 1 mL PVS2 solution inside the cryovial, but
much lower (about 43%) than the droplet-vitrification protocol.
Figure 3.5. Mint shoot tip survival and re-growth percentages after different cryopreservation techniques and a 4-week recovery period. Means of survival or re-growth with the same letter are not significantly different according to the Duncan�’s Multiple Range Test at alpha = 0.05. Bars: standard error.
The observed percentages can be directly related to physical characteristics of specimens
and experimental systems employed (containers and cryoprotecting solutions), especially their
mass, heat capacity and heat conductivity. For example, those systems with a larger mass will
27
3. MEASUREMENT OF COOLING AND WARMING RATES
have a higher inertia towards temperature change. The more relevant factor would be the heat
amount that the system requires to absorb or to yield for causing a temperature change, i.e. its
total heat capacity. The contribution of the different heat capacities of the system components
must be accounted for. A higher thermal inertia can be expected when the water content (with
a large specific heat capacity) is high, while the mass of cryovials and aluminium foil would
represent a lesser influence, due to its lower heat capacity.
On the other hand, the temperature change is determined not only by the heat amount to be
exchanged, but by the rate of this transfer. The different experimental systems employed
present many diverse characteristics on this respect. The presence of containers always
represents an additional barrier for heat exchange. The position of the specimens inside the
cryovial partly determines this behaviour, as depending on the extent of its contact with its
walls, heat transfer could be very different. This may explain the complex effect of the
presence of vitrification solutions inside the cryovial. On one hand, the thermal mass
increases and the total heat amount to be transferred is higher. But this solution could
constitute a thermal bridge between the cryovial walls and the specimen, improving heat
exchange and so fastening the temperature change process.
The data obtained and showed in figures 2-4 show continuous temperature variation on
time. As it can be seen, the slope of the curves (instantaneous rate) changes continuously,
decreasing as the driving heat gradient declines. In order to provide a single figure rate to
enable comparisons, a �“temperature window�” must be defined. This also produces a
parameter more interesting than the temperature change rate, for cryopreservation purposes:
the time of permanence in the ice formation risk area.
Although the purpose of cryopreservation is to keep samples free from ice formation risk
in the glassy state, and the procedures previous to plunging into LN are endeavouring to
reduce the likelihood of ice formation in any case (mainly by increasing viscosity and
reducing the water available), the risk of ice formation during the cooling or warming
processes crossing the area below the freezing point and over the glass transition temperature,
is ever existing (Angell, 1982; Franks, 1986).
A good knowledge of a given system to be cryopreserved would allow setting a more
adjusted temperature window. For comparison purposes, here we have defined two windows:
from 0 to -150ºC and from -20 to -120ºC. The first one, spanning from pure water equilibrium
freezing point (the higher temperature where ice can exist, in any aqueous solution) to -150ºC,
28
3. MEASUREMENT OF COOLING AND WARMING RATES
well below any expected glass transition in a living system, is largely overestimated over the
real ice formation risk zone for systems in cryopreservation, as its Tf is always much lower
than 0ºC, due its high solute and low water contents. The variability in the glass transition
temperature is high, being very sensitive to the content of water and other small size
molecules (Roos & Karel, 1991; Zamecnik et al., 2007), but, for plant viable germplasm, is
usually well over -150ºC. Moreover, the thermal gradient ( T, the difference between sample
and surrounding media temperatures) is similar in the initial and final windows extremes, for
both cooling and warming (using a 40ºC bath). For example, for the 0 to -150ºC window, T
would evolve from 196ºC at the initial point, to 46ºC at the window end-point, for cooling,
and from an initial value of 190ºC to a final one of 40ºC, for warming. This contributes to
generate similar cooling and warming rates, as other contributing phenomena will have a
more reduced weight, due to the high speed of the process (such as convention layer changes
or geometrical alterations of sample and containers).
The narrower temperature window, from -20 to -120ºC, was fixed taking into consideration
the freezing point observed for mint shoot tips in previous stages of the droplet
cryopreservation protocol (Teixeira et al., 2013). The specimen at the final stage of the
protocol, actually those plunged into LN, was reported to present no freezing event in
calorimetric conditions (at a cooling rate of 10ºC min-1), but we have considered that -20ºC
would be a safe limit for ice formation, considering also the usual reduction of the nucleation
temperature with respect to the equilibrium freezing one. The -120ºC limit follows the
reported TG for this system on -119ºC (Teixeira et al., 2013).
Table 3.1 and 3.2 show the calculated cooling and warming rates and permanence times
calculated for these two windows for experiments of Figures 3.2 to 3.4. In all cases, the
narrower (-20 to -120ºC) window gives higher rates. This is due to the larger temperature
gradient, as the region closer to the end point (always showing slower rate) is left out of the
calculation. The more adjusted the window is to the real ice formation probabilities, the more
relevant the rate and times calculated would be. Anyhow, it must be noted that the probability
of ice formation is not constant for the whole window. Its dependence with temperature is
complex and difficult to study, but it increases as temperature descends from the equilibrium
freezing point, being counterweighted by the reduction on molecular mobility, also driven by
lower temperatures. Near the TG, for time spans of the order of seconds, this probability is
most likely very small. A region of about 20ºC over the TG could be considered also safe for
these purposes (for short permanence, not for long term storage), as it has been reported that
29
3. MEASUREMENT OF COOLING AND WARMING RATES
its high viscosity grants that any scale change (such as ice nucleation) would take place very
slowly (Mishima & Stanley, 1998).
Table 3.1. Cooling and re-warming rate measurements obtained following the vitrification protocols.
Cooling Warming Method Window
(ºC) Time1
(s) Rate1
(ºC s-1) Time1
(s) Rate1
(ºC s-1) 0/-150 21.0 ± 0.2 7.14 ± 0.01 27.8 ± 0.4 5.4 ± 0.6 Control: cryovial
LN bath 40ºC -20/-120 10.0 ± 0.1 10.0 ± 0.1 16.3 ± 0.3 6.1 ± 0.7
0/-150 52 ± 1 2.88 ± 0.02 95 ± 1 1.58 ± 0.01 Cryovial with 0.5 mL PVS22 LN bath 40ºC -20/-120 31.43 ± 0.5 3.18 ± 0.04 55.1 ± 0.9 1.81 ± 0.02
0/-150 65 ± 1 2.31 ± 0.02 86 ± 1 1.74 ± 0.01 Cryovial with 1 mL PVS21 LN bath 45ºC -20/-120 41.9 ± 0.9 2.39 ± 0.03 54.0 ± 0.8 1.85 ± 0.02
1Average values and standard deviation for at least three repeats. 2Experiment performed with shoot apices in the cryovial. Window: interval selected for time and rate calculation. Time: that spanning between the recording of the window temperature interval (either during cooling of warming). Rate: calculated in the window temperature interval.
The main difference between the methods of cryopreservation used in this work is the
amount of PVS2. The advantage of using the droplet is the possibility of achieving very high
cooling/warming rates due to the very small volume of cryoprotective medium in which the
explants are placed. The signification of the highest rates reported must be taken with
moderation. The directly measured parameter is (apart from temperature), the time between
two temperature readings. In the faster processes, the fraction of this time that could
correspond to manipulations of the operator when transferring samples can be non-negligible,
and when the rate is calculated by division of these two quantities, very high differences can
be so generated, perhaps unduly.
Resorting to fast temperature changes in cryopreservation has been broadly studied and
employed to reduce ice crystal size. Luyet and co-workers (1965) pioneered the use of rapid
(non-equilibrium) cooling as a means of restricting the size of ice crystals during
cryopreservation of hydrated samples. For example, cooling rates in excess of 100°C s-1 are
reported to facilitate the preservation of microorganisms (Wesley-Smith et al., 2001). In spite
of the crucial role of thermal change rate on cryopreservation, few studies include detailed
measurements of the evolution of temperature in these conditions (Sakai et al., 1968;
30
3. MEASUREMENT OF COOLING AND WARMING RATES
Vertucci, 1989, 1990; Wesley-Smith et al., 1992, 2001). High viabilities in recovered tissues
have been reported at elevated cooling rates (40-400ºC s-1; Pennycooke & Towill, 2000; Kim
et al., 2006b). The rates reported by these workers with potato shoot tips show good
agreement (6ºC s-1) with our results for cooling inside a cryovial, while for cooling on
exposed AFS, a lower value (130ºC s-1) was reported, when compared to our 350-500ºC s-1
data.
Table 3.2. Cooling and re-warming rate measurements obtained following the droplet-vitrification protocols.
Cooling Warming Method Window
(ºC) Time1
(s) Rate1
(ºC s-1) Time1
(s) Rate1
(ºC s-1) 0/-150 2.32 ± 0.05 65 ± 1 0.09 ± 0.03 1700 ± 500 Thermocouple
LN bath 40ºC (3-5 s) -20/-120 1.38 ± 0.05 72.5 ± 2 0.05 ± 0.03 1887 ± 700
0/-150 0.43 ± 0.03 350 ± 25 1.59 ± 0.05 94 ± 2 AFS LN 1.2M sucrose room temperature -20/-120 0.20 ± 0.03 500 ± 70 0.32 ± 0.03 310 ± 30
0/-150 25.10±0.30 6.00 ± 0.05 40.0 ± 0.4 3.75 ± 0.03 AFS inside cryovial LN bath 40ºC
(3-5 s) -20/-120 11.80 ± 0.10 8.5 ± 0.1 26.5 ± 0.4 3.77 ± 0.04
0/-150 1.35± 0.05 110 ± 3 4.58 ± 0.08 32.8 ± 0.4 AFS with 5 x 2 µl PVS2 droplets2 LN 1.2 M sucrose room temperature
-20/-120 0.94 ± 0.04 106 ± 3 2.03 ± 0.04 49 ± 1
0/-150 20.6 ± 0.3 7.28±0.06 26.4 ± 0.5 5.68 ± 0.03 AFS with 5 x 2 µl PVS2 droplets2 in a cryovial LN bath 40ºC (3-5 s)
-20/-120 11.00 ± 0.02 9.1 ± 0.1 15.4 ± 0.2 6.49 ± 0.08 1Average values and standard deviation for at least three repeats. 2Experiment performed with shoot tips. Window: interval selected for time and rate calculation. Time: that spanning between the recording of the window temperature interval (either during cooling of warming). Rate: calculated in the window temperature interval.
The need to employ a high rate to avoid ice formation during cooling (and warming) has
especial importance when the specimens�’ water content is high. However, the rates required
to avoid ice formation cannot be practically reached when water content is very high, for a
sample of the size required for plant germplasm successful manipulation and cultivation. It is
often necessary to act on the specimen to reduce the probabilities of ice formation, generally
decreasing water content, with the multiple effect of reducing the ice formation risk window
31
3. MEASUREMENT OF COOLING AND WARMING RATES
(both by lowering Tf and, at the same time, increasing TG), while the increase in intracellular
viscosity limits the ice formation probability, even within this window. So, partial
dehydration reduces the requirements of very fast cooling.
Relatively slow temperature variations, as those reported in this study for procedures using
cryovials (1-7ºC s-1) would be inadequate for application to high water content systems.
However, the higher rates (100-500ºC s-1) obtained in some methods using aluminium foil
strips, would be appropriated for specimens with higher water contents. The rate differences
associated to the shoot tip water content, would be, however, small in cryovials-based
methods (differences between 2 and 4ºC s-1), being the limiting effect the thermal diffusion
and the own cryovial mass.
32
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
4. GLASSY STATE AND CRYOPRESERVATION OF MINT
SHOOT TIPS
4.1. Introduction
Biologically material, most frequently that being used for plant propagation (i.e.,
germplasm) can be stored at low temperature for long periods. Plant germplasm, particularly
seeds, pollen or reproductive tissues, is often kept at liquid nitrogen (LN) temperature to
avoid deterioration (Towill, 1990). Cryopreservation is currently an important tool for
germplasm long-term storage, requiring minimal space and maintenance, and the number of
species and cultivars cryopreserved quickly increases (Sakai & Engelmann, 2007). Low-
temperature storage of living tissues and cells must avoid ice crystal formation (especially
intracellular) during the whole process (cooling, storage and final re-warming), as ice results
lethal for cells, impairing specimens viability (Muthusamy et al., 2005; Nadarajan et al.,
2008).
Most techniques employed include an initial step designed to reduce the tissues water
content (with the limit of avoiding dehydration damage) and increase the cytoplasm internal
viscosity. This step may include the incorporation of extrinsic viscosity enhancing substances
(such as sugars, glycerol or ethylene glycol) and the induction of cold adaptation and defence
mechanisms by the plant cells. A second step consists in quick cooling by plunging into LN,
to avoid ice formation. The chances of ice crystallization are limited by both a decrease in the
amount and mobility of intracellular water and an increase in cooling rates, hindering the
rearrangement of water molecules into ice, a necessary previous nucleation step. Cooling to
LN temperature places the system well into the glassy state. Once vitrified, despite some
time-depending phenomena still taking place, relatively large molecular reorganizations, as
those required for ice formation, are not possible and specimens can be stored for
hypothetically indefinite periods, without ice being formed (Zamecnik et al., 2007). A third
step consists in the re-warming process, which must be performed quickly enough to avoid ice
nucleation while crossing by the temperature region comprised between the glass transition
temperature (TG) and the equilibrium freezing point (Tf) (Niino et al., 1992; Reinhoud, 1996;
Volk & Walters, 2006). Progressive dehydration and rapid cooling has been found to avoid
the formation and growth of lethal intracellular ice in plant cells (Wesley-Smith et al., 2001).
33
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
Many apparently independent physical properties undergo large changes associated to the
glass transition within similar temperature ranges. For example, the TG values determined by
calorimetry, thermal expansivity or viscosity are often nearly identical (Kauzmann, 1948).
This is a result of all these properties being parallelly influenced by changes in molecular
mobility occurring during the glass transition.
The initial works on mint cryopreservation by vitrification included gradual dehydration
using a mixture of ethylene glycol, dimethyl sulfoxide (DMSO) and polyethylene glycol
(Towill, 1990). A successful method among those usually employed for plant germplasm
cryopreservation is that named droplet-vitrification (Senula et al., 2007). It is derived from
the droplet-freezing technique (Kartha et al., 1982) developed for preserving shoot tips, which
are placed in droplets of cryoprotective medium and cooled slowly in a programmable
freezer. In the droplet-vitrification method, the specimen is initially incubated in solutions
with increasing concentration of cryoprotecting and dehydrating agents, for different periods.
Then it is treated and included in a small drop of cryoprotecting agents solution (containing
high concentrations of glycerol, ethylene glycol, DMSO and sucrose (Sakai et al., 1990)), and
deposited on a small strip of aluminium foil. Finally, the strip with the droplets is quickly
plunged into LN, and then stored in a cryovial in a LN container. Warming is carried out
similarly, plunging the strip plus droplets into warm liquid medium containing 1.2 M sucrose
and, after 20 min unloading, shoot tips are retrieved and placed on recovery medium. The
results of this method, in terms of viability, have been good for a variety of systems (Sakai &
Engelmann, 2007; Senula et al., 2007)
The main advantage of this technique is enabling very high cooling and warming rates, due
to the small volume of medium in which the explants are placed (Sakai & Engelmann, 2007).
Many protocols involve exposure of plant shoot apices to PVS2 at 0°C (Sakai et al., 1990;
Reed, 2008). To prevent cellular damage, cryoprotectant solutions both dehydrate and
penetrate cells to stabilize proteins and membranes. However, the mode of action of each
component may differ with cell type, species, temperature, and other solution components
(Dereuddre et al., 1990; Jouve et al., 2004) A particular component of the cryoprotectant
solutions employed, DMSO, enhances chemical penetration into cells and, in spite of its own
cytotoxicity -for which testing the treatment duration is essential- it has been found to
promote survival of cryoexposed cells (Fahy et al., 1984; Benson, 1999; Volk & Walters,
2006).
34
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
The interplay between exogenous and endogenous carbohydrates plays an important role
on plant resistance to stresses. For example, a two-step preculture treatment of gentian buds
with increasing sucrose concentrations produced a raise of abscisic acid, followed by an
increase of endogenous sucrose concentration, all of which resulting in a higher dehydration
tolerance (Suzuki et al., 2006). Various high-performance liquid chromatography (HPLC)
techniques have been developed to facilitate carbohydrates analyses in an effort to better
understand their role in biochemical processes. High-performance ion chromatography (IC)
coupled with pulsed amperometric detection (PAD) is an efficient method to quantify
carbohydrates in natural samples and food products (Carpenter & Dawson, 1991; Lee, 1996;
Masuda et al., 2001; Schiller et al., 2002; Cheng & Kaplan, 2003).
Differential scanning calorimetry (DSC) is a well-known technique used for studying
thermal properties of thermally driven processes. It has been used for long in cryopreservation
studies, as it yields interesting information on freezing and vitrification processes (Yamada et
al., 1991; Matsumoto et al., 1994; Volk & Walters, 2006; González-Benito et al., 2007;
Zamecnik et al., 2007). Water freezing latent heat is large, so calorimetry is a sensitive
method to monitor ice formation or thawing, even in small quantities. Besides the amount of
ice formed, after knowledge of the system�’s water content, the ratio of frozen-unfrozen water
may be also obtained. DSC yields also Tf, and its reduction speaks of increasing solute
concentrations. TG is also acquired, if scans are performed at sufficiently low temperature,
although DSC is not particularly sensitive for this purpose.
Scanning electron microscopy is a powerful and user-friendly microscopic technique, able
to provide a wealth of structural information on a wide range of samples. When combined
with a cryo-stage (cryo-SEM), it is especially interesting to study the microstructure of
biological systems, not altered in sample preparation (Craig & Beaton, 1996). Low pressure
LN frozen samples preserve most structural relations present in the original sample, with little
or no artefacts. Cryo-SEM allows sample observation without need of prior chemical fixing or
drying. Ice crystals formed are small and not altering tissue structures. Ice formed inside cells
can be observed, after fracture in the cryo-stage and a suitable etching process, as black
�“void�” regions surrounded by grey ridges formed by highly cryo-concentrated solution either
frozen as euthectic, unfrozen or even vitrified.
In this work, these three powerful techniques, DSC, Cryo-SEM and HPLC, have been
applied to follow the behaviour towards LN cooling of specimens of plant germplasm -mint
35
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
(Menta ×piperitha) shoot tips- in different steps of the droplet-vitrification protocol. To date,
no publication provides data obtained during this cryopreservation protocol, but only of its
final outcome. Actually, very little physical information exists on actual cryopreserved or
vitrified plant specimens. Besides, the combined use of information resulting from these
physicochemical techniques allows us to better understand the phenomena taking place
between the normal and the vitrified states of plant tissues and back, to restore its viability as
germplasm. Interesting relations among sucrose and cryoprotectant content in cells and its
freezing and vitrification behaviour are found, as well as with the other factors ruling over the
physical control of ice formation: water content and cooling rate. The data here presented are
expected to help to design improved cryopreservation procedures, involving less damaging
cryoprotectants and/or suitable for problematic species.
4.2. Materials and methods
4.2.1. Plant material pre-culture and shoot tips extraction
Shoot tips were extracted from in vitro shoots of Mentha ×piperita (genotype �“MEN 186�”
obtained from the IPK Genebank, Gatersleben, Germany). In vitro plants were monthly
subcultured on medium MS (Murashige & Skoog, 1962) with 3% sucrose, and incubated at
constant temperature (25°C) with a photoperiod of 16 h, and an irradiance of 50 µmol m-2 s-1
from fluorescent tubes. One-node segments were obtained from these shoots, transferred to
fresh medium and incubated at alternating temperatures of 25°C (day) and -1°C (night),
always with 16 h photo- and thermoperiod, 50 µmol m-2 s-1irradiance, provided by fluorescent
tubes (stage a, see Figure 4.1). After 3-weeks of culture under these conditions, shoot tips (1-
2 mm) were excised from axillary buds.
4.2.2. Incubation and dehydration of shoot tips
A protocol based in that of Senula et al. (2007) was followed. Five excised shoot tips were
pre-cultured overnight with 2 mL of liquid MS medium containing 0.3 M sucrose at 25ºC,
over filter paper (stage b, see Figure 4.1). Thereafter, the explants were transferred to a Petri
dish with 2 mL of loading solution (2 M glycerol + 0.4 M sucrose) over filter paper, for 20
min, at room temperature (stage c). Finally, they were osmotically dehydrated in 2 mL PVS2
36
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
(plant vitrification solution 2: 30% w/v glycerol, 15% w/v ethylene glycol, 15% w/v DMSO
and 0.4 M sucrose in plant growth medium (Sakai et al., 1990)) in a Petri dish, on filter paper,
for 30 min at 0ºC (stage d). Immersion in LN was carried out in the cryo-SEM or inside the
aluminium pans of DSC, depending on the experiment. A scheme of these steps can be seen at
Figure 4.1.
Figure 4.1. Scheme of the steps of the droplet cryopreservation protocol employed, see text for more details on media and solutions employed.
4.2.3. Plant recovery and viability
Treated and control shoot tips were warmed from LN temperature by plunging into warm
liquid medium with 1.2 M sucrose for 20 min. After the unloading of cryoprotectants, shoot
tips were cultured on re-growth medium, composed of solid MS medium supplemented with
0.5 mg/L BAP + 3% sucrose, cultivated in the dark for 24 h and finally placed into the culture
room at 22-25ºC and 16 h illumination. For control samples (-LN), all steps were carried out,
including the incubation in PVS2 for 30 min. After that, the explants were rinsed in 1.2 M
sucrose solution for 20 min and then placed on the re-growth medium.
Survival and re-growth were calculated as rates over the total number of shoot tips used.
Survival was defined including all forms of visible viability (evidence of green structures or
callus) observed 1-2 weeks after re-warming. Re-growth was defined as the formation of
small plantlets, 4 weeks after re-warming.
37
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
4.2.4. Low temperature scanning electron microscopy
Low temperature scanning electron microscopy (cryo-SEM) observations were performed
with a Zeiss DSN 960 scanning microscope equipped with a Cryotrans CT-1500 cold plate
(Oxford, UK). Three shoot tips in the same stage of the cryopreservation protocol were fitted
on a special bracket with their axes vertically aligned. The geometry of sample and sample
holder required optimization for both freezing and fracturing procedures: these steps require
stable mechanical mounting of the sample on a support. This piece was plunged into liquid
nitrogen (LN) under low pressure, physically fixing tissues for microscopic observation. This
cooling step was considered equivalent to the cooling process taking place in the actual
cryopreservation process, by LN immersion. The holder was, then, inserted in the pre-
chamber of the Cryotrans cold plate, at -180ºC, and specimens were fractured perpendicularly
to their axis, to obtain a suitable observation surface (a cartoon of the tips before introduction
in the microscope and after fracture can be seen in Figure 4.2.
a
b
Figure 4.2. Cartoon showing the three mint tips on the microscopy holder before insertion in the microscope (a) and after equatorial freeze fracture (b).
Later, samples were inserted in the microscope and etching (partial ice sublimation,
induced to provide contrast) was performed for three minutes at -90ºC. Once etched and re-
cooled, the sample was retrieved into the cryo-preparation chamber, and coated with high
38
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
purity Au, which acts as a conductive contact for electrical charge. The etched and coated
sample was reinstated into the cooled SEM stage and observed using an accelerating voltage
of 15 kV, at -150/-160ºC.
4.2.5. Dry matter and water content determination
Six sets of five tips were used for determining the dry mater content (dm) in shoot tips at
the different stages of the cryopreservation protocol. It resulted from weighing after oven-
drying (~85°C, 72 h) and was expressed as relative to the total tip mass. The water content
(Wc) of shoot tips could be calculated for stages a and b, directly as the difference between its
total mass and dm. It was expressed as relative to the total shoot tip mass (Wc(s)) or the dry
mass (Wc(dm)).
4.2.6. Differential scanning calorimetry
Differential scanning calorimetry (DSC) experiments were performed with a Mettler-
Toledo DSC 30 instrument (Mettler-Toledo, Griefsen, Suiza). Cooling was performed using
the calorimeter control (10°C min-1), or for higher rates, by quenching the samples in LN (see
below). The response of shoot tips apices to each step in the mint protocol was evaluated.
Five shoot tips in the same stage of the cryopreservation protocol were introduced in a DSC
pan, which was sealed and weighed. Shoot tips had been sequentially treated with MS
medium, loading solution and PVS2. Samples were submitted to a cooling scan from room
temperature to -150ºC and a short 5 min equilibration at this temperature, followed by a
warming scan (at 10ºC min-1), back to room temperature. Calorimetric data were collected
from two replicates per treatment.
For the quenching method shoot tips, sealed into a DSC pan, were quickly plunged into
LN. Pans were subsequently cold-loaded into a pre-chilled DSC (-150ºC) for analysis.
Later, pans were punctured and dried in an oven at 85ºC, for 72 hours, when they showed
constant weigh. Thermograms were analysed using the standard procedures provided in the
Mettler-Toledo STARe software. Ice thawing thermal events (more precise than freezing
events), were used for extraction of Tf. The routine produced the temperature corresponding
39
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
to the event onset, Tf(onset), corresponding to the equilibrium freezing temperature and that to
the peak, Tf(peak).
The equilibrium freezing temperature can be related to the water activity (aw) of the
system, to its osmolality ( ) and to its solute content (Fullerton et al, 1994; Elliott et al.,
2007). For comparative purposes, a �“sucrose equivalent�” concentration (Suceq) and a �“sodium
chloride equivalent�” (NaCleq) were determined from Tf, assuming that the only solute present
were sucrose or sodium chloride, respectively. Tf(peak) data were used for the determination of
these parameters, as it was considered to better represent the behaviour of the whole phase
change. Eq. 1 (Fullerton et al., 1994) was followed for aw determination:
Eq. 1 ln aw = Hf(w) Aw (Tf - Tf *) / (Tf Tf *)
where Hf(w) is the pure water freezing enthalpy (specific, per water gram), Aw water
molecular weight, and Tf * the equilibrium freezing point of pure water. The osmolality was
calculated after eq. 2 (Elliott et al., 2007):
Eq. 2 = Tf /1.86
where Tf is the freezing point depression, which for water at atmospheric pressure, being the
pure substance freezing point 0ºC, equals to Tf with a positive sign.
Suceq and NaCleq could not be calculated from Raoult�’s law, using analytical equations
(Guignon et al., 2008), only valid for more diluted solutions. Experimental data from
literature (Wolf et al., 1985; Fullerton et al., 1994) and calorimetric determinations performed
on concentrated sucrose solutions (data not shown) were employed to relate these hypothetic
solute concentrations with the observed cryoscopic temperature decrease.
The process associated enthalpy, phase change enthalpy Hf, proportional to the event
area, was also obtained. It was expressed as relative to either the total shoot tip mass ( Hf(s)),
its dry mass ( Hf(dm)) or its water content Hf(w), for allowing easier data comparison.
Water fractions: frozen water (Wf) and unfrozen water (Wu), were calculated by
comparison of Hf and the pure water freezing enthalpy ( Hf(w)* = 333.4 J/g at 0ºC), together
with the total water contents previously determined, after eq. 3:
40
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
Eq. 3 Wf= ( Hf/ Hf(w)*); Wu=Wc-Wf
Specific enthalpy values (per sample gram) were always used, after the corresponding
oven dry pan weighs. Enthalpy values measured at temperatures different from the
corresponding phase change equilibrium temperature, 0ºC, are underevaluated, as a result of
the difference between water and ice heat capacities (Cpw and Cpi, respectively). So, the
value of Hf(w)* was corrected after eq. 4, before applying eq. 3. Single freezing temperatures,
Tf(peak), were used, and Cpw and Cpi data were obtained from a data calculation routine
produced in this laboratory (Otero et al., 2002), after interpolation when required, and other
sources (Angell et al., 1973; Angell et al., 1982).
Eq. 4 - (
Frozen (Wf) and unfrozen (Wu) water contents were expressed as relative to the total shoot
tip mass (Wf(s),Wu(s)), its dry mass (Wf(dm), Wu(dm)) or its total water content, when available,
(Wf(w), Wu(w)).
The glass transition can be observed as a small step in the heat capacity thermogram
baseline. The corresponding heat capacity increment is very small and, in our experiments,
only could be appreciated by using 30 shoot tips in the same DSC pan.
For comparative purposes, calorimetric experiments were performed with samples of the
cryoprotectant solutions employed in each stage of the droplet protocols (i.e., liquid MS
medium with 0.3 M sucrose, loading solution and PVS2). Experiments were performed in the
same conditions than those carried out with shoot tip specimens.
4.2.7. Sucrose content assessment in shoot tips
Specimens were analysed after each stage of the cryopreservation procedure for its sucrose
content. Five shoot tips were weighed (average fresh weight 3.0 ± 0.1 x 10-3 g) and mortar-
ground into a fine powder in LN. As grinding proceeded, the vessel was slowly warmed.
Immediately, 1.5 mL Milli-Q water was added and the mixture was filtered through a syringe-
driven filter (Millex-GS, 0.22 m).
= * )
41
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
The determination of sucrose was carried out by an Ion Chromatography 817 Bioscan
(Metrohm, Herisau, Switzerland) system equipped with a Metrosep Carb 1-250 column and a
pulsed amperometric detector (PAD) with a gold electrode. A three-step PAD protocol was
used with the following time intervals (ms) and potentials (mV): t1, 400/E1 = +0.05
(detection); t2, 200/E2 = +0.75 (cleaning); t3, 400/E3 = 0.15 (regeneration). Samples (1.5
mL) were injected using an autosampler (model, 838 Advanced Sample Processor, Metrohm,
Herisau, Switzerland) and the flow rate through the column was 1mL min-1, the eluent was
100 mM NaOH.
Identification of sucrose was performed by comparison with the retention time of the pure
standard sucrose (Merck) and the data was acquired using IC Net 2.3 software. Sucrose
content was expressed as ppm and the data were the mean of three replicates. Results (Suc)
are expressed as p.p.m. of the total tip mass.
4.3. Results and Discussion
4.3.1. Detection of glassy state and ice by cryo-SEM
Two different observation approaches were compared: secondary electron and
backscattered electron image. Basically the same information was obtained with both,
however, the cellular structures could be better appreciated using backscattered electrons, and
this was employed in the observations and micrographs here presented.
Figure 4.3 shows micrographs showing shoot tips cooled in LN in the microscope cryo-
unit as described in the Methods section, in different moments of the droplet cryopreservation
protocol: control (A); stage a, cold treatment (B); stage b, preculture (C, D); stage c, loading
(E, F), and stage d, dehydration (G, H). C, E and G show lower magnification images of the
shoot tips axial plane. The concentric cell distribution in the equatorially fractured tips could
be appreciated. There were no noticeable differences among central or more external cells,
indicating that the fracture was close to the apical dome, so effective homogeneity for
temperature and solute and water concentrations could be assumed for tip cells.
Ice crystals formed in the more concentrated solutions were smaller, due to the limitations
to crystal growth caused by the higher solution viscosity. On the other hand, conversion of
water into ice during cooling increased the concentration of solutes in the more concentrated
42
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
regions. The etching procedure applied gave rise to a contrast in the SEM micrograph,
removing part of the ice formed by sublimation and leaving the darker regions separated by
sector or �“ridges�” of freeze-concentrated solution that can be appreciated in micrographs A-F.
This behaviour allowed visualization of individual crystals. Sublimation is only taking place
at an appreciable rate out of pure ice crystals, while water in glassy state has a sublimation
rate almost negligible (Umrath, 1983; Franks, 1986). So, when solutions become vitrified,
they cannot be etched in this way. Micrographs G and H show a complete lack of the
structural details revealed by etching.
4.3.2. Determination of pre-culture effect by cryo-SEM
Figure 4.3 A and B show, respectively, typical cryo-SEM micrographs after 3 weeks of
culture at 25ºC (control apices) or at 25º/-1ºC. Widespread cellular disruption occurred in
control apices, as it can be observed in micrograph 3 A, where ice can be seen as dark areas
inside the cell�’s cytoplasm and vacuole, meanwhile clearer parts would correspond to the
supercooled cryo-concentrated solution matrix. Cold-treated shoot tips (4.3 B) showed the
formation of smaller ice crystals, mostly in the vacuole space, which indicates a higher solute
cytoplasmic concentration with respect to control apices. The observed size crystal reduction
is understood as due to an increase in the amount of endogenous cryoproctecting agents,
developed by the plant during the cold hardening procedure, confirming the positive influence
of tips cold hardening prior to cryopreservation observed by other workers (Senula et al.,
2007).
4.3.3. Observed plant recovery and viability
Both survival and recovery reached a 96% value for specimens treated with the complete
droplet cryopreservation protocol (i.e., quenched in LN after stage d, dehydration in PVS2).
Meanwhile, survival and recovery of previous stages, a to c, were 0% for all cases. The
control samples (-LN) showed a 100% survival and recovery.
43
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
A B
C D
E
G
F
H
Figure 4.3. Cryo-SEM micrographs of shoot tips cooled in LN in the microscope cryo-unit (see Methods). Control (A); stage a, cold treatment (B); stage b, preculture (C and D); stage c, loading (E and F), and stage d, dehydration (G and H). The bar corresponds to 10 µm (A, B, D, F, H) or 50 µm (C, E, G).
44
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
4.3.4. Analysis of the droplet-vitrification protocol steps by cryo-SEM
Micrographs of specimens in the stages b (preculture) (C, D), c (loading) (E, F) and d
(dehydration) (G, H) of the droplet-vitrification protocol can be seen in Figure 4.3.
Specimens in both b and c stages showed a clearly visible tissular and cellular structure, with
some organelles and vacuolar membrane elements present. Specimens in stage b showed
larger ice crystals and a higher degree of alteration of cellular structures by ice than those in
stage c. Tips treated with alternating temperatures (see previous section) had a similar aspect
to those in step b (preculture). The ice crystal size difference could be due to concentration
differences among samples, as the crystals formed in the more diluted solution can grow to a
larger final size, while those occurring in concentrated solutions, where molecular mobility is
impaired, would be smaller.
During the treatment in the loading solution, the cells of specimens in stage c of the
protocol accumulated exogenous glycerol and other solutes of endogenous origin. These cells
were clearly partly plasmolyzed as a result of the osmotic dehydration imposed by the loading
treatment, which was related to the progressive acquisition of tolerance toward freezing
during the protocol.
PVS2, the cryopreservation solution employed in the last step, affected cellular freezing
properties (Volk & Walters, 2006). It has components that are penetrating (dimethyl
sulfoxide, glycerol and ethylene glycol) and non-penetrating (sucrose) for the cellular
membrane of most cell types (Benson, 2008). This complex solution served to dehydrate
shoot tips and to change the behaviour of the water remaining within shoot tips.
Micrographs of specimens in the stage d of the protocol, after osmotic dehydration in
PVS2 solution, presumably with a lower water content and more concentrated in solutes
induced by permeation from the cryopreservation solution, showed a grey continuous, little
detail surface, indicating vitrification (4.3 G and H).
4.3.5. Changes in the dry mass and water content during the droplet-vitrification
protocol
The dry mass and water content of shoot tips, derived of its weight-difference after drying,
are shown in Table 4.1. For step a and b, where water was the only volatile substance
45
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
(overseeing very small quantities of organic compounds), Wc could be obtained by this
procedure, and is presented in Table 4.1, expressed as related to the total sample mass Wc(s) or
to the dry mass Wc(dm).
The water content of tips undertaked a small decrease with the first steps of the
cryopreservation protocol, associated to sucrose incubation in stage b. Water content for
stages c and d could not be derived from oven-drying data, as the amount of volatile
substances apart from water was high. Nevertheless it could be assumed that there was a large
decrease in the water content associated to step c, as inferred from the increase in dm, which
was in good agreement with the reported microscopic observations showing a smaller ice
crystal size in these samples and with the sucrose content data (see below).
Table 4.1. Compositional and thermal parameters (mean standard deviation) of the thawing events obtained from DSC thermograms for mint shoot tips specimens at different stages (a, b, c and d) of the droplet cryopreservation protocol. Cold treatment
(stage a) Preculture (stage b)
Loading (stage c)
Dehydration (stage d)
dm (gdm gsample-1) 0.117 0.009 0.151 0.009 0.344 0.049 0.252 0.013
Wc(s) (gwater gsample-1) 0.883 0.009 0.849 0.009 -------- --------
Wc(dm) (gwater gdm-1) 7.56 0.65 5.64 0.39 -------- --------
Tf (onset) (ºC) -2.96 0.06 -4.9 1.8 -7.93 0.79 --------
Tf (peak) (ºC) -0.48 0.08 -0.58 0.21 -5.5 1.1 --------
Hf(s) (J g sample
-1) 241 16 209.85 0.12 93.0 7.2 --------
Hf(dm) (J gdm-1) 1800 650 1394 83 274 + 60 --------
Hf(w) (J gw-1) 272 14 247.1 2.5 -------- --------
Wf(s) (gwater gsample
-1) 0.724 0.47 0.632 0.000 0.290 0.023 0 Wf(dm) (gwater gdm
-1) 6.20 0.88 4.20 0.25 0.86 0.19 0 Wf(w) (gwater gwater
-1) 0.820 0.045 0.744 0.007 --------- 0
Wu(s) (gwater gsample-1) 0.159 0.038 0.217 0.009 -------- --------
Wu(dm) (gwater gdm-1) 1.34 0.22 1.44 0.14 -------- --------
Wu(w) (gwater gwater-1) 0.180 0.045 0.256 0.007 -------- --------
aw 0.9954 0.001 0.9944 0.002 0.9470 0.001 ---------
(Osm) 0.26 0.04 0.31 0.12 2.97 0.60 ---------
Stage d, following the same arguments, would show the complex result of water
osmotically driven out of the cell by the strong potential of the very concentrated PVS2
solution, rich in the non-penetrating sucrose, entrance of the penetrating agents DMSO,
ethylene glycol and glycerol, and water retention inside the cell by the increased internal
presence of these cryoprotectors (Schäfer-Menhur et al., 1997; Hubálek, 2005; Panis &
46
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
Lombardi, 2006; Benson, 2008; Volk & Walters, 2008). Notwithstanding the increasing
presence of endogenous cryoprotecting (and often water binding) agents, as a result of the
plant response to the induced stress.
4.3.6. Measurement of ice heat of fusion using DSC
Figure 4.4 shows typical re-warming DSC thermograms for the same specimens types (at
stages a, b, c and d), previously cooled at a rate of 10ºC min-1. Table 4.1 shows the thermal
parameters derived from DSC experiments: Tf(onset) and Tf(peak), respectively, the onset
temperature of the melting endotherm (corresponding to the equilibrium freezing temperature)
and its peak temperature. The freezing enthalpy ( H) is also included. The information that
both enthalpy and equilibrium freezing temperature can provide is of the highest interest, as it
yields information on the water content within cells and, in a context where it is difficult to
know the detailed cytoplasm composition and interacting effects of endogenous and
exogenous solutes, to draw conclusions on the availability of this water, to form ice, to permit
diffusion-driven processes and even to keep the stability of protein and membranes.
Specimens both in stages b and c of the cryopreservation protocol showed freezing (not
shown) and melting events in the respective cooling and re-warming scans.
The equilibrium freezing temperature is a good raw data to determine accurately solution
characteristics, such as water activity and frozen and unfrozen water (Guignon et al., 2008).
However, the precision yielded by DSC for melting temperatures, especially in complex
systems, such as natural tissues, is limited. The onset temperature is considered usually as the
best estimation of the equilibrium freezing point. But, in complex solutions,
compartmentalized in different isolated pools, with markedly different composition and water
contents (cytoplasm, vacuoles, intercellular space, different cells and tissues) the onset
temperature would rather correspond to the melting of the most concentrated pool, which
would thaw at the lowest temperature.
We have considered that, for these sample types, the peak temperature was giving a better
estimation of the average melting of the specimens studied. Moreover, the DSC-measured
temperature was affected by the thermal differences (as well as the compositional ones)
within the sample, not negligible at a relatively fast scanning speed, as 10ºC min-1.
47
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
The temperatures determined were decreasing steeply with the progress of the
cryopreservation protocol, which spoke about the increase in the �“number�” solute molecule
concentration. The last stage showed no thawing event, and there would be no frozen water at
all, in these conditions. From Tf, the aqueous solution parameters aw and were derived and
shown in Table 4.1.
Figure 4.4. Typical DSC thermograms corresponding to re-warming processes of shoot tips in different stages of the droplet cryopreservation protocol: cold treatment (stage a), preculture (stage b), loading (stage c) and dehydration (stage d). Each experiment was performed with five shoot tips. The insert shows an expanded thermogram section which allows the glass transition, obtained with 30 tips, to be appreciated. Scanning rate was 10ºC min-1. (See Materials and Methods and Figure 4.1. for more details). The ordinates scale of the hermograms baseline is arbitrary.
t
The thawing enthalpy is, in practice, a much more accurate parameter than freezing
enthalpy, depending less on kinetic, geometric or compartmentalization issues. The phase
change enthalpy, that is presented in Table 4.1 as relative to either the total sample mass,
Hf(s), the dry mass, Hf(dm), or the total water content, Hf(w), decreased as the protocol
progresses. After correction for the different value of the pure water melting enthalpy at the
actual phase change temperature for each of these processes, the amount of thawed water in
48
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
each process could be calculated, by simple comparison with this quantity. The resulting
frozen water content, presented in Table 4.1 relative to either the total sample mass, Wf(s), the
dry mass, Wf(dm), or the total water content, Wf(w), again markedly decreased as the protocol
progressed. The last step could also be comprised in this trend, as Wf would equal to zero.
The unfrozen water fraction (derived from the frozen water fraction) can help to visualize
the changes undergoing in the cryopreserved tissues, and is also showed in Table 4.4 relative
to the total sample mass, Wu(s), the dry mass, Wu(dm), or the total water content, Wu(w).
Traditionally, frozen water was understood and often named as free water, while the unfrozen
fraction was considered as bound water (Robinson, 1931). These denominations are much
criticised nowadays (Franks, 1986), as the differences found in the actual binding of these two
water fractions are very small, if any at all (Hills et al., 1990; Wiggins, 1990; Hills et al.,
1991; Slade et al., 1991; Hills et al., 1998; Wolfe et al., 2002).
For comparative purposes, Figure 4.5 shows typical calorimetric thermograms obtained
during re-warming for the cryoprotective solutions employed (at stages b, c and d), in the
same conditions than those applied in the experiments of Figure 4.4.
Figure 4.5. Typical DSC thermograms corresponding to re-warming processes of the cryopreservation solutions employed in different starges of the droplet cryopreservation protocol: preculture (stage b): liquid MS medium containing 0.3M sucrose, loading (stage c): loading solution and dehydration (stage d): PVS2. The insert shows an expanded thermogram section which allows the glass transition to be appreciated. Scanning rate was 10ºC min-1. (See Materials and Methods and Figure 14 for more details). The ordinates scale of the thermograms baseline is arbitrary.
49
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
Hf(s) for stage b (liquid MS medium with 0.3 M sucrose) was 228 ± 2 J g sample-1, while for
stage c (loading solution) it was 138 ± 2 J g sample-1. Stage d (PVS2) showed no melting
exotherm. Hf(w) for stage b amounted to 250 ± 3 J gw-1. The Tf (onset) were -3.97 ± 0.2ºC for
stage b and -4.68 ± 0.3ºC for stage c.
4.3.7. Effects of quench-cooling on heat of fusion of ice
In Figure 4.6, thermograms of the re-warming DSC stage of mint shoot tips, cooled by
quenching are compared with those at a rate of 10ºC min-1 in specimens of step a. It can be
seen that, in the quench cooled sample, the thawing temperature decreased and the enthalpy
increased slightly. These results, though consistent in all repeats and for experiments in
different conditions (data not shown) were unexpected, and of difficult explanation. A
possibility is that the fast cooling might trap solute molecules within the ice phase, which
would later contribute to an average effective melting at lower temperatures (Obbard et al.,
2003; Ohnoa et al., 2005; Hashimoto et al., 2011).
Figure 4.6. DSC scans showing warming at 10ºC min-1 for shoot tips treated at the preculture stage (b) of the droplet cryopreservation protocol. Cooling rate was 10ºC min-1 or quenching (direct immersion of the DSC pan in LN).
50
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
Nevertheless, this behaviour, although may be further from equilibrium than the observed
in the slow cooling, would correspond more closely to the real events taking place in
cryopreservation practice.
4.3.8. Detection of glass transition by DSC
Specimens at the last stage of the cryopreservation protocol did not show any freezing
event, in any case, either at the cooling (not shown) or melting runs of DSC studies.
Nevertheless, in many experiments, the step in the heat capacity baseline showing the actual
glass transition in DSC thermograms could not be appreciated. Cooling rates of 10ºC min-1,
controlled by the calorimeter itself, or externally driven, by quick introduction into LN
(quenching) were tested, but the glass transition could only be observed (for both cooling
rates) for samples containing 30 shoot tips. Calorimetry is often claimed to be a rather
insensitive method to observe the glass transition (Verdonck et al., 1999), and this is
especially applicable here, as the dehydrated tips have a very small weight (total average
sample mass for each DSC experiment -5 tips- for d-stage specimens was approximately 3.5
mg and the total mass of water in these samples 2.5 mg).
As shown in the insert of Figure 4.5, PVS2 solution is also showing a glass transition in
these conditions, centred at -120ºC.
4.3.9. Changes in sucrose content during the droplet-vitrification protocol
The actual composition of the cellular contents in cryopreservation conditions is critical for
the formation of ice or the vitrification of the system. Most studies rather equate this
composition (or the physical behaviour of the cytoplasm) to that of the cryopreservation
solutions applied, which is not necessarily correct. A simple extraction procedure and a
general chromatographic method for small carbohydrate molecule quantification calibrated
for sucrose were applied, as a first approach to work out this composition. The sucrose
content data obtained are presented in Table 4.2, expressed as related to the total sample
mass, Suc(s), or to the dry mass, Suc(dm), reflecting the increase in sucrose content within
tissues, not only as a result of the actual increase of the content of this compound, but of the
decrease in that of water.
51
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
There was a strong increase in its level between stages b and c (Table 4.2). Meanwhile the
sucrose incubation of the preculture leading to stage b only implied a moderate increase
(about 10%) of the internal sucrose level, the loading with glycerol gave rise to a dramatic
increase of this concentration (7 fold). The stage d, dehydration, after PVS2 treatment,
underwent a further increase, but much more moderate. This could be interpreted as a
reflection of the actual cellular water content, and was in good agreement with the data of
water contents and its frozen and unfrozen fraction showed in Table 4.1.
Other sources for changes in sucrose levels, different from the alteration in the water
content, were possible, such as the actual penetration of sucrose, which could be facilitated by
the presence of DMSO in the PVS2 solution, an agent known for promoting the penetration of
other solutes through membranes (Sakai & Engelmann, 2007).
Equivalent solute concentrations (isoosmotic) can be obtained from the freezing
equilibrium temperatures, obtained by DSC (Buckley et al., 1958). The observed decrease in
this temperature can be related by Raoult�’s law to the molal concentration of the solution. In
the case of tissues it would rather be an average of the different solution pools in the different
cell types and sub-cellular compartments of the specimens studied. The analytical equations
are only valid for solutions much more diluted than those considered here, and their
application would overestimate the solvent concentration required to induce the observed
cryoscopic decrease. The Tf(peak) obtained were, so, compared with tabulated data sets,
complemented when necessary with experimental DSC determinations for concentrated
solutions (data not shown). Equivalent sucrose concentration, Suceq, and equivalent sodium
chloride concentrations, NaCleq, were calculated and presented in Table 4.2. These coarse
estimations could not describe the complex composition of cellular fluids. Nevertheless, as
the observed decreases in Tf were caused by the increased number of molecules in solution,
the amount of small molecular weight compounds (either dissociated, as NaCl, or non-
dissociated, as sucrose), would have a more determinant role than the presence of larger
macromolecules and other specific plant-generated substances.
When comparing the chromatographic sucrose content with these isoosmotic solute
estimated concentrations, it could be seen that the total solute content of cellular
compartments must be much higher than the sucrose amount reported. Especially, a high
increase in the estimated solute concentrations could be appreciated after incubation with the
loading solution, may be reflecting the entrance of significant amounts of glycerol in cells,
52
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
apart form the already commented dehydration. It must be noted, when comparing Table 4.2
parameters that, while the sucrose content chromatographically determined refers to the total
or dry sample mass of the shoot tips, the estimated sucrose and sodium chloride contents, as
originated from the cryoscopic decrease, refer to the solution mass (only considering solutes
and solvent).
Table 4.2. Solute content (mean standard deviation) of mint shoot tips at the different stages of the droplet cryopreservation protocol. Cold
treatment (stage a)
Preculture (stage b)
Loading (stage c)
Dehydration (stage d)
Suc(s) (p.p.m. total
sample weight) 5900 600 6500 300 46900 700 53000 4000
Suc(dm) (g gdm
-1) 0.059 0.006 0.044 0.002 0.137 0.002 0.209 0.015
1Suceq (p.p.m.) 8800 10800 470000 ---------
1NaCleq (p.p.m.) 8000 10000 91000 ---------
1These concentrations, as derived from the freezing point depression, refer to the solution mass (mass of solutes plus mass of solvent), not taking into consideration any non-dissolved substances.
4.4. General discussion
The combined results of DSC and cryo-SEM are in good agreement, implying that part of
the water contained in the system for specimens on steps a and b becomes frozen when
cooled in LN (with less amount of ice formation in the latter) while specimens in step c would
avoid ice formation, presumably getting vitrified in the cooling process, as they showed no ice
trace, either microscopically or calorimetrically. This is also in good agreement with our data
for viability and recovery and those of literature, resulting of applying the droplet method to
several systems, including mint apices (Keller & Dreiling, 2003; Halmagyi et al., 2005;
Leunufna & Keller, 2005; Panis et al., 2005; Kim et al., 2006a; Kryszczuk et al., 2006;
Senula et al., 2007; Sant et al., 2008; Halmagyi et al., 2010; Vujovic et al., 2011).
The different experimental approaches employed agree in a reduction of the cellular water
content with the cryopreservation protocol progress. The mayor dehydration would take place
during the loading solution incubation (stage c, showing much lower water content than b).
This agrees well with the dry weight evolution, as well as the calorimetric frozen water
content and the observed decrease of the equilibrium freezing temperature. The decrease in
53
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
the size of ice crystals observed by cryo-SEM and the increases in the sucrose content
obtained by ionic chromatography would confirm this result.
Regarding the last and most interesting stage d, there is less information available, as on
one hand, the presence of large quantities of volatile substances precludes the measurement of
a reliable water content by differential weighing, and on the other, there were no freezing
event observed that could yield information about water fractions or cryoscopic decrease.
Nevertheless, the sucrose content data could imply a further dehydration degree over stage c,
unless a significant amount of the sucrose contained in PVS2 solution could have penetrated
cells facilitated by DMSO. The evidences of vitrification derived of the cryo-SEM
observations and the glass transition detected in DSC (as well as the very lack of freezing or
thawing events at any scanning rate) justify a higher cytoplasm viscosity in stage d than in
stage c specimens, evidently not vitrified. This viscosity increase could be originated either by
a further water content reduction or by the entrance in cells of the viscosity enhancing PVS2
components (or both).
The incubation with glycerol, a known penetrating agent (Schäfer-Menhur et al., 1997;
Hubálek, 2003; Volk & Walters, 2006; Panis & Lombardi, 2006; Benson, 2008) has been
reported to promote water exit from cells, driven by the osmotic gradient, but also a
substitution of part of this water by glycerol inside cells (Benson, 2008). The comparison of
total shoot tips specimen and dry mass (Table 4.1) with water content data from other
workers (Volk & Walters, 2006), whose mint shoot tip protocol is not equivalent but similar
enough, allow calculate that for stage c specimens, at a water content of approximately 1.8
gwater gdm-1, the amount of glycerol possible incorporated to shoot tips would be small (about
0.1 gwater gdm-1). While in this case most of the effect of the protocol step would be, then,
simple osmotic dehydration, the same type of calculation applied to stage d yields, for a water
content of approximately 0.7 gwater gdm-1, an entrance of extrinsic compounds corresponding to
nearly 60% of the final specimen mass (about 2.3 gwater gdm-1).
A general reflection on these data may acknowledge the natural variability of the
properties here determined. In spite of the efforts to prepare equivalent start point materials
and to treat them in a similar way, differences in size, permeability, microscopic scale damage
and all short of small compositional and microstructural differences would exists, in a way not
easy to monitor. Comparison with other similar experiments (data not shown), it can be
observed that this variability would have a larger impact on the equilibrium freezing
54
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
temperature data, while the enthalpy and associated water fractions would be more constant.
And, an absolute observation is that for all repeats, sets of experiments, and even cooling rates
applied, the final stage of the cryopreservation protocol is never producing calorimetrically
detectable formation of ice. This is in good agreement with the viability results found for this
protocol (Senula et al., 2007), viability which is incompatible with the formation of ice.
The calorimetric properties (melting enthalpies and equilibrium freezing and glass
transition temperatures) of the cryoprotecting solutions are not too different of those of the
specimens treated with them. In addition to some differences being evident, this calorimetric
similarity cannot be taking as a proof of equal composition and/or behaviour in other respects.
The probabilities of ice formation in specimens under TG and over TG but at temperatures
not too elevated (roughly, no more than 20ºC over TG) are similar, if a short frame time is
considered, such as those corresponding to cooling or warming processes in cryopreservation.
But this may be not so for longer time periods, such as those corresponding to storage.
Although storage is normally carried out below -150ºC, well under these systems TG, it must
be remembered that, when over TG, at long storage periods, ice formation is always a real
possibility (Angell, 1982; Franks, 1986).
A further consideration can be derived from the lack of ice observations in DSC at the last
stage of the cryopreservation protocol, when the cooling is carried out at 10ºC min-1, a rather
slow speed. This, generally observed for this samples and this protocol (data not shown),
would be indicative of a possible excessive amount of cryoprotecting agents, although, for the
biological material here employed, we find that there is no appreciable damage, at least in
terms of viability and recovery. Cooling at 10ºC min-1 would mean that the samples are in the
ice formation window (roughly, without considering the differences in ice formation
probabilities for different temperature regions, between -20ºC to -110ºC), for about 9-10 min.
Quench cooling would mean a permanence in this ice formation possible temperature window
for only a few seconds (data not shown). Considering only the effects of the cryopreservation
protocol in the formation of ice (not valuating here the possible membrane or protein
protection towards low temperature or dehydration, for example) a reduction in the
concentration of the vitrification solution employed could produce specimens that may be ice-
formation prone at low cooling rates but, would not form ice at quenching rates, which are
those actually used, in practical cryopreservation. This observation should be taken into
consideration, especially when the effect of some of the cryoprotecting components of these
55
4. GLASSY STATE AND CRYOPRESERVATION OF MINT SHOOT TIPS
solutions, such as DMSO, are criticised for its possible mutagenic or cytotoxic effects
(Harding, 2004).
56
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
5. CRYOPRESERVATION OF MINT SHOOT TIPS BY
ENCAPSULATION-DEHYDRATION: DETERMINATION OF
THE GLASSY STATE BY CRYO-SEM AND DSC
5.1. Introduction
Mint is an economically important crop presenting, together with its related wild species,
both types of material susceptible to be preserved as germplasm: seeds and vegetative
material. Although the initial studies on mint were mainly focused to its chemical
composition, cultivation systems and essential oil extraction and product quality, currently the
preservation, especially long term, of mint genetic resources is getting an outstanding
attention. This trend is comprised in the current general interest for preservation and the boost
of cultivation of medicinal and aromatic plants. Mint belongs to the genus Mentha L.
(Lamiaceae), a plant native of mild climatic areas but showing a cosmopolitan character
(Towill, 2002).
Cryopreservation offers long-term storage capability, maximal stability of phenotypic and
genotypic characteristics of stored germplasm, minimal storage space and maintenance
requirements, and has been considered to be an ideal means for long-term conservation of
genetic resources of vegetatively propagated plants (Engelmann, 1997, 2000; Shibli, 2000). It
is usually performed in liquid nitrogen (LN) at -196ºC, and it is the only method currently
available ensuring the safe, efficient, and cost-effective storage of germplasm of many plant
species. Cryopreservation results in arrested metabolic and biochemical processes, such as
cell division and growth (Towill, 1996; Engelmann, 2000). Other biophysical processes
leading to protein and nucleic acid degradation would also be halted. Thus, the plant material
can be stored without deterioration or modification for unlimited periods (Ashmore, 1997),
maintaining its genetic stability and regeneration potential (Rajasekaran, 1996; Matsumoto et
al., 1998; Towill, 2002).
To be successful, cryopreservation must avoid the formation of ice crystals inside cells
during immersion in LN. This is achieved by reducing cell water content prior to rapid
cooling. Therefore, treatments resulting in low cell water content without causing cell death
are key factors for successful cryopreservation (Matsumoto et al., 1998). Several pre-
57 57
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
treatments, including cold acclimation, exposure to ABA, immersion in concentrated sugar
solutions, and extensive dehydration, have been reported to enhance the survival after
cryopreservation in plant cells and tissues (Shibli et al., 1999; Tahtamouni & Shibli, 1999;
Shibli, 2000). The result is an increase of the cytoplasmic microviscosity, slowing the water
reorganization process required for ice nucleation. Viscosity, which is also increasing steeply
as the system temperature decreases, can reach such high values that movement is virtually
stopped. In this situation, named glassy or vitreous state, most chemical reactions and
physical processes are considered to be detained, and ice formation is deemed as impossible
(Sakai & Engelmann, 2007).
Cryopreservation techniques include vitrification (Moukadiri et al., 1999), encapsulation-
vitrification (Hirai & Sakai, 1999), and encapsulation-dehydration (González-Arnao et al.,
1996, 1999; Sakai et al., 2000; Shibli et al., 1999: Shibli, 2000). Among these methods,
encapsulation-dehydration is widely used because it is applicable to many species (Shibli,
2000). Encapsulation-dehydration cryopreservation methods, used with a variety of plant
germplasm (Rao et al., 1998), are based on the successive osmotic and evaporative
dehydration of plant cells, previous to the LN cooling step (Swan et al., 1999). Dehydration
techniques allow more flexibility when handling large sample numbers because the
processing is less time-critical than the vitrification techniques sensu stricto (Sakai et al.,
2000). Encapsulation-dehydration also avoids the use of toxic cryoprotectants as compared to
other methods (Shibli et al., 2006).
DSC has been frequently employed to study ice freezing and vitrification (Rajasekaran,
1996; Ashmore, 1997). Calorimetric data include information on the amount of frozen and
unfrozen water, as well as a clear proof of vitrification and its corresponding glass transition
temperature, TG. Thermal analysis of samples during pre-treatment prior to cryopreservation
in LN has only been employed previously to examine the processes occurring in alginate-
encapsulated Ribes nigrum shoot tips (Benson et al., 1996). The larger volume of alginate
relative to the encapsulated sample, together with the physical changes and processes within
the bead itself during pre-treatment and cryopreservation, may exert a significant influence on
the success of the cryopreservation protocol, and hence on the survival of plant samples.
In spite of the recognized importance of performing both cooling and warming steps
extremely fast for success of the cryopreservation process (Wesley-Smith et al., 2001), there
have been no previous reports of the measurement of cooling and warming rates for alginate
58 58
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
encapsulated samples. The closer data available refer to other plant cryopreservation protocols
(Kaczmarczyk et al., 2011; see Chapter 3).
Low-temperature scanning electron microscopy (cryo-SEM) allows observation of
microstructural features with no alteration due to preparation procedures, such as fixation and
dyeing (Craig & Beaton, 1996). Intra- and extracellular ice can be observed, after fracture and
etching, and be differentiated from vitrified tissues.
The aim of this paper is to study the effects of different dehydration times of alginate beads
on mint shoot tip viability (survival and re-growth) after cryopreservation and on the water
content and vitrification events, the latter studied using thermal analysis techniques (DSC)
and cryo-SEM.
5.2. Materials and Methods
5.2.1. Plant material and shoot tips extraction
Plant material was prepared as described elsewhere (Teixeira et al., 2013). Shoot tips were
extracted from in vitro shoots of Mentha ×piperita (genotype �“MEN 186�” obtained from the
IPK Genebank, Gatersleben, Germany). In vitro plants were monthly subcultured on medium
MS (Murashige & Skoog, 1962) with 3% sucrose, and incubated at constant temperature
(25°C) with a photoperiod of 16 h, and an irradiance of 50 µlmol m-2 s-1 from fluorescent
tubes. One-node segments were obtained from these shoots, transferred to fresh medium and
incubated at alternating temperatures of 25°C (day) and -1°C (dark), always with 16 h photo-
and thermoperiod, 50 µlmol m-2 s-1irradiance, provided by fluorescent tubes. After 3-weeks of
culture under these conditions, shoot tips (1-2 mm) were excised from axillary buds.
5.2.2. Shoot tip cryopreservation procedure
Ten excised shoot tips were precultured overnight with 2 mL of liquid MS medium
containing 0.3M sucrose at 25ºC, over filter paper (Figure 5.1). Thereafter, the explants were
placed in 3% (w/v) low viscosity alginic acid (Sigma, USA) in liquid MS without calcium, at
pH 5.7. Beads were formed by suspending shoot tips in alginate and dripping them, with the
help of a Pasteur pipette, into a calcium chloride solution (MS medium with 100 mM CaCl2
59 59
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
and 0.4 M sucrose). Beads were allowed to polymerize for 30 min. Encapsulated shoot tips
were cultured in liquid MS medium with 0.75 M sucrose on a rotary shaker (130 rpm)
overnight. Beads were blotted dry on sterile filter paper, transferred to an open glass Petri dish
and dehydrated for 1, 2, 3, 4, 5 and 6 h in a laminar-flow hood. Dehydrated beads (30 per
treatment) were placed in 1.0 mL cryovial (10 beads per cryovial) and plunged into NL
(Figure 1). The cryovials were rewarmed in water bath at 45ºC for 1 min and then at 25ºC for
one more minute (Uchendu & Reed, 2008). Subsequently, beads were placed on the re-growth
medium (MS, with 3% sucrose and 0.5 mg/L BAP), incubated in the dark for 24 h and
thereafter under 16 h photoperiod, at 25ºC. For control samples (-LN), all steps were carried
out except immersion in LN and rewarming.
Survival and re-growth were calculated as percentages over the total number of beads used,
4 weeks after culture. Survival was defined including all forms of visible viability (evidence
of green structures or callus). Re-growth was defined as the formation of small plantlets.
Three replicates (of 10 beads each) were used per treatment. Data were subjected to analysis
of variance and Duncan�’s Multiple Range Test (alpha = 0.05) was used for comparison of
means.
Figure 5.1. Scheme of the steps of the encapsulation-dehydration protocol employed, see text for more details on media and solutions used.
5.2.3. Water content determination
Four sets of three specimens were used for determining the dry mater content (dm) of
either shoot tips (control and after 0.3 M sucrose incubation step) or beads after the different
drying times. It resulted from weighing after oven-drying (~85°C, 72 h). The water content
(Wc) was calculated as the difference between the total mass and dm. It was expressed as
relative to the fresh weight mass (Wc(s)) or the dry mass (Wc(dm)).
60 60
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
5.2.4. Differential scanning calorimetry
DSC experiments were performed with a Mettler-Toledo DSC 30 instrument (Mettler-
Toledo, Griefsen, Suiza). Cooling was performed using the calorimeter control (10°C min-1),
or for higher rates, by quenching the samples in LN (see below). The response of beads after
each step of the cryopreservation protocol was evaluated; three beads were included in a DSC
pan, which was sealed and weighed. Samples were submitted to a cooling scan from room
temperature to -150ºC and a short 5 min equilibration at this temperature, followed by a
warming scan (at 10ºC min-1), back to room temperature. Calorimetric data were collected
from two replicates per treatment. For study of the quenching method, beads, sealed into a
DSC pan, were quickly plunged into LN. Pans were subsequently cold-loaded into a pre-
chilled DSC (-150ºC) for analysis. Later, pans were punctured and dried in an oven at 85ºC,
for 72 hours. Thermograms were analysed using the standard procedures provided in the
Mettler-Toledo STARe software. Ice thawing thermal events (more precise than freezing
events), were used for determination of Tf. The routine produced the temperature
corresponding to the event onset, Tf(onset) (corresponding to the equilibrium freezing
temperature) and the peak temperature, Tf(peak).
The melting enthalpy Hf, proportional to the event area, was also obtained. It was
expressed as relative to either the total sample mass ( Hf(s)), its dry mass ( Hf(dm)) or its water
content Hf(w), for allowing easier data comparison. Water fractions: frozen water (Wf) and
unfrozen water (Wu), were calculated by comparison of Hf and the pure water freezing
enthalpy ( Hf(w)* = 333.4 J/g at 0ºC), together with the total water contents previously
determined, after eq. 1:
Eq. 1 Wf= ( Hf/ Hf(w)*); Wu=Wc-Wf
Specific enthalpy values (per sample gram) were always used, after the corresponding
oven dry pan weighs. Enthalpy values measured at temperatures different from the
corresponding phase change equilibrium temperature, 0ºC, are underevaluated, as a result of
the difference between water and ice heat capacities (Cpw and Cpi, respectively). So, the
value of Hf(w)* was corrected after eq. 2, before applying eq. 1. Single freezing temperatures,
Tf(peak), were used, and Cpw and Cpi data were obtained from a calculation routine produced
61 61
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
in this laboratory (Otero et al., 2002), after interpolation when required, and other sources
(Angell et al., 1973, 1982).
Eq. 2 - (
Frozen (Wf) and unfrozen (Wu) water contents were expressed as relative to the total
sample mass (Wf(s),Wu(s)), its dry mass (Wf(dm), Wu(dm)) or its total water content (Wf(w),
Wu(w)).
5.2.5. Temperature measurement and cooling and warming rates
Thin T-type thermocouples (copper-constantan) (TC Medida y Control, S.A., Madrid,
Spain), with good performance at LN temperatures, were employed to measure cooling and
warming rates, using a fast data acquisition equipment MW100-UNV-H04 (Yokogawa,
Tokio, Japan), with a frequency of 20 data per second, basically as described previously (see
Chapter 3). Liquid nitrogen and ice were used as controls (-195.8 and 0.2ºC, respectively),
calibration being performed frequently between measurements. Cooling and warming rates
were defined as the ratio of a predetermined temperature interval T1-T2, over the time elapsed
between the reported measurement of T1 and T2. The determination of cooling and warming
rates was carried out, at least in triplicate, during the cooling and warming phases of the
described protocols. Thermocouples were calibrated as a part of the measurement routine.
A cartoon of the experimental set employed for cooling and warming rate determination is
shown in Figure 5.2. Cryovials, containing 10 alginate beads, were handled by means of the
attached thermocouple wire, with isolated tongs, to reduce heat losses. For cooling rate
determination cryovials were tempered at 1-4ºC before immersion in LN, and data collection
took place from 0 ºC till near -196ºC. For warming, cryovials were immersed and stirred in a
40ºC water bath (for 1 min) followed by immersion and stirring in another bath at 25ºC (for 1
min). Measuring took place from the exit from LN till room temperature equilibrium. Rates
were calculated after adopting a suitable ice risk temperature window.
= * )
62 62
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
Figure 5.2. Experimental set up for thermal change rate measurement for encapsulation-dehydration.
5.2.6. Low temperature scanning electron microscopy
Low temperature scanning electron microscopy (cryo-SEM) observations were performed,
basically, as previously described (Teixeira et al., 2013), with a Zeiss DSN 960 scanning
microscope equipped with a Cryotrans CT-1500 cold plate (Oxford, UK). Cryo-SEM allows
sample observations without the need of prior chemical fixing or drying processes.
Three shoot tips or beads containing tips in the same stage of the cryopreservation protocol
were fitted on a special bracket with their axes vertically aligned (a cartoon of the tip-
containing beads, before placing inside the microscope and after fracture can be seen in
Figure 5.3), and this piece was immersed into liquid nitrogen (LN) under low pressure,
physically fixing tissues for microscopic observation. This cooling step was considered
equivalent to the cooling process taking place in the actual cryopreservation process, by LN
immersion. The holder was, then, placed in the pre-chamber of the Cryotrans cold plate, at
-180ºC, and specimens were fractured perpendicularly to their axes, to obtain a suitable
observation surface. Later, samples were inserted in the microscope and etching (partial ice
sublimation, induced to provide contrast) was performed for three min at -90ºC. Once etched
and re-cooled, the samples were retrieved into the cryo-preparation chamber and coated with
high purity Au, which acts as a conductive contact for electrical charge. Finally, the etched
and coated samples were reinstated into the cooled SEM stage and observed under secondary
electron mode, at -150/-160ºC, using an accelerating voltage of 15 kV.
63 63
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
Figure 5.3. Cartoon showing the three mint tips-containing beads on the microscopy holder before insertion in the microscope (a) and after equatorial freeze fracture (b).
The initial water content of beads was lower than that of precultured tips, as a result of the
culture of beads in 0.75 M sucrose. For increasing drying times water content decreased
asymptotically, with the highest water reduction taking place between 1 and 3 hours of drying
time. Longer drying periods were associated to slight or non-significant water content
variations, when compared with the experimental error. An associated bead size decrease and
hardening could also be observed.
The dry mass (dm) and water content of shoot tips (control and 0.3 M sucrose precultured
tips) and beads containing shoot tips (after different drying times: 0 to 6 hours), derived from
weight-difference after extensive drying, are shown in Table 5.1. Water content was
expressed in relation to the total sample mass (Wc(s)) and the dry mass (Wc(dm)). The water
content of tips underwent a small decrease with the preculture in medium with 0.3 M sucrose.
5.3.1. Water content reduction during the drying period
5.3. Results and Discussion
a
b
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5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
65 65
Table 5.1. Compositional and thermal parameters (mean standard deviation) of the thawing events obtained from gravimetry and DSC thermograms for mint shoot tips specimens at different stages of the encapsulation-dehydration cryopreservation protocol. Preculture Dehydration (h) Control 0.3M suc. T0 T1 T2 T3 T4 T5 T6 dm (gdm gsample
-1) 0.12 ± 0.01 0.18 ± 0.10 0.25 ± 0.01 0.35 ± 0.02 0.57 ± 0.02 0.77 ± 0.12 0.77 ± 0.01 0.78 ± 0.01 0.79 ± 0.01 Wc(s) (gwater gsample
-1) 0.88 ± 0.01 0.82 ± 0.01 0.75 ± 0.08 0.66 ± 0.20 0.43 ± 0.02 0.23 ± 0.12 0.27 ± 0.13 0.22 ± 0.01 0.21 ± 0.01 Wc(dm) (gwater gdm
-1) 7.30 ± 0.60 4.70 ± 0.40 3.00 ± 0.10 1.90 ± 0.10 0.74 ± 0.06 0.30 ± 0.20 0.30 ± 0.01 0.28 ± 0.02 0.27 ± 0.02
Tf (onset) (ºC) -3.10 ± 0.30 -3.80 ± 0.06 -8.90 ± 0.30 -12.50 ± 1.90 -28.20 ± 0.40 --- --- --- --- Tf (peak) (ºC) 4.10 ± 0.60 -0.17 ± 0.06 2.40 ± 0.80 -3.00 ± 4.00 -12.60 ± 3.10 --- --- --- ---
Hf(s) (J g sample
-1) 237 ± 8.00 210 ± 11.00 180 ± 3.00 148 ± 10.00 64 ± 3.00 --- --- --- --- Hf(dm) (J gdm
-1) 1940 ± 160.00 1200 ± 150.00 714 ± 33.00 420 ± 50.00 112 ± 9.00 --- --- --- --- Hf(w) (J gw
-1) 264 ± 1.00 255 ± 9.00 242 ± 3.00 228 ± 10.00 151 ± 1.00 --- --- --- --- Wf(s) (gwater gsample
-1) 0.69 ± 0.01 0.63 ± 0.03 0.540 ± 0.01 0.46 ± 0.04 0.21 ± 0.01 --- --- --- --- Wf(dm) (gwater gdm
-1) 5.80 ± 0.50 3.50 ± 0.20 2.14 ± 0.09 1.30 ± 0.20 0.37 ± 0.02 --- --- --- --- Wf(w) (gwater gwater
-1) 0.79 ± 0.01 0.77 ± 0.02 0.73 ± 0.02 0.70 ± 0.05 0.49 ± 0.01 --- --- --- ---
Wu(s) (gwater gsample-1) 0.19 ± 0.01 0.19 ± 0.02 0.21 ± 0.01 0.20 ± 0.02 0.22 ± 0.01 0.30 ± 0.20 0.30 ± 0.01 0.28 ± 0.02 0.27 ± 0.02
Wu(dm) (gwater gdm-1) 1.50 ± 0.20 1.20 ± 0.02 0.86 ± 0.05 0.60 ± 0.05 0.37 ± 0.03 0.30 ± 0.20 0.30 ± 0.01 0.28 ± 0.02 0.27 ± 0.02
Wu(w) (gwater gwater-1) 0.21 ± 0.05 0.23 ± 0.02 0.27 ± 0.01 0.30 ± 0.06 0.51 ± 0.01 1.00 1.00 1.00 1.00
TG (ºC) --- --- --- --- --- -68.70 ± 0.30 -51.00 ± 2.00 -43.00 ± 2.00 -44.00 ± 2.00
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
5.3.2. Calorimetric studies on water status
Calorimetric measurements were carried out for shoot tips (control and precultured) and
tip-containing beads for 0 to 6 hours drying times. Figure 5.4 shows typical scans obtained
for beads at the different drying times. Inserts help visualizing the small glass transitions,
when present. The parameters derived from DSC data analysis (melting exotherm
temperatures and enthalpies and the derived frozen water contents) appear in Table 5.1.
Clearly, samples could be divided in two sets, corresponding to higher and lower water
contents. Experiments with naked shoot tips and for beads desiccated for 0, 1 or 2 hours
presented a melting exotherm, implying that part of the water in these systems became frozen
upon cooling. Meanwhile, samples at drying times 3 to 6 hours showed no trace of melting
events, meaning no water was frozen. Instead, these samples exhibited glass transitions,
proving these beads became vitrified upon cooling.
Figure 5.4. Typical DSC thermograms corresponding to re-warming processes of shoot tips with beads of the encapsulation-dehydration protocol. Beads were desiccated under the air flow of a laminar-flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h and T6 = 6 h. Data are the mean of at least three repeats. Each experiment was performed with three beads. The inserts show an expanded thermogram section which allows seeing the glass transition. Scanning rate was 10ºC m-1. The ordinates scale of the thermograms baseline is arbitrary.
66
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
Data analysis (Table 5.1) allowed drawing a perfect parallelism between water content and
the calorimetric behaviour. The temperatures of the melting process (when taking place) were
decreasing as water content did, showing that the solute concentration was increasing. The
area of the curves, i.e., the melting enthalpy, was also decreasing with water content, as well
as the derived frozen water content. While Wf(w) for control tips was approx. 80 %, this
amount decreased to nearly 50% in beads, after 2 h desiccation, which was the longer drying
time showing freezing processes. This, added to the approx. 40% reduction in total water
content at T2, implies that the mass of frozen water was only 20% of the sample total mass.
The ratio of frozen to unfrozen water decreased from control and precultured tips and with
increasing drying times, to reach a value of 1 at the highest drying time showing a freezing
event, i.e. 2 h desiccation (Figure 5.5).
Glass transition temperatures (Table 5.1) showed an increase with the progress of the
drying process. It increased from 3 h to 5 h desiccation, while there was no difference from 5
to 6 h, which corresponded to the very small or constant water content reduction at these
times.
Figure 5.5. Ratio Wf(dm) Wu(dm)-1 for mint shoot tips (control and precultured on 0.3 M
sucrose) and shoot tip-containing beads (for drying times 0 to 6 hours). Bars: standard error.
Beads and tips studied by DSC in a similar way but after LN quench cooling presented a
very similar behaviour (data not shown), but their freezing events displaced to lower
67
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
temperatures. Three to 6 h dried beads showed no freezing processes and the glass transitions
observed in these cases were unchanged, within experimental error.
5.3.3. Cooling and warming rate determination
An ice-risk formation window of 0ºC to -150ºC was considered in this work, for cooling
and warming rate calculation. This window comprises the regions were ice formation can take
place, though with a very low probability or in a very long time (i.e., in very slow cooling or
warming processes). The selected window is widely over-dimensioned compared to the actual
ice formation risk for cryopreserved systems, as its equilibrium freezing temperature is always
well under 0ºC, due to its high solute and low water contents. Per contra, the glass transition
temperature, under which the possible ice formation area ends, although highly variable, tends
to be located well over the -150ºC limit. Other narrower windows would yield higher rates
(see Chapter 3), while considering the duration of the process all the way down to LN
temperature (-196ºC) would yield meaningless and exceedingly long times.
Cooling (Figure 5.6) and warming (Figure 5.7) processes followed an approximately
symmetrical behaviour with not too strong differences among samples. It must be noted that
natural differences in samples and manipulation variations during normal cryopreservation
procedures were responsible for a higher dispersion in data (see Chapter 3).
The cooling rates obtained with beads dried for different times (Table 5.2) showed an
increase easy to be related to the water content decrease (Table 5.1). Rates could be related to
the samples physical characteristics: mass, heat capacity and thermal conductivity. Besides
the dependence with mass, larger water contents imply larger global heat capacities. In this
case, both sample mass and its heat capacity decreased with drying time. Heat conductivity
also plays a role in determining cooling and warming rates. Factors as the position of beads in
the cryovials and the contact between the wall and beads are important. These thermal
contacts are favoured by the higher water content on beads.
The larger variations in rates were found between 2 and 3 hours drying time, and at longer
periods the variation was quite small, also correlating with the asymptotic water content
reduction. Warming rates behaved in a similar way, although the observed warming rates
were smaller than the cooling ones.
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5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
Table 5.2. Cooling and warming rate measurements obtained following the encapsulation-dehydration protocol.
Cooling Warming
Sample Rate
(ºC min-1)
Time
(s)
Rate
(ºC min-1)
Time
(s)
Sample temperature at
the end of protocol3
(ºC)
Control 3.83 ± 0.60 39.90 ± 6.65 3.41 ± 0.30 44.28 ± 3.80 9.93 ± 1.20
T0 1.64 ± 0.10 91.83 ± 5.60 1.28 ± 0.01 111.87 ± 0.08 -7.30 ± 0.70
T1 1.60 ± 0.04 94.02 ± 2.10 1.27 ± 0.01 110.87 ±1.12 -9.80 ± 1.35
T2 1.83 ± 0.08 82.10 ± 3.55 1.29 ± 0.01 112.93 ±0.14 -3.93 ± 0.85
T3 2.78 ± 0.11 53.93 ± 2.16 1.97 ± 0.02 76.08 ± 0.81 8.50 ± 0.28
T4 2.47 ± 0.13 60.93 ± 3.23 1.95 ± 0.08 77.25 ± 3.20 8.13 ± 0.57
T5 2.76 ± 0.04 54.40 ± 0.87 2.06 ± 0.06 73.07 ± 2.08 8.80 ± 0.72
T6 2.73 ± 0.05 54.95 ± 0.92 2.49 ± 0.10 60.42 ± 2.47 11.70 ± 0.60
Average values and standard deviation for at least three repeats. Experiments performed with shoot tips included in beads, inside a cryovial. Rates and permanence times calculated for the ice formation risk window 0ºC to -150ºC. Beads were desiccated under the air flow of a laminar flow bench T0 = 0 hours (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = cryovial without beads. 3Temperature of beads after warming for 1 min at 45ºC and 1 min at 25ºC.
Warming process was performed after the encapsulation-dehydration protocol, which
included a 1 min warming in a 45ºC water bath and another minute in a 25ºC bath. This time
was, for beads with the highest water contents, insufficient for them to reach temperatures
over 0ºC (Table 5.2). To complete the warming process at room temperature would lengthen
the time under 0ºC and could increase the risk of ice formation.
69
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
Figure 5.6. Temperature evolution, during cooling by immersion in LN, of cryovials with 10 alginate beads containing mint shoot tip and treated after the encapsulation-dehydration protocol. Beads were desiccated under the air flow of a laminar flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = cryovial without beads. Data are the mean of at least three repeats.
Figure 5.7. Temperature evolution, during warming in a 45ºC water bath for 1 min and then in 25ºC for 1 min of cryovials with 10 alginate beads containing mint shoot tip and treated after the encapsulation-dehydration protocol. Beads were desiccated under the air flow of a laminar flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = cryovial without beads. Data are the mean of at least three repeats.
70
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
5.3.4. Low temperature scanning electron microscopy
The microstructure of alginate gel beads submitted to the extreme conditions of the drying
process (6 h) was observed by cryo-SEM, and compared to that of non-desiccated ones.
Observation with secondary electrons and backscattered electrons was compared, and
although the same basic information was obtained with both, cellular structures appear clearer
with backscattered electrons, which were used in the micrographs presented.
Figure 5.8. Cryo-SEM micrographs of alginate beads. View of beads external surface at drying time 0 (A) and 6 hours (B), and internal cryofracture surface, at drying time 0 (C) and 6 hours (D). The bars correspond to 10 µm.
Figure 5.8 shows pictures corresponding to these conditions (sections not including shoot
tips). The water content reduction from A-C to B-D beads implied a significant increase of
solute concentration in the remaining water. Although not directly measured, it could be
roughly estimated, considering an initial 0.75 M sucrose concentration in the T0 beads, which,
for an approximate 10 fold decrease in Wc(dm) between T0 and T6, would give a final 7.5M
sucrose. This would give rise to a strong reduction in molecular mobility for high drying
71
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
times, which caused the system to vitrify when quickly cooled at LN temperatures (in the
microscope stage).
The etching procedure employed for creating contrast in cryo-SEM removes part of the ice
formed in the cooling of the sample in the microscope (always small crystals, due to the high
cooling rate and the small size of the sample) by sublimation, leaving darker �“void�” regions
separated by cleared �“ridges�” of freeze-concentrated solution. Glassy state has a near null
sublimation rate, so vitrified solutions do not present enhanced contrast after the etching
process (Angell et al., 1982; Umrath, 1983). This was considered a suitable method for
studying vitrified tissues and their microstructure and was employed in previous works of this
group (Teixeira et al., 2012, 2013).
Figure 5.9 shows cryo-SEM micrographs corresponding to mint shoot tips at different
stages of the encapsulation-dehydration protocol. A to G correspond to the progressive drying
times. It can be seen that A to D show microstructural intracellular details resulting from the
etching ice sublimation procedure, and showing that in the conditions of microscopic
observation, assumed to be equivalent to those of the rapid cooling in LN used in normal
cryopreservation protocols, there would be intracellular ice and hence, damage to cell
function. On the other hand, E to G, corresponding to the longer drying times, show no
structural details, implying that water could not sublimate during etching and so that these
tissues were in glassy state.
The four first pictures (A to D) show intracellular ice crystals of decreasing size as the
cytoplasm concentration increased with water reduction. Apices after 1 and 2 h desiccation
(T1 and T2; B and C) exhibited extracellular solutions partially vitrified that could be also
associated to the higher sucrose concentration.
Meanwhile, cells at T3 (Figure 5.9 D) show a mix of intracellular ice with vitrified
regions. The ice crystals size reduction is a direct effect of the increase in viscosity, allowing
more ice nuclei to grow instead of favouring the growth of only a few crystals.
The cells shown in Figure 5.9. H correspond to a recovered specimen, after
cryopreservation, warming and one week of successful recovery. Its water and solute contents
would be similar to that of material in the starting point of the process (as those found in
studies conducted by Teixeira et al., 2012), and ice crystals, larger than in picture A, can be
observed, as corresponds to a higher water content.
72
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
Figure 5.9. Cryo-SEM micrographs of mint shoot tips in different stages of the encapsulation-dehydration protocol. Drying times (hours): A (0), B (1), C (2), D (3), E (4), F (5), G (6). H, tip growing for one week, after warming. The bars correspond to 10 m.
D
A B
C
E F
G H
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5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
5.3.5. Cryopreserved shoot tips survival and re-growth
Survival and re-growth after 4 weeks culture on recovery medium were studied for shoot
tips cryopreserved following the encapsulation�–dehydration protocol with different air-
dehydration times and subsequent cooling in liquid nitrogen (Figure 5.10). In most cases
viability and re-growth rates were fairly similar within treatment. Control specimens, not
immersed in liquid nitrogen (LN), present nearly a 100% survival, proving the good initial
performance of shoot tips.
The T0 and T1 high water content samples showed complete lack of survival or re-growth.
Intermediate water contents (T2 and T3) had rates of less than 50% for both these parameters,
while the dryer samples (T4, T5 and T6) showed high and similar survival and re-growth
figures. Re-growth percentages for beads exposed for six hours to dehydration were as high as
93.3%.
5.4. General discussion
The general behaviour of mint shoot tips following the encapsulation-dehydration
cryopreservation protocol at different air-drying times is clear and consistent, in spite of the
inherent variability of plant samples and of the whole process. Water content in beads (and
hence in the encapsulated tips) decreased with drying time, reaching soon (T3) to
approximately stable values (Wc(s) ~ 0.25 gwater gsample-1, Wc(dm) ~ 0.30 gwater gdm
-1). These water
contents are in the range of critical values reported for encapsulated shoot tips (Dereuddre et
al., 1990; Uragami et al., 1990). Damages associated to water contents of these orders have
been reported in different plant germplasm systems (González-Benito et al., 1998; Sun, 1999;
González-Benito et al., 2004).
74
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
a a a a a a a a
b b b c
c d c d
Figure 5.10. Mint shoot tip survival and re-growth percentages after dehydration for different times following the encapsulation-dehydration protocol. Observations were made after a 4-week recovery period. Beads were desiccated under the air flow of a laminar flow bench for different times: T0 = 0 h (no desiccation); T1 = 1 h; T2 = 2 h; T3 = 3 h; T4 = 4 h; T5 = 5 h; T6 = 6 h; and Control = before cooling in LN. Data are the mean of at least three repeats. Means of survival or re-growth with the same letter are not significantly different according to the Duncan�’s Multiple Range Test at alpha = 0.05. Bars: standard error.
Most of the other parameters and observations behaved in parallel to water content. Both
cooling and warming rates increased as the drying time did (Figure 5.6 and 5.7). Fast cooling
and warming processes reduce the probability of ice formation and increase that of
vitrification. Nevertheless, the increase in these rates for the different drying times is
relatively small and quite likely not the main source of the different success of the
cryopreservation process.
A water content reduction would mean an increased solute concentration in the remaining
water. This results, for both bead and shoot tip, in higher viscosity (reduced molecular
mobility) and hence, an increased probability of vitrification as they get quickly cooled down.
Low temperature scanning electron microscopy showed (Figure 5.8 and 5.9) how samples at
higher water content suffered ice crystallization but after 4 hours drying vitrification took
place, avoiding any ice trace (with the T3 samples in a intermediate situation).
The water freezing parameters obtained from DSC (Table 5.1) agreed with the previous
observations by showing melting processes fully compatible with increasing solute
concentrations (decrease of freezing temperature and reduction of frozen water content) up to
3 h desiccation when no ice formation could be appreciated. The calorimetric glass transitions
75
5. ENCAPSULATION-DEHYDRATION: DETERMINATION OF THE GLASSY STATE BY CRYO-SEM AND DSC
that can be visualized after this drying time confirm the cryo-SEM observations and, as their
temperature increased up to 5 h desiccation, indicate the progress of the dehydration process.
Viability of cryopreserved specimens (survival and recovery, as a global physiological
evaluation of the success of the procedure), is in good agreement with the results reported for
this protocol (Uchendu & Reed, 2008) and confirmed the thermodynamic results and the
images observed.
The encapsulation-dehydration procedure submits specimens to prolonged dehydration and
high sucrose concentration stresses. This may be reflected in damages to recovered plants, not
evaluated in the viability analysis, such as genetic alterations (Martín et al., 2011). A
conclusion of this study is that the asymptotic reduction of water content and the associated
variation of most parameters makes unnecessary to employ too prolonged times to protect
samples from ice formation. DSC observations would imply that 3 hours drying could be
enough to avoid the formation of ice (even operating at a slow cooling rate: 10ºC min-1).
Nevertheless, cryo-SEM showed how the shoot tips cytoplasm at 3 hours drying time might
be not completely ice-free, as some crystal evidence is observed. This is confirmed by the
viability data, in which samples desiccated for 3 h showed an intermediate response, implying
that for some of the specimens, lethal ice formation took place.
A more complex picture is completed by the glass transition temperature evolution in the
lower water content samples. Although water content data indicated no further changes from
3 h desiccation onwards, there was a distinct increase of TG from 3 h to 5 h desiccation, where
a stable situation was reached.
76
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF
PLANT VITRIFICATION SOLUTIONS
6.1. Introduction
Glass, the state of matter where molecular mobility is so reduced that most
physicochemical processes (including ice formation) are actually detained, is a basic state of
matter for cryopreservation. The temperature at which glass initially forms from supercooled
liquid is known as the glass transition temperature (TG). It is rather a range of temperatures
than a single defined one. The glass transition is also characterized by a change in heat
capacity ( Cp), which is measured as the baseline difference at the transition inflection point.
At temperatures above TG, there is an increase in molecular mobility and a decrease in
viscosity which enables the crystallization of water, if sufficient time to carry out the process
is granted (Roos & Karel, 1991). Per contra, when temperature is below the glass transition,
the system can be considered stable. Most physical and chemical processes (especially those
that are diffusion driven) are impeded, in a physically accessible time frame. Actually, there is
movement in the glassy state: many reports describe the existence of a significant degree of
short range mobility, associated to solvent molecules (water, in this case) (for example Ablett
et al., 1993; Mestre et al., 2002). However, the relatively large-scale molecular
reorganizations required for ice crystal nucleation are certainly not possible in these
conditions of greatly reduced molecular mobility (Angell et al., 1978).
Consequently, glassy state allows living tissues preservation with no or very limited
physicochemical changes and completely free of ice formation, which is associated with lethal
freeze injury (Muthusamy et al., 2005; Nadarajan et al., 2008). Since the knowledge that cells
treated with specific cryoprotecting solutions survive exposure to cryogenic temperatures was
achieved, numerous variations on solution composition were developed for plant cells (Volk
& Walters, 2006). Different vitrification solutions were developed by various research teams
worldwide (Sakai et al., 1990; Steponkus et al., 1992). Sakai�’s group developed the PVS
(Plant Vitrification Solution) series. PVS1 has been employed for work with asparagus cell
suspensions (Uragami et al., 1989, 1990). PVS2, originally developed for treating citrus cell
suspensions (Sakai et al., 1990), has been successfully used for different explants of around
200 species (Sakai, 2003), including garlic (Niwata, 1995). PVS3 has been notably employed
77
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
with wasabi (Matsumoto et al., 1995), asparagus (Nishizawa et al., 1993) and garlic
(Makowska et al., 1999) shoot tips. The cryopreservation of garlic germplasm, an
economically important crop, has been profusely studied by several authors using different
approaches (Niwata, 1995; Makowska et al., 1999; Keller 2002; Volk & Walters 2006, 2007).
The glass transition temperature of aqueous systems is very sensitive to the proportion of
water and other small molecules, and small changes in compositions can give rise to large
variations in TG. When taking advantage of the glassy state for preservation purposes, often
the difficulty lies in the prediction and control of this temperature. Another problem is how to
reach temperatures below TG, from room temperature, without ice being formed during the
cooling process (the same problem arises when warming specimens to room temperature)
(Roos & Karel, 1991; Zamecnik et al., 2007). The probability of ice formation increases with
the time that the system is at a temperature comprised between Tf (the equilibrium freezing
temperature) and TG. Actually, the ice nucleation temperature should be considered, instead
of Tf. This nucleation temperature has not a fixed value, being dependent on Brownian
movement, but is often placed well below Tf. On the other hand, the region �“close�” above TG
is considered too viscous for anything like ice nucleation to happen, in a short time frame. A
zone of 20ºC above TG is often accepted as having a very low probability of ice formation
(Mishima & Stanley, 1998).
Glass is known to undergo relaxation when stored at temperatures below TG and this
process might affect the preservation quality of biological materials embedded in the glassy
matrix (Gao et al., 2005). Most available literature reports are concerned with the glass
forming or nucleation properties of polyalcohols/water mixtures (Alberola et al., 1983; Shaw
et al., 1997; Murthy, 1997), and little attention was given to their relaxation behavior. Some
authors reported the glass transition and (or) enthalpy relaxation of pure polyalcohol
compounds, such as glycerol (Chang & Baust, 1991; Takeda et al., 1998), propylene glycol
(Mehl, 1996; Takeda et al., 1998), ethylene glycol (Angell & Smith, 1982; Takeda et al.,
1998) and their binary mixture systems (Takeda et al., 1998), but there is a lack of
information about relaxation of plant vitrification solutions below TG.
The glass transition is not a phase transformation in the full thermodynamic sense. Its
occurrence is determined by the history of the material, and is dependent on the exact
conditions of the experiment (Yavin & Arav, 2007). The change between liquid and vitreous
state has also been described as a second order phase change, not characterized by an enthalpy
78
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
increment as first class changes are, but by a step in some of the physical properties, such as
the heat capacity, specific volume and apparent viscosity (Walstra, 2002). Differential
scanning calorimetry (DSC) is a powerful tool to investigate glass transition, heat capacity
and annealing behaviors of plant vitrification solutions (Leprince & Walters-Vertucci, 1995).
The present study aimed the characterization of the calorimetric properties of the most
common plant vitrification solutions, and of garlic shoot tips treated with one of such
solutions, under a wide range of cooling and warming rates and the investigation of the
stability of their glasses under conditions relevant to their use as cryopreservation agents.
6.2. Materials and methods
6.2.1. Standard compounds
Dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) of
chromatographic grade were purchased from Sigma Aldrich (Heidelberg, Germany); glycerol,
sorbitol and sucrose used were manufactured by Penta (Chrudim, Czech Republic) and
isothiazolinone by Schulke & Mayr (Norderstedt, Germany). All other chemicals used were
of the highest commercially available purity. See Annex I for the molecular formulas of the
compounds.
6.2.2. Solutions
The vitrification solutions were (Table 6.1): (1) PVS1 (Plant Vitrification Solution 1: 19%
w/v glycerol, 13% w/v EG, 13% w/v PG, 6% w/v DMSO in half-strength MS liquid medium
+ 0.5 M sorbitol; Uragami et al., 1989), (2) PVS2 (30% w/v glycerol, 15% w/v EG, 15% w/v
DMSO, and 0.4 M sucrose in half-strength MS liquid medium; Sakai et al., 1990), (3)
modified PVS2 (PVS2 mod: 37.8% w/v glycerol, 16.7% w/v EG, 16.5% w/v DMSO and 0.4
M sucrose in half-strength MS liquid), (4) PVS3 (50% w/v glycerol, 50% w/v sucrose in
water; Nishizawa et al., 1993), (5) modified PVS3 (PVS3 mod: 50% w/v glycerol, 50% w/v
sucrose, 5% DMSO in water; Nishizawa et al., 1993).
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6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
Table 6.1. Composition of vitrification solutions. Component (% w v-1) Solution Sorbitol EG DMSO PG Glycerol Sucrose Water
PVS1 9.1 13 6 13 - - 64.1 PVS2 - 15 15 - 30 13.7 40.3
PVS2 mod - 16.5 16.5 - 37.8 13.7 31.3 PVS3 - - - - 50 50 28.6
PVS3 mod - - 5 - 50 50 24.1
6.2.3. Plant material
Experiments were carried out with Alium sativum L. sativum cv. ´Djambul 2´. Microbulbils
were rinsed with 1% isothiazolinone solution for two hours. Subsequently, the shoot apex (1
mm) in each microbulbil was excised with a scalpel blade and forceps. Shoot apices were
placed in 75% isothiazolinone solution for 2 s and then transferred to 1% isothiazolinone
solution for 10 min. Finally, batches of 20 apices were directly placed onto MS medium
(Murashige & Skoog, 1962) with 10% sucrose overnight.
Excised shoot tips were immersed into the loading solution (13.7% w/v sucrose + 18.4%
w/v glycerol; Sakai et al., 1990) for 20 min at 25ºC. Shoot tips were immersed in PVS3 at
25ºC for 2 h (Sakar & Naik, 1998).
6.2.4. Differential scanning calorimetry conditions
Thermal processes in vitrification solutions were measured using a TA 2920 DSC (TA
Instruments, New Castle, DE, USA). Hermetic aluminum pans (TA) were used in all DSC
experiments and an empty pan was used as reference. The furnace block of DSC was flushed
with dry nitrogen gas to avoid condensation of moisture from the air. Helium gas (99.999%)
was used as sample purge at a rate of about 33 ml min-1. The temperature scale of the
instrument was calibrated with cyclohexane (-87°C), mercury (-38.87°C), water (0.01°C) and
indium (156.6°C) and with heat fusion of water (334 J g-1). All calibrations were performed
by using scanning rates of 5, 10 and 20°C min-1. Calorimetric data were collected from two
replicates per treatment.
Samples within pans were either approx. 10 mg of vitrification solutions or three garlic
shoot tips, immediately after treatment with the vitrification solution PVS3. In work with
shoot tips, pans were weighed before and after DSC; in the second case pans were punctured
80
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
and oven-dried (~85°C, 72 h), to obtain the water content. Calorimetric data were collected
from two replicates per treatment.
6.2.5. Glass transition characterization for PVS
The glass transition parameters (TG and Cp) were determined for the different
vitrification solutions considered, in both cooling and warming scans. Pans with samples were
cooled at standard rates (10ºC min-1) from 30 ºC to -145ºC. After 5 min equilibration at this
temperature, they were warmed back to 30ºC, also at 10ºC min-1. This cycle was repeated four
times. Two different samples were studied for each solution.
6.2.6. Cooling rate test
For this test, cooling was performed at different rates: either using the calorimeter control
(5, 10 and 20°C min-1), or, for higher rates, by quickly immersing the closed pan with the
sample in LN (either naked or previously included inside a cryovial). The pan was then
transferred to the calorimeter sample chamber, pre-cooled to -150°C, where a short
equilibration time was allowed. Glass transition temperature and the corresponding heat
capacity change were observed upon warming from -145°C to room temperature, at a
standard warming rate of 10°C min-1. The samples were either vitrification solutions or garlic
shoot tips, after treatment with the vitrification solution PVS3.
6.2.7. Warming rate test
For investigating the effect of the warming rate, cooling was performed at two fixed rates:
10°C min-1 (using the calorimeter control) or by quenching in LN (without cryovial).
Quenched pans were transferred to the calorimeter sample chamber, pre-cooled to -150°C,
where a short equilibration time was allowed. Glass transition temperature and the
corresponding heat capacity change were measured upon warming pans with samples from
-145°C to room temperature, at different warming rates: 5, 10 and 20°C min-1. The samples
were either vitrification solutions or garlic shoot tips, after treatment with the vitrification
solution PVS3.
81
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
Crystallinity, for garlic shoot tips was evaluated using TA proprietary software (TA
Instruments, undated), using a value for the specific fusion heat of water of 334 J g-1. The
routine basically evaluates the thermal event enthalpy and, by comparing with the water
specific fusion heat and considering the water content of the sample (obtained by differential
weighing, after drying in an oven after the DSC experiment), calculates the fraction of water
that had crystallized into ice.
6.2.8. Storage test
To test the effect of storage time, DSC pans with solution samples were plunged directly
into LN and kept immersed (at -196ºC) for five different time lapses (0, 1, 7, 30 and 60 days).
Pans in LN were, after the storage period, transferred to the calorimeter sample chamber, pre-
cooled to -150°C, where a short equilibration time was allowed. Glass transition temperature
and the corresponding heat capacity change were measured upon warming pans with samples
from -145°C to room temperature, at a standard warming rate of 10°C min-1.
6.2.9. Annealing test
The annealing thermal behavior of PVS1 and PVS3 was studied. These two solutions were
chosen as their vitrification parameters were distinct (see Results). The samples were (Figure
6.1.) first cooled at 10°C min-1 (standard cooling rate) from room temperature (A) to -145°C
(well below the vitreous transition of the solutions), and kept at this temperature for 5 min
(B). Then, the samples were heated to a temperature over its TG, (-90ºC for PVS1 and -70°C
for PVS3) at a rate of 20°C min-1 and kept for 5 min at these temperatures (C). Later, the
samples were cycle cooled again at 20°C min-1 to a target temperature decreasing one degree
per cycle (from -100 to -120ºC for PVS1 and from -77 to -100°C for PVS3), and kept at these
temperatures for 30 min (D). Finally, the samples were cooled again at 20°C min-1 to -145°C,
kept for 5 min (E) and warmed to 30°C, at 20°C min-1 (F). The DSC thermogram of the last
step was recorded and the annealing areas measured. In order to reduce experimental scatter
due to differences in heat transfer between the calorimeter and the sample, it was left
undisturbed inside the furnace for a set of experiments. All experiments were repeated twice.
82
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
Figure 6.1. Scheme of the steps of the annealing protocol used; see text for more details on solutions employed.
6.2.10. Statistical analysis
The results were analyzed by analysis of variance (ANOVA), and means were compared
by Duncan�’s multiple range test (P 0.05) using STATISTICA version 10, (StatSoft, Inc.,
2011).
6.3. Results and Discussion
6.3.1. Vitrification behavior of different vitrification solutions
Table 6.2 shows the results of determination of the glass transition parameters for the five
PVS employed, at standard rates (10ºC min-1). Glass transitions temperatures were fairly
reproducible and showed clear and consistent differences among vitrification solutions
(confirmed by the statistical analysis), which were related to different composition and water
contents (Table 6.1). TG values calculated for each solution were identified by the statistical
analysis as distinct. The value of TG generally decreased as the water content of the solutions
increased. This behavior was not followed for PVS1 and PVS2, as the latter solution TG was
the lowest, while PVS1 had the highest water content.
83
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
Table 6.2. Glass transition parameters of different pla-1
nt vitrification solutions (cooling and warming rates were both 10°C min ).
Type of PVS TG Cp
(°C) (J g °C-1)
PVS1 -112.15 ± 0.38b 1.22 ± 0.03a
PVS2 -114.31 ± 0.38a 1.12 ± 0.04a,b
PVS2 m c
b
od -109.42 ± 0.11 d
1.07 ± 0.04 b
b
PVS3 -89.85 ± 0.29 e
0.87 ± 0.01
PVS3 mod -86.84 ± 0.65 0.83 ± 0.01
Average values and standard deviation TG and Cp, in a column, with the sa er are tly ording to the Duncan�’s Multiple Range Tes 0.05
olecules available per each solute molecule was low
and even lower the num
t -89ºC (PVS3 and PVS3 mod). The heat
capacity changes measured were less distinct than the corresponding T values, but a
0) reported a TG value of -115ºC (equal for both cooling and
warm
for at least four repeats. Means of not significanme lett different acc
t at alpha = .
These vitrification solutions contained large amounts of solutes able to form hydrogen
onds with water. The number of water mb
ber of water molecules per potential hydrogen bond in solute
molecules. Nevertheless, in such concentrated and complex mixtures not all these potential
bonds could be expected to bind water, as many intersolute (and intramolecular) bonds would
arise (see Annex I for molecular formulas of the components of the cryopreservation
solutions and tables of their molar composition).
The different TG found could be divided into two groups: one centered around -112ºC
(PVS1, PVS2 and PVS2 mod) and the other a
G
decrease with the water content in solutions could be appreciated. However, PVS solutions
could be distributed according to Cp in the same groups. The prediction of glass transition
behavior for complex mixtures as those studied here, is, currently, not a solved problem (see,
for example, Angell et al., 2000). Nevertheless, water content seems to be the dominant factor
over the solute composition.
Very few data on glass transition parameters of PVS have been reported. The initial PVS2
publication (Sakai et al., 199
ing scans), within experimental error of our data. It must be noted that, in the same
publication, the existence (upon warming at 10ºC min-1, following 80ºC min-1 cooling) of a
devitrification event (at -75ºC), subsequently followed by melting (at -36ºC) was reported.
84
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
After extensive, repeated and reproducible DSC experiments, we have been unable to find any
ice formation event, at any of the combinations of cooling and warming rates considered, and
for any of the PVS studied. The reported devitrification event took place upon warming at
10ºC min-1, twice as fast as our 5ºC min-1 slowest experiment (where ice formation would
have been more likely). PVS3 was also previously reported to have a TG in good agreement
with those reported here (Záme ník et al., 2012).
Pure water glass transition temperature, though difficult to physically measure, is believed
to take place at 135K (-138ºC) (Johari et al., 1990; Johl et al., 2005), actually not so far away
fro
on vitrification behavior
Vitrification in cryopreservation protocols is achieved, without sophisticated cooling
ph
rec
G
nce observed among different solutions. The TG of a
sol
m the TG found here for PVS solutions. Other authors even rise this temperature to 165 K
(-108ºC) (Velikov et al., 2001; Angell, 2008), or even higher (Swenson & Teixeira, 2010;
McCartney & Sadtchenko, 2013). Clearly, the question is far from being settled.
6.3.2. Effect of cooling rate
equipment, by simply plunging specimens into liquid nitrogen (LN) after a set of
ysicochemical treatments, designed to increase their cytoplasmatic microviscosity and
enhance tissue resistance to cold and dehydration. Fast cooling is required to achieve
vitrification avoiding the ice formation. In a similar way, warming after the storage period
must be carried out quickly, and it is practically performed by plunging samples (naked or
inside cryovials) directly from LN into a water bath or warm culture medium.
Figure 6.2 shows details of the thermograms obtained from testing the effect of cooling
time on the vitrification behavior of PVS3, after the method described. The glass transition
orded in the warming scan (always carried out at 10°C min-1) was similar both in
temperature and heat capacity change. The width of the glass transition process was estimated
to be on an average of 7ºC.
Figure 6.3 shows TG and Cp for the five solutions studied. T variation with cooling
rates was much smaller than the differe
ution did not significantly change within a wide range of cooling rates (from 5 to 20°C
min-1, under the DSC control, and the faster resulting from plunging the sample pan into LN,
with the aluminum pan naked or inside a cryovial). A total change in TG inflexion point of
1.2°C, as was observed between the �“slow�” (5-20ºC min-1) methods and the �“faster�” one
85
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
(direct into LN), did not influence considerably the glass transition region, as compared with
the width of the whole transition interval.
PVS3 thermograms showing the glass transition, performed at a warm and after cooling at different rates. Quenching: direct immersion of the DSC pan in
quenching
cryovial
20ºC min-1
5ºC min-1
10ºC min-1
Figure 6.2. ing rate of 10ºC min-1
LN; cryovial: pan included in a cryovial and then in LN. White arrows mark the glass
ow a larger variation, no clear trend with the cooling rate could be
observed (Figure 6.3). Besides, statistical analysis on both TG and Cp, showed that the
dif
expected value (3-5ºC per rate magnitude order corresponds to 9-15ºC, for the more
transition inflection point.
Although Cp values sh
ferences observed among cooling rates were not significant. This was an unexpected
finding, as both TG and the Cp are generally considered to be dependent on cooling rate (e.g.
Angell et al., 1978; Debenedetti & Stillinger, 2001). After this, the slower a liquid is cooled,
the longer time would be available for configuration of samples at each temperature, and
hence the colder it could become before falling out of liquid-state equilibrium. This would be
reflected in TG increasing with cooling rate (Moynihan et al., 1976; Brüning & Samwer,
1992), as the properties of the glass depend on the process by which it is formed. In practice,
the reported dependence of TG on the cooling rate is weak: TG changes by 3�–5°C when the
cooling rate changes by an order of magnitude (Ediger et al., 1996; Debenedetti & Stillinger,
2001).
Possible explanations for the discrepancy between the small variation (1.2ºC) reported here
and the
86
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
tha
eters measured at fast cooling rates were evaluated always at the ensuing
wa
measured. They range from the very slow processes of
tho
n three orders between 5 and 6600ºC min-1 of our experiments) may lie on that the
experimental observations in which TG undergoes these changes with cooling rate refer in
most cases to much simpler systems, either pure liquids or binary mixtures. These ternary,
quaternary and pentary solutions may present a compensating behavior, with different
components having contributions of different sign to the glass transition displacement with
cooling rate.
Another explanation may lay on the fact that, in the present work, the reported glass
transition param
rming scan. Actually, very few experimental techniques can perform a proper glass
transition measurement while cooling at fast rate. Faster cooling would cause the glass
transition to take place at earlier, higher temperatures (giving rise to a less stable glass). Upon
warming, the glass transition would occur as soon as the lower TG (of the more stable glass,
found at slow warming processes) was reached. In that way, the influence of the previous
cooling rate would be ignored.
Cooling rates present a wide variation in practical plant cryopreservation, though they have
not been very frequently accurately
se methods relying on extracellular freezing for induced intracellular vitrification (using
temperature control equipment, with rates as low as 0.3ºC min-1; Kaczmarczyk et al, 2011), to
those of he vitrification method (approx. 2.3°C s-1), the encapsulation-dehydration protocol
(approx. 2.7°C s-1) and the faster droplet-vitrification method (110ºC s-1) (Kaczmarczyk et al,
2011; see Chapters 3 and 5). Other faster approaches have being tested, such as those
avoiding the Leidenfrost effect that limits the cooling rate in LN by using other cryogenic
fluids, such as liquid propane or ethane, or even using mechanical implements to make faster
the immersion of the sample in the cryogenic medium. The two quench cooling procedures
used here had rates similar to those of the droplet-vitrification (naked DSC pan) and the
encapsulation-dehydration method (inside the cryovial), i.e. approximately 110ºC s-1 (6600ºC
min-1), and 2.7ºC s-1 (160ºC min-1), respectively.
87
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
Figure 6.3. Glass transition temperatures (a) and glass transition heat capacity changes b) for the different PVS and garlic shoot tips (after treatment with PVS3), obtained in warming
he behavior of the vitrification parameters of garlic shoot tips in the last stage of the
cryopreservation protocol (after treatment with PVS3) was different from that of the
vitr
a
b
*
*
(
(10ºC min-1) DSC experiments, after being cooled at different rates. * Rate not tested for garlic samples. Bars: standard error.
T
ification solutions (Figure 6.3). Shoot tips present a much more marked dependence of
88
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
both TG and Cp with the cooling rate than solutions. This may be due to the fact that the
shoot tips have higher cytoplasmic water content or to the presence of other cellular
components, apart form their structural and compartmentalization elements.
6.3.3. Effect of warming rate on vitrification behavior
ing rates after
usi
e order of those reported
for
6.3.4. Crystallinity of garlic shoot tips
ication procedure exhibited both freezing and glass
tra
such as water content or permeability to solutes, may sustain alternative explanations.
Figure 6.4 shows the glass transition parameters obtained for different warm
ng two different cooling rates: 10°C min-1 (a and c) and LN quenching (b and d). The
variations in both TG and Cp were not significant and, again, showed no dependence of the
warming rate, but a clear relation with the solution composition and for all the cooling and
warming conditions studied, with a similar response of the group PVS1-PVS2-PVS2 mod, on
one hand and the group PVS3-PVS3 mod, on the other.
Practical warming rate values for cryopreservation are of the sam
cooling rates, as most cases heat exchange is driven by stirring the sample (naked,
encapsulated or inside a cryovial) in a warm water bath or culture medium, being the
temperature gradients induced comparable to those created by plunging room temperature
specimens into LN. Nonetheless, warming rates slightly lower than those found for cooling
have been reported (see Chapters 3 and 5): approx. 1.8, 2 and 40°C s-1, for mint shoot tips
following the vitrification, encapsulation-dehydration and droplet-vitrification protocols,
respectively.
Garlic shoot tips subjected to the vitrif
nsition events when cooled slowly (10ºC min-1) (Figure 6.5). Other systems treated by the
vitrification method did not produce water freezing when, in the last step of the protocol, were
cooled at this slow rate. This is the case of mint shoot tips (Teixeira et al., 2013) and this
might be due to their smaller size, when compared with garlic shoot tips. Mint shoot tip
average weight after the vitrification dehydration step was 0.7 mg (Teixeira et al., 2013),
while garlic shoot tips in the same stage weighed 3.9 ± 0.4 mg. Sample size is one of the most
important determinants of vitrification probability (Yavin & Arav, 2007). Other differences,
89
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
Figure 6.4. Glass transition temperature (a and b) and heat capacity (c and d) obtained in DSC during warming at different rates (5, 10 or 20°C min-1). DSC pans had been cooled either at 10°C min-1 in the calorimeter (a and c) or by quenching in LN (b and d). Bars: standard error.
e of this (always very small) melting endotherm allows calculation of the
action of crystallized water. The onset of the melting process and the crystallinity calculated
after the TA proprietary software, are presented in Table 6.3 for different warming rates. The
crysta
The presenc
fr
llinity decreased with increasing warming rates, while the transition onset grew. The
corresponding glass transition temperatures were almost constant with warming rate. In
similar systems, it has been reported that Cp decreases while crystallinity increases (Gao et
al., 2005), which agrees with the observations of Figure 6.5 and Table 6.3. Although, at
quenching rates (those employed in practical cryopreservation) there was no formation of ice,
these findings could imply a potential amount of freezable water that could result in ice
formation.
90
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
Figure 6.5. Typical scan of shoot tips of garlic after treatment with PVS3 for 2 h. Both cooling and warming rates were 10ºC min-1. The black arrows mark to the glass transition inflection point in cooling and warming scans, while gray arrows signal the freezing and melting events.
d warming scans at different rates. Warming rate Onset Crystallized
Table 6.3. Crystallization parameters (mean ± standard deviation) of garlic shoot tips after treatment with PVS3 for 2 h. Onset of the melting endotherm and crystallinity as crystallization percentage, both measured in the warming scan. Cooling scans were performed at 10ºC min-1 an
(°C min-1) (°C) (%) 5 -19.87 ± 9.40 1.96 ± 2.51 10 -16.49 ± 3.73 1.21 ± 0.25 20 -14.61 ± 0.62 0.37 ± 0.51
6.3.5. Effect of the stora time on on b VS
Table 6.4 and 6.5 w no nges TG or Cp, respectively,
ssociated to the period of storage in LN, up to 60 days. Although the storage period of
e maximum storage
period used here, it was considered to be a reasonable period and should enable the
ge the vitrificati ehavior of P
sho significant cha either in
a
cryopreserved material, in many practical instances, exceeds well th
observation of any change with time in the vitrified sample. The fact that quenched cooled
samples did not show any variation in these properties with storage time confirmed that the
system had reached a stable vitreous state, suitable for cryopreservation.
91
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
Table 6.4. Cryopreservation solutions glass transition temperature (mean ± standard deviation) versus storage time.
TG (°C)
Storage time (days) Type of PVS
1 7 30 60
PVS1 -112.38 ± 0.10 -112.35 ± 3.01 -111.33 ± 0.001 -111.81 ± 0.11
PVS2 -112.59 ± 0.86 -112.72 ± 1 3.88 ± 0.52 -114.19 ± 0.66
-108.81 ± 1.64 -108.7 ± 0.06 -109.97 ± 0.09
PVS3 -88.63 ± 2.17 -88.92 ± 0.04 -88.52 ± 0.98 -88.38 ± 0.13
PV d
.55 -11
PVS2 mod 1 ± 0.73 -108.88
S3 mo -85.47 ± 0.93 -85.80 ± 1.53 -85.43 ± 0.15 -86.29 ± 0.01
able 6.5. Cryopreservation solutions heat capacity change (mean ± standard deviation) ersus storage time.
Cp (J g°C-1)
Tv
Storage time (days) Type of PVS
1 7 30 60
PVS1 1.06 ± 0.06 1.08 ± 0.38 0.95 ± 0.15 1.22 ± 0.05
PVS2 0.65 ± 0.14 1.10 ± 1.16 ± 0.05
0.78 ± 0.09 1.26 0.09 1.28 ± 0.01
PVS3 0.83 ± 0.24 0.97 ± 0.02 0.69 ± 0.20 0.98 ± 0.11
PV d
0.20 1.07 ± 0.33
PVS2 mod ± 0.03 1.01 ±
S3 mo 0.68 ± 0.15 0.97 ± 0.12 0.63 ± 0.02 0.94 ± 0.05
.3.6. Annealing of PVS1 and PVS3
Considering the previously reported grouping of the vitrification solutions, the annealing
ehavior of PVS1 and PVS3 were studied, as representative of each group. Figure 6.6 shows
typical thermogram of PVS1 with a detail of the step E-F of the annealing method depicted
6
b
a
92
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
in Figure 6.1, where areas over the curve were measured. These areas are represented in
Figure 6.7, versus the corresponding target temperature (D), for PVS1 and PVS3. The
behavior observed is clearly distinct for the two solutions: for PVS1 the curve slope increased
with decreasing target temperature, implying quickly growing annealing areas. On the other
hand, the slope was much shallower for PVS3, and large temperature variations were reflected
in a reduced area increase.
Figure 6.6. Typical thermogram of PVS1 on the step E-F of Figure 6.1. The annealing areas were comprised between the curve and the extended baseline.
93
6. GLASS TRANSITION AND ANNEALING BEHAVIOR OF PLANT VITRIFICATION SOLUTIONS
Figure 6.7. Annealing area versus temperature (D in Figure 6.1) of the solutions PVS1( ) and PVS3(�•).
Increases in the annealing areas are considered as proof of a glass to be unstable,
susceptible to evolve to form more stable vitreous structures at temperatures close to TG (Gao
et al., 2005). In this case, PVS3 would be forming more stable glasses and this could indicate
a better behavior for storage of cryopreserved samples. This would be relevant for storage at
temperatures well over that of LN. The difference between the LN temperature (-196ºC) and
the annealing temperatures employed here is nearly 100ºC, and stability at LN conditions
would be guaranteed. A different case would be for storage in -80ºC freezers, at temperatures
too close to TG, of these order (-87 to -114ºC). Interestingly, it has also been reported an
influence of the annealing time on the glass transition temperature (Chan & Baust, 1991). In
our experiments, an annealing time as long as four hours did not produce a noticeable
difference over the customary 30 min employed.
94
7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
7. THE APPLICATION OF CHITOSAN FILMS ON
ALGINATE BEADS FOR CRYOPRESERVATION USES
7.1. Introduction
Chitosan, a natural-origin product with a wide range of interesting properties, comprising
antimicrobial, protective, antioxidant, plant defense promoting and ligand binding activities,
has become in the last few decades an ubiquitous molecule and the number and variety of its
applications are continuously increasing. They range from its now common use as a
pharmaceutical inert phase, either involved in the modulated delivery of drugs or as a
protecting cover (Uhrich et al., 1999; Morris et al., 2010), to medical uses, for example, as a
wound healing promoter (Ravi-Kumar, 2000), and to its extended inclusion as food
component, with a variety of functions (Knorr, 1984).
Chitosan is a natural linear bio-polyaminosaccharide derived from chitin, which is the
principal component of protective cuticles of crustaceans (No et al., 2003; Rinaudo, 2006).
Chitosan (Figure 7.1) is obtained from chitin by N-deacetylation (Andres et al., 2007) and
consequently is rich in amino groups, which makes chitosan a cationic polyelectrolyte (pKa
~6.5). In aqueous acidic media chitosan has high positive charge on �–NH3+ groups, having the
ability to aggregate to negatively charged molecules (Krajewska et al., 2011). Chitosan is
most frequently characterized by its molecular weight (M) and its degree of acetylation (DA)
(Goy et al., 2009) but there are other important variables, such as the nature of the salt
counterion, pH, ionic strength and the addition of a non-aqueous solvent.
Chitosan (and also chitin) have been shown to have several useful activities for biological
and biotechnological applications, such as null toxicity, biocompatibility and biodegradability
(Sinha & Kumria, 2001). Its properties are especially relevant in relation to plant biology:
they include antiviral, antibacterial and antifungal, as well as mineral chelating activities and
plant natural defences enhancement (El Hadrami et al., 2010).
The mechanisms underlying the effects of chitosan against plant disease are little known,
but would include direct microbial toxicity and metal chelation, as well as the formation of
physical barriers. Additionally, it would trigger signalling cascades leading to defence
activation. The plant defence enhancing properties of chitosan include the induction of
95
7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
lignification, pH cytoplasm changes, chitinase activation, reactive oxygen species scavenging
and other biochemical and genetic responses (Barber et al., 1989; Kuchitsu et al., 1995;
Minami et al., 1996). Chitosan has a role in early elicitation of plant defence (Amborabé et
al., 2008), promoting the accumulation of its related metabolites. A possible way of action is
the recognition of chitosan, among other cell wall polysaccharides, by specific intruder
receptors (Dangl et al., 2001).
Figure 7.1. Schematic representation of the chemical structures of the chitin and chitosan (adapted from Goy et al., 2009).
Chitosan has been used to promote the germination of plants of several species, such as
maize, peanut or rice (Reddy et al., 1999; Ruan & Xue, 2002; Zhou et al., 2002).
Additionally, it promotes wound healing by means of its binding properties (Hirano et al.,
1999) and its applicability to fresh product storage is promising. Chitosan also has been
reported to have an interesting antioxidant activity (Xie et al., 2001; Sun et al., 2006, 2008),
and it can function as an oxidative molecule scavenger.
96
7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
The antimicrobial activity of chitosan against different groups of microorganisms has
received considerable attention in recent years (Goy et al., 2009; El Hadrami et al., 2010).
This polysaccharide owns antimicrobial activity against both Gram-positive and Gram-
negative bacteria (Krajewska et al., 2011). Chitosan is employed to limit microbial
proliferation on many substrates, for example, as a food protecting layer, while also
modulates permeability to chemicals. Its antimicrobial activity has been shown to depend on
its molecular weight (El Hadrami et al., 2010).
Besides the effect of chitosan on fungi by promoting the host plant defence responses, it
has been shown to directly inhibit growth of many fungi such as Fusarium oxysporium and
Botrytis cinerea (Leuba & Stössel, 1986; Benhamou, 1992; El Ghaouth et al., 1994;
Benhamou et al., 1998; Romanazzi et al., 2002; Ait Barka et al., 2004; El Hassni et al., 2004).
Exceptions to the wide antimicrobial and antifungal activity of chitosan (Muzzarelli et al.,
1990) are some fungi with chitosanolytic activity (Palma-Guerrero et al., 2008). Chitosan has
been successfully employed as a treatment to prevent fungi damage to seeds (Reddy et al.,
1999; Rabea et al., 2003) and it has been applied to control plant diseases by foliar application
and soil amendment. As foliar spray, it reduced mildew infection severity (Faoro et al., 2008),
while as soil amendment, acted against fungi of the genera Fusarium (Rabea et al., 2003) and
Aspergilus (El Ghaouth et al., 1992). In spite of this wide-spectrum antimicrobial properties,
the understanding the underlying mechanism is poor.
Alginate is another naturally occurring polysaccharide of sea origin, also with growing and
widespread applications. It is produced by three brown sea algae species (Laminaria
hyperborea, Ascophyllum nodosum and Macrocystis pyrifera). Alginic acid and its salts are
anionic linear polysaccharides containing 1,4-linked D-mannuronic acid and L-glucuronic
acid residues. It can form irreversible hydrogels in the presence of some multivalent metal
cations, such as the divalent calcium ions (Coviello et al., 2007; Draget et al., 2011). A
common use of alginate is as gel beads that are easily formed by releasing drops of a solution
of suitable concentration of alginic acid into the counterion solution (drops of a cation
solution dispersed in the alginic acid solution also produce gel beads). Cations diffuse into the
alginate drops promoting its gelation, and the gel strength and the beads consistency increases
with time. The counterion employed is often calcium, as this divalent ion allows the formation
of strong and flexible gels for low alginate concentrations (2-3%), at reduced diffusion times.
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7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
The consistency of the alginate beads grows with time, as the calcium diffusion within the
alginate drop progresses, allowing cross-linking. The maximum gel hardness occurs in the
bead surface, as the ions concentration is higher there (King, 1983; Draget et al., 1997;
Draget, 2000).
The biocompatible and inert nature of alginate has allowed the use of its gelling properties
in several contexts, such as the development of new food forms and the delivery of nutrients
(Coviello et al., 2007; Deladino et al., 2008; Anbinder et al., 2011) and pharmaceutical
products (Pasparakis & Bouropoulos, 2006), or the encapsulation of plant germplasm as a
way of protection against environmental fluctuations (Phunchindawan et al., 1997;
Engelmann et al., 2008; Rai et al., 2009). The sometimes called �“artificial seeds�” are
elaborated by simply enclosing a somatic embryo in a drop of the alginate solution, and
dispensing it in the calcium solution (Senaratna, 1992; Khor & Loh, 2005).
The gelation of alginate is compatible with a variety of dissolved substances, so the
required nutritive media can be added. Once the gel is formed, it behaves mostly as an inert
solution sustaining network, so it can be permeated by other substances, and its water content
can be altered by evaporation. These properties are currently used in cryopreservation
protocols.
Cryopreservation of plant germplasm often includes steps in which the viscosity of the
solutions of the different tissue compartments, including cytoplasm, is reduced in order to
decrease the probability of ice formation and increase the glass transition temperature. This is
achieved either by evaporative dehydration, induced by air flow, and/or by incubation in
highly concentrated sucrose solutions. Alginate beads allow easy equilibration by both means
(Engelmann et al., 2008).
The permeability of alginate beads to water and solutes as well as their mechanical
resistance has been modulated by addition of other polysaccharides (Deladino et al., 2008;
Anbinder et al., 2011). The combination of alginate and chitosan polymers, preserving their
intrinsic biocompatibility, non-toxicity, and biodegradability characteristics, is increasingly
employed for uses such as drug delivery and enzyme and cell immobilization (George &
Abraham, 2006; Pasparakis & Bouropoulos, 2006; Kim et al., 2008; Rajendran, 2009).
Chitosan, being a cationic polysaccharide, can externally bind to alginate beads or diffuse into
three-dimensional alginate gel network, which has an anionic character (Hyland et al., 2011).
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7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
With the purpose of studying the possible application of chitosan to improve the
performance of the encapsulating substrates employed, the permeability and water interaction
properties of chitosan-covered alginate beads were investigated. Additionally, scanning
electron microscopic techniques were applied to observe the chitosan layer on the beads.
7.2. Materials and Methods
7.2.1. Chitosan types and hydrodynamic characterization
Three different chitosan commercial forms were obtained from Sigma Aldrich (USA):
chitosan C (from shrimp shells, Sigma No. C3646), medium molecular weight chitosan
(MMW) (Sigma No. 448877) and low molecular weight chitosan (LMW) (Sigma No.
448869). The producer specifications indicated that all of them were 75-85% deacetylated and
had been extracted from crustaceous shells. All samples were prepared at a concentration of
3.0 mg/mL dissolved in 0.2 M acetate buffer pH 4.3.
Sedimentation velocity determinations were performed with chitosan solutions using an
Optima XL-I analytical ultracentrifuge (Beckman Instruments, Palo Alto, USA). Samples
were centrifuged at 45000 rpm, at a temperature of 20.0°C. Data were analyzed using the
least-squares c(s) method included in the SEDFIT software (Dam & Schuck, 2005).
Sedimentation coefficients, s, were extrapolated to zero concentration, to correct for non-
ideality effects (Patel et al., 2007).
The extended Fujita approach (Fujita, 1962; Harding et al., 2011) was applied to calculate
approximate molecular weights for chitosan, from the determined sedimentation coefficients,
using the eq. 1:
(Eq. 1) so20,W = s Mb
where M is the molecular weight and so20,W, the sedimentation coefficient at infinite dilution
and 20oC. The pre-exponential factor s, a constant for a particular polymer under a defined
set of conditions, and the exponent b, defining the hydrodynamic behavior (ranging from 0.4�–
0.5 for a coil, ~0.15�–0.2 for a rod and ~0.67 for a sphere), were taken as 0.1 and 0.24,
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7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
respectively, from data fitted for chitosan samples of similar acetylation degree (Harding et
al., 2011).
7.2.2. Alginate beads
Calcium alginate beads were made with 2 or 3 % (w/v) low viscosity alginic acid (Sigma,
USA) in liquid MS (Murashige & Skoog, 1962) without calcium, at pH 5.7. Droplets of
alginate were pipetted onto 100 mM calcium chloride (CaCl2) solution prepared also in MS
liquid medium, using a 2 mL plastic disposable Pasteur pipette, in a flow chamber, at room
temperature. Beads were allowed to stand in the CaCl2 solution for 30 min. Then, this solution
was filtered to remove the formed alginate beads.
7.2.3. Chitosan solution and alginate bead treatment
2% C type chitosan solutions were prepared in 1% acetic acid (Protanal, Norway), diluted
in MilliQ water (Millipore, Billerica, USA) and stirred overnight. Then, pH was adjusted to
5.6 with NaOH (Sánchez-Dominguéz et al., 2007). Chitosan-covered alginate beads were
prepared by immersion of the preformed calcium alginate beads in a container with the
selected chitosan solution and stirred at 150 rpm, for 30 min. The chitosan solution was
filtered to recover the beads.
7.2.4. Water content determination and air dehydration studies.
Four sets of three beads were used for determining the dry mater and water content. Dry
matter resulted from differential weighing before and after oven-drying (~85°C, 72 h) and
was expressed as relative to the total mass (dm). The beads water content (Wc) could be
calculated for dehydration stages, directly as the difference between its total mass and dm. It
was expressed as relative to the total sample mass -fresh weight- (Wc(s)).
To study the beads air-dehydration kinetics, freshly made beads were transferred to an
open glass Petri dish and dehydrated for 1, 2, 3, 4, 5 and 6 h in a laminar-flow hood.
Afterwards, bead weight was determined as described above.
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7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
7.2.5. Differential scanning calorimetry and frozen and unfrozen water fractions
DSC experiments were performed with a Mettler-Toledo DSC 30 instrument (Mettler-
Toledo, Griefsen, Switzerland). Three beads with the same treatment were placed in a DSC
pan, which was sealed and weighed. Samples were submitted to a cooling scan from room
temperature to -150ºC and a short 5 min equilibration at this temperature, followed by a
warming scan (at 10ºC min-1), back to room temperature. Calorimetric data were collected
from two replicates per treatment. Later, pans were punctured and dried in an oven at 85ºC,
for 72 hours, when they showed constant weight.
Thermograms were analyzed using the standard procedures provided in the Mettler-Toledo
STARe software. Ice thawing thermal events (more precise than freezing events), were used
for extraction of Tf. The routine produced the temperature corresponding to the event onset,
Tf(onset), corresponding to the equilibrium freezing temperature and that to the peak, Tf(peak).
The melting enthalpy Hf, proportional to the event area, was also obtained. It was expressed
as relative to either the total bead mass ( Hf(s)), its dry mass ( Hf(dm)) or its water content
Hf(w), to allow easier data comparison. Water fractions, frozen water (Wf) and unfrozen
water (Wu), were calculated by comparison of Hf and the pure water freezing enthalpy
( Hf(w) = 333.4 J/g at 0ºC), together with the total water contents previously determined, after
eq. 2:
Eq.2 Wf= ( Hf/ Hf(w)); Wu=Wc-Wf
Specific enthalpy values (per sample gram) were always used, after the corresponding
oven dry pan weights. Frozen (Wf) and unfrozen (Wu) water contents were expressed as
relative to the total bead mass (Wf(s), Wu(s)), its dry mass (Wf(dm), Wu(dm)) or its total water
content (Wf(w), Wu(w)).
7.2.6. Low temperature scanning electron microscopy observations.
Cryo-SEM studies were performed, basically as previously described (Teixeira et al.,
2013), using a Zeiss DSN 960 scanning microscope equipped with a Cryotrans CT-1500 cold
plate (Oxford, UK). Cryo-SEM allows sample observations without the need of prior
chemical fixing or drying processes. Three beads were fitted on a special bracket and this
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7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
piece was immersed into liquid nitrogen (LN) under low pressure, physically fixing tissues for
microscopic observation. The holder with the samples was placed in the microscope and
etching (partial ice sublimation, induced to provide contrast) was performed for three minutes
at -90ºC. Afterwards, the samples were coated with high purity Au, which acts as a
conductive contact for electrical charge. Finally, they were inserted in the Cryotrans cold plate
and observed under secondary electron mode, at a temperature of -150/-160ºC, using an
accelerating voltage of 15 kV and a working distance of 10-25 mm.
7.2.7. Environmental scanning electron microscopy
Environmental SEM analysis was performed using a Jeol JSM-6360 (Japan) microscope.
Beads were attached to stubs using a two-sided adhesive tape, then coated with a layer of gold
(40 nm-50 nm) and examined using an acceleration voltage of 10 kV. The observation
temperature was 5-8ºC and the pressure at the sample chamber was 6 torr. Some beads were
air dehydrated for five hours before observation.
7.3. Results and Discussion
7.3.1. Chitosan hydrodynamic characterization
The sedimentation coefficients for each chitosan type were determined and represented as a
function of the concentration (Figure 7.2), to obtain the infinite dilution extrapolated
sedimentation coefficients, so20,W, which are shown in Table 7.1. The molecular weights
calculated using the extended Fujita approach, as described (eq. 1), are also presented in this
Table. The most common commercially available chitosan presentations are often highly
polydisperse and their molecular weight can present large differences among batches.
Employing this relatively easy approach, the size of the chitosan polymers studied can be
estimated (also presented in Table 7.1), with the help of fitting parameters obtained for
similar samples (Harding et al., 2011).
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7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
0.0 0.5 1.0 1.5 2.0 2.50.0
0.2
0.4
0.6
0.8
1.0
1.2
Chitosan (MMW)
(1/s 2
0,W) (
sec-1
)
Concentration (mg/ml)0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Chitosan (LMW)
(1/s 2
0,W) (
sec-1
)
Concentration (mg/ml)
0.0 0.5 1.0 1.5 2.0 2.50.0
0.2
0.4
0.6
0.8
1.0
1.2
Chitosan (C)
(1/s 2
0,W) (
sec-1
)
Concentration (mg/ml)
a b
c
0.0 0.5 1.0 1.5 2.0 2.50.0
0.2
0.4
0.6
0.8
1.0
1.2
Chitosan (MMW)
(1/s 2
0,W) (
sec-1
)
Concentration (mg/ml)0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Chitosan (LMW)
(1/s 2
0,W) (
sec-1
)
Concentration (mg/ml)
0.0 0.5 1.0 1.5 2.0 2.50.0
0.2
0.4
0.6
0.8
1.0
1.2
Chitosan (C)
(1/s 2
0,W) (
sec-1
)
Concentration (mg/ml)
a b
0.0 0.5 1.0 1.5 2.0 2.50.0
0.2
0.4
0.6
0.8
1.0
1.2
Chitosan (MMW)
(1/s 2
0,W) (
sec-1
)
Concentration (mg/ml)0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Chitosan (LMW)
(1/s 2
0,W) (
sec-1
)
Concentration (mg/ml)
0.0 0.5 1.0 1.5 2.0 2.50.0
0.2
0.4
0.6
0.8
1.0
1.2
Chitosan (C)
(1/s 2
0,W) (
sec-1
)
Concentration (mg/ml)
0.0 0.5 1.0 1.5 2.0 2.50.0
0.2
0.4
0.6
0.8
1.0
1.2
Chitosan (MMW)
(1/s 2
0,W) (
sec-1
)
Concentration (mg/ml)0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Chitosan (LMW)
(1/s 2
0,W) (
sec-1
)
Concentration (mg/ml)
0.0 0.5 1.0 1.5 2.0 2.50.0
0.2
0.4
0.6
0.8
1.0
1.2
Chitosan (C)
(1/s 2
0,W) (
sec-1
)
Concentration (mg/ml)
a b
c
Figure 7.2. Plots for extrapolation of the sedimentation coefficient of the different chitosan types to infinite dilution: a = MMW, b = LMW and c = C chitosan.
7.1. Analytical ultracentrifugation-derived and chitosan size parameters (mean ± standard deviation).
Type of chitosan So20,W (s) ks M (x 103) No.
monomers length (nm)
Chitosan (MMW) 2.32 ± 0.07 608 ± 35 490 ± 50 2600 ± 300 1200 ± 700 Chitosan (LMW) 1.85 ± 0.02 297 ± 7 190 ± 8 1020 ± 40 450 ± 20 Chitosan (C) 2.50 ± 0.20 650 ± 72 670 ± 200 3580 ± 1000 1600 ± 500
The sedimentation coefficient concentration dependence factor, ks, accounts for non-
ideality, i.e. interparticle interactions (Morris et al., 2009). This effect arises from the
increased viscosity of the solution at higher concentrations, and from the fact that sedimenting
solute particles must displace solvent backwards in the process. Both effects become
vanishingly small as concentration decreases. It contains information on the particle size,
shape and conformation, as well as on their degree of intermolecular interactions. ks is small
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7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
for globular particles, but becomes much larger for elongated molecules (Ralston, 1993). It is
extracted from the slope of the plots in Figure 7.2 and it is also show in Table 7.1.
The data obtained here for sedimentation coefficients and molecular weight of chitosans
are of a similar order to those reported, for different origin samples, by other authors (Berth et
al., 2002; Morris et al., 2009; Harding et al., 2011). These authors found that data treatment
derived from semi-flexible rod particles fitted well the experimental determinations from
chitosan. At the experimental conditions at which these experiments were performed (pH 4.3),
the ionisable groups of chitosan would be positively charged and the repelling charges would
keep the molecule in a nearly rigid, straight conformation.
From the molecular weights obtained the number of monomeric units can be also derived.
Taking as monomer weights 221 for N-acetyl glucosamine (the acetylated monomer) and 179
for glucosamine (the deacetylated subunit), a pondered average monomer weight of 187 can
be considered, for chitosans of this deacetylation degree (approximately 80 %). The number
of monomers obtained for each chitosan type is also showed on Table 7.1.
Although further information would be required to calculate a flexibility degree or a
persistence length, the simple contour length, not so different here from the length of the
extended chain, can be calculate with a mass per unit length value of 420 g mol-1 nm-1 (Vold,
2004; Morris et al., 2009). Results show that chitosan molecules are quite large, almost
macroscopic (approximately 1 µm).
In spite the lack of clear understanding of the mechanisms by which chitosan produces its
different effects and activities, such as antimicrobial, antioxidant, plant defence responses
eliciting (El Hadrami et al., 2010; Kong et al., 2010), references to the size-dependence are
frequent. Most reports ascribe a more pronounced effect to larger size chitosan molecules, for
the same acetylation degree (No et al., 2002; El Hadrami et al., 2010), while some others
report activities, such as chitosan plant antiviral action, that seem to be inversely correlated to
molecular weight (Kulikov et al., 2006). However, most of these differences are among low
condensation-degree oligomers, rather than referring to chains as large as those compared
here (No et al., 2002; Cabrera et al., 2006). Although the size differences among the three
chitosan types employed are not too extreme, the higher molecular mass type (�“C�”) was
chosen to perform further experiments. Besides, C chitosan is a cheaper product with
extensive use.
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7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
7.3.2. Water content determination and air dehydration
Table 7.2 shows the dry mass (dm) and water content of 2 or 3% calcium alginate beads,
naked (AB) or covered by chitosan (ACB). Water content was expressed relative to total mass
(Wc(s)). Both dry matter and water contents showed little differences between AC and ACB.
Table 7.2. Water and dry matter content (mean ± standard deviation) for alginate beads (AB) and chitosan-covered alginate beads (ACB). See Materials and Methods for parameter description.
AB ACB 2% alginate 3% alginate 2% alginate 3% alginate
dm (gdm gsample-1) 0.03 ± 0.01 0.05 ± 0.01 0.04 ± 0.01 0.03 ± 0.01
Wc(s) (gwater gsample-1) 0.97 ± 0.03 0.95 ± 0.03 0.96 ± 0.03 0.97 ± 0.01
The water content decay upon air flow dehydration was similar for all bead types except
for 2% alginate beads, whose dehydration was slower during the first hour. The plateau
where most water has been eliminated was reached between 2 and 3 hours drying time.
Figure 7.3. Evolution of the water content over total sample mass, Wc(s), with air flow drying time, for alginate beads covered with chitosan or not, prepared with 2 and 3% sodium alginate concentration. Bars: standard error.
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7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
7.3.3. Calorimetric studies on water status
Calorimetric measurements designed to obtain information on the water status on beads
were performed for 3% alginate AB and ACB. Table 7.3 shows the resulting parameters.
Calorimetric parameters showed data corresponding to the fully hydrated beads, again
showing little differences between them. Melting temperatures were found to be similar. The
melting enthalpy obtained, which allowed the calculation of water frozen and unfrozen
fractions, was roughly similar for both bead types. The slightly higher unfrozen water content
of ACB may reflect the intake of an additional water amount on the chitosan covering step,
but the presence of this water content after equilibration would imply additional interactions
with the electrostatically charged chitosan. This could mean that this additional water amount
would not be frozen when temperature was reduced, which is not sustained by these
observations. Alternatively, the slightly higher frozen water content of ACB may be due to a
lower amount of water interacting with alginate, resulting from a saturation of the alginate
charged groups by the chitosan-alginate electrostatic interaction.
Table 7.3. Calorimetric parameters (mean ± standard deviation) obtained for 3% alginate beads (AB) and chitosan-covered alginate beads (ACB). See Materials and Methods for parameter description.
AB ACB Tf (onset) (ºC) -5.1 ± 0.1 -4.5 ± 0.1 Tf (peak) (ºC) 4.9 ± 1 5.8 ± 0.2
Hf(s) (J g sample-1) 255 ± 2 277 ± 2
Hf(w) (J gw-1) 284 ± 5 296 ± 7
Wf(s) (gwater gsample-1) 0.76 ± 0.02 0.83 ± 0.02
Wf(dm) (gwater gdm-1) 7.5 ± 0.8 12.9 ± 2.6
Wf(w) (gwater gwater-1) 0.85 ± 0.01 0.89 ± 0.02
Wu(s) (gwater gsample-1) 0.13 ± 0.01 0.11 ± 0.02
Wu(dm) (gwater gdm-1) 1.3 ± 0.3 1.7 ± 0.6
Wu(w) (gwater gwater-1) 0.15 ± 0.01 0.11 ± 0.02
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7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
7.3.4. Scanning electron microscopy of alginate beads with and without chitosan
Figure 7.4 shows the aspect of the external surface of AB and ACB under cryo-SEM. As
the purpose of the experiment was to examine the external bead surface under liquid nitrogen
temperature, etching was not performed. No evident differences could be appreciated in bead
examination: chitosan would form a homogeneously distributed film, completely covering the
surface of alginate beads, and the aspect of both AB and ACB would be similar. There were
no evident changes, such as patches or cracks, on the chitosan layer, although cooling to
liquid nitrogen temperature was performed by quenching, a process that could induce
mechanical stress on the bead surface.
Figure 7.5 compares the surfaces of AB and ACB, as observed under an environmental
scanning electron microscope, i.e., at room temperature and reduced pressure. Again, the
surface aspect of both bead types was not especially different for non-dehydrated beads.
Nevertheless, beads after an advanced dehydration time (5 hours) showed a wrinkled surface
in ACB, probably resulting from the chitosan layer adaptation to the bead reduced volume
after drying.
Figure 7.4. Cyro-SEM micrographs showing the external AB (right) and ACB (left) bead surface after quench cooling to liquid nitrogen temperature. The bar corresponds to 5 µm.
Chitosan exhibits favourable characteristics for its use in biological and biotechnological
applications, such as its null toxicity, its biocompatibility and biodegradability (Sinha &
Kumria, 2001) and a wide spectrum antimicrobial, antipathogen and antioxidant activity (No
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7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
et al., 2002; El Hadrami, 2010). Alginate beads enclosing plant structures help to buffer
environmental conditions and specially water content while the preliminary procedures to
cryopreservation take place (Benson, 2008). These may include air dehydration and
incubation on concentrated solutions, which diffuse into the gel matrix and also promote its
dehydration and that of the enclosed tissues (Fabre & Dereuddre, 1990; Paulet et al., 1993;
Sakai & Engelmann, 2007).
The transfer properties of alginate beads would be critical for its use, either for its stability
and gel integrity, or for the convenient treatment of contained tissues for cryopreservation.
The addition of an external layer of chitosan was considered that would have an influence on
the matter transfer properties of AB. Chitosan has been applied, for example, to modulate
plant water interchanges and transpiration, by creation of an anti-transpirant film (Bittelli et
al., 2001; Iriti et al., 2009). However, in the formulation employed here, chitosan appears not
to significantly alter the alginate beads water interaction behaviour: neither water initial
retention, water loss upon air-flow dehydration or water freezing behaviour have presented
important changes when the chitosan layer has been added to alginate beads.
a b
c d e
Figure 7.5. Environmental SEM micrographs showing bead surfaces after different air-flow dehydration times: a) AB and c) ACB (0 hours -not dehydrated beads); b) AB, d) and e) ACB (5 hours dehydration). The bar corresponds to 50 m for a) and e) and to 100 m for b), c) and d).
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7. CHITOSAN FILMS ON ALGINATE BEADS FOR CRYOPRESERVATION
The alginate beads employed in this study behave quite differently from the beads actually
employed in cryopreservation practice. In the encapsulation-dehydration protocol (Chapter 5)
the alginate beads had been cultured in 0.75 M sucrose. Meanwhile, the beads in this work
had a much lower solute concentration, only that corresponding to the salts and other nutrients
of MS medium, with a total ionic strength of 94.25 mM (Murashige & Skoog, 1962; McCown
& Sellmer, 1987). This causes for example, that water content decreases to almost nought
upon 6 hours drying, in contrast with the less dramatic reduction in solute-loaded beads
(Chapter 5). Also, the low solute content caters for the lack of detection of a vitrification
calorimetric signal here, in spite of the extreme water content reduction.
Although, following other preparation procedures, alginate and chitosan can give raise to a
three-dimensional network (Hyland et al., 2011), the process followed here caused chitosan to
form an external layer around alginate beads. The chitosan layer of ACB beads behaved well
towards liquid nitrogen cooling, not showing any sign of cracking or other type of alteration
when inspected by cryo-SEM. However, environmental SEM revealed that the chitosan
external layer gets wrinkled, in association to the changes in water content and bead volume
caused by dehydration. A stripped or wrinkled bead surface was reported also by Anbinder et
al. (2011), when observing ACB.
Alginate beads can, additionally, be considered as a model of food (and other systems)
behavior. Microbial proliferation may often be considered a surface affair and polyssacharide
gel beads can constitute a suitable model for the study of the interface microbial growth and,
in this case, be helpful to study the mode of action of chitosan towards microorganisms.
Results obtained suggest the applicability of chitosan to the alginate beads employed in
cryopreservation as the most important gel properties, such as its water interaction behaviour,
were found to be scarcely altered. The antimicrobial protection conferred by chitosan and
their antioxidant properties can be also of great interests, as often cultivation and growth after
cryopreservation can be impaired by microbial contamination or cellular oxidative damages.
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8. DISCUSIÓN GENERAL/GENERAL DISCUSSION
8. DISCUSIÓN GENERAL
La crioconservación constituye un conjunto de técnicas de aplicación práctica relativamente
sencilla, que permiten, con un coste comparativamente pequeño, en términos tanto de mano de
obra especializada, como de material o espacio, mantener colecciones de celulares, ápices y
embriones somáticos durante periodos indefinidos, sin que se observen importantes problemas
de deterioro de su viabilidad (Engelmann, 2004; González-Arnao et al., 2008) y manteniendo la
estabilidad genética del material (Harding, 2004).
Existen en la actualidad protocolos apropiados para una gran diversidad de especies y tipos
de tejido (Sakai, 1997; Engelmann & Takagi, 2000; González-Arnao & Engelmann, 2006;
Reed, 2008). Se han desarrollado también protocolos con técnicas específicas, como la
vitrificación (Langis et al., 1989; Sakai et al., 1991; Yamada et al., 1991) y la encapsulación-
deshidratación (Scottez et al., 1992; Bouafia et al., 1996). Además, la elaboración de
protocolos que permitan la crioconservación de nuevos materiales es un campo de intensa
actividad investigadora (Saragusty & Arav, 2011). Sin embargo, muchos tejidos y especies de
interés no pueden aún ser conservados de esta manera. Las causas de la mortalidad o deterioro
de estos especímenes pueden ser atribuidas ya sea al daño derivado de la formación de hielo en
el proceso, o aquel ocasionado por los procedimientos llevados a cabo para evitar la
congelación (Engelmann, 2008).
En las condiciones de almacenamiento empleadas (temperatura muy baja, estado vítreo)
cualquier actividad biológica, incluso las reacciones bioquímicas que llevarían al
envejecimiento o a la muerte celular, resultan efectivamente paralizadas (Mazur et al., 1984).
Tanto es así, que se ha considerado con frecuencia la posibilidad de que este almacenamiento
pudiera prolongarse por periodos indefinidos (si se evitan los daños producidos por radiación)
(Meryman, 1966a). Sin embargo, los procesos de enfriamiento y calentamiento pueden dar
lugar a daño y muerte celular, o inducir mutaciones en los individuos supervivientes, lo que
puede cuestionar el éxito del proceso.
La formación de hielo en los tejidos vegetales es frecuentemente causa de severos daños,
denominados globalmente “daños por frío” (“cold injury”). Las implicaciones de la nucleación
de hielo intra y extracelular fueron discutidas por Muldrew et al. (2004) y Mazur (2004). El
hielo intracelular es generalmente considerado letal, especialmente en plantas (Levitt, 1956;
Meryman, 1966a; Wesley-Smith et al., 1992; Benson, 2008), si bien en el reino animal, algunos
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insectos y nemátodos soportan cierto de grado de formación de cristales de hielo intracelulares,
de crecimiento frecuentemente limitado por la acción de proteínas anticongelantes (Ramløv et
al., 1996; Wharton, 2011). También resulta letal, en muchos casos, el crecimiento de cristales
de hielo en el espacio intercelular, en especial, para complejos tejidos multicelulares (Benson,
2008). Esta localización es, en realidad, la más proclive a la formación de hielo, pues la
concentración de los fluidos extracelulares es siempre menor que la del citoplasma. Esto facilita
la formación y crecimiento del hielo doblemente, ya que, cuanto menor es la concentración de
solutos, mayor es la temperatura de equilibrio de congelación (y también la temperatura de
nucleación de cristales de hielo) y menor la viscosidad de los fluidos, lo que, a su vez, facilita
el crecimiento de los núcleos de hielo inicialmente formados. Se ha mencionado incluso un
papel activo de agentes nucleadores de hielo extracelulares, como coadyuvantes de este
fenómeno (Tomashow, 1998).
La deshidratación celular puede ser ocasionada por la formación de cristales de hielo tanto
intra como extra-celulares, ocasionando el correspondiente daño fisiológico (Meryman, 1966a,
b). Si bien se puede producir ruptura de las membranas celulares por la acumulación de hielo
(Levitt, 1980), la extrema deshidratación celular que la congelación ocasiona se considera que
es la principal responsable del daño celular (Levitt, 1980; Steponkus & Webb, 1992). A
temperaturas suficientemente negativas, el potencial químico del agua sólida es menor que el
de la líquida y esto ocasiona que el agua migre y se adicione a los cristales en crecimiento. Aún
si la formación de hielo es externa a las células, la diferencia de potencial hídrico ocasiona la
salida del agua (Mazur, 1963; Mazur et al., 1984) y la extrema deshidratación celular: la
formación de hielo extracelular, a temperaturas tan poco extremas como -10ºC, ocasiona la
salida del 90% del agua celular (Tomashow, 1998).
El agua que se incorpora al hielo procede, entre otras localizaciones, de aquella necesaria
para estabilizar proteínas y solutos, que pueden desnaturalizarse o precipitar en su ausencia, y
especialmente, para mantener la conformación de las membranas celulares. Dependiendo del
tejido y de la temperatura se han encontrado diversos tipos de lesiones en las membranas,
responsables del daño observado (Steponkus & Webb, 1992; Steponkus et al., 1993;
Tomashow, 1998). Es de destacar que el daño por deshidratación es también responsable de las
lesiones por frío en plantas a temperaturas bajas, pero a temperaturas sobre cero (Salinas,
2002).
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8. DISCUSIÓN GENERAL/GENERAL DISCUSSION
La formación de hielo no ocurre inmediatamente cuando se alcanza la temperatura de
equilibrio de congelación, sino que este proceso está controlado cinética y no
termodinámicamente. Así es necesario un paso previo de nucleación que ocurre con creciente
probabilidad (en la región cercana al punto de congelación) a medida que la temperatura
disminuye (Meryman, 1966b; Franks, 1972; Mazur, 2004). La formación de cristales de hielo
intracelulares puede ocurrir por nucleación homogénea (sin que los cristales de hielo se formen,
en el enfriamiento del citoplasma, alrededor de partículas preexistentes, nucleadoras) o por
nucleación heterogénea (mediada por partículas nucleadoras). La temperatura de nucleación
homogénea del agua pura es de -38ºC. Es decir, que por debajo de esta temperatura la
formación de hielo sería espontánea. Más aún, en el citoplasma celular, multitud de agregados
moleculares y elementos microestructurales pueden dar lugar a la formación de núcleos de
hielo. Por consiguiente, esta temperatura sería, en la práctica, aún mayor. Así, el enfriamiento
de células y tejidos hasta la temperatura de vitrificación requiere un comportamiento
significativamente diferente del que exhibe el agua pura.
La transición vítrea de los sistemas acuosos puede ser concebida como el proceso
correspondiente del agua pura, desplazada hacia temperaturas más altas por el efecto de
reducción de la movilidad inducida por las sustancias en solución. De hecho, la misma
temperatura de transición vítrea del agua es difícil de medir, debido a las complicaciones
asociadas a realizar medidas experimentales en la región líquida metaestable cercana a TG que
llega hasta los 240 K (-33ºC) y es usualmente denominada como “inaccesible” (Angell, 2008,
2011). Gyan Johari, Erwin Mayer y Andreas Hallbrucker (Johari et al., 1990; Johari, 2003,
2005; Kohl et al., 2005 a,b) propusieron en diversos estudios y publicaciones el valor de 136 K
(-137ºC) para la TG del agua pura. Sin embargo, posteriormente, el prestigioso investigador del
estado vítreo Charles Austen Angell, propuso un valor más alto: 165 K (-108ºC) (Velikov et al,
2001; Angell, 2004; Yue & Angell, 2004, 2005). Esta controversia ha sido replanteada por
otros autores, pero el valor de 136 K parece el más probable, según las consideraciones más
recientes (Capaccioli & Ngai, 2011). A pesar de los intentos de Angell para reconciliar estos
diferentes puntos de vista (Angell, 2008), también han sido propuestos valores tan altos como
228 K (-45ºC) (Swenson & Teixeira, 2010) y 205 K (-68ºC), obtenido recientemente, de la
extrapolación de datos de soluciones de solutos orgánicos (McCartney & Sadtchenko, 2013).
Este baile de temperaturas de transición vítrea del agua pura, publicados en unos pocos años,
dan una imagen del furioso debate existente sobre el estado vítreo del agua. Por una parte, se
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8. DISCUSIÓN GENERAL/GENERAL DISCUSSION
puede observar que es una cuestión que está aún por resolver de una manera completamente
satisfactoria. Por otra parte, se observa que es un tema candente en la actualidad, ya que las
publicaciones de estudios sobre esta materia tienen lugar en fechas contemporáneas.
En relación con los estudios presentados en este trabajo, llaman la atención los valores de TG
propuestos para el agua pura. Como se ha indicado, la interpretación más extendida implica que
la transición vítrea de sistemas acuosos ocurriría a temperaturas crecientes, a medida que la
concentración de solutos, relativa a la del agua, aumenta (por ejemplo, Roos & Karel, 1991;
Arvanitoyannis et al., 1993). Así, las TG de todos los sistemas biológicos y soluciones aquí
estudiados deben, necesariamente, tener valores más altos que la del agua pura. Los valores de
TG medidos y reportados en este trabajo, varían entre los -120ºC y -88ºC para las soluciones de
crioconservación, y entre los -118ºC y -88ºC encontrados para tejidos biológicos preparados
para la crioconservación (Capítulos 4 y 6). Estos valores son muy cercanos a los -137ºC más
aceptados, lo que es algo sorprendente, ya que las concentraciones de solutos de estos sistemas
son muy altas. Más sorprendentes aún son las propuestas para el agua pura de valores tan altos
como -108ºC, -68ºC ó -45ºC, claramente incompatibles con nuestros datos, según la
interpretación aceptada de este fenómeno.
El valor de TG para el agua representa una temperatura límite, bajo la cual el
almacenamiento indefinido de material crioconservado sería seguro. A pesar de ello, la
probabilidad que fenómenos tales como la formación de hielo puedan suceder, en un periodo de
tiempo limitado, se considera muy baja hasta 20ºC por encima de TG (Mishima & Stanley,
1998). Actualmente se considera seguro, fuera de toda discusión, el almacenamiento en NL, a
-196ºC. Por el contrario, el almacenamiento en ultracongeladores tradicionales (a -80ºC), es
controvertido, pues no se garantizaría el estado vítreo de los sistemas almacenados. La
aceptación de mayores temperaturas para la transición vítrea del agua entraría en conflicto con
estas consideraciones.
El empleo de temperaturas intermedias, alrededor de -150ºC (propias del vapor de NL o de
algunos congeladores de temperaturas extremas), sería considerado también como seguro, por
debajo de la TG del agua (y desde luego, de las concentradas soluciones citoplasmáticas), si
bien, como se refleja también en este trabajo (Capítulo 6), esta temperatura podría ser
demasiado alta para evitar reorganizaciones moleculares dentro del estado vítreo, a prolongados
tiempos de almacenamiento. En estudios realizados con semillas ortodoxas, se ha determinado
que la temperatura “segura” de conservación (a aquella que no habrá deterioro a largo plazo)
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8. DISCUSIÓN GENERAL/GENERAL DISCUSSION
estaría a 70ºC por debajo de la TG (see Pritchard & Dickie, 2003). El efecto de estas
transiciones o reorganizaciones sobre la viabilidad y propiedades del material almacenando es
básicamente desconocido, si bien podría ser responsable del deterioro y la dificultad de
almacenamiento en crioconservación observado en algunos sistemas (Walters et al., 2004).
Desde que la acción crioprotectora del glicerol fue descubierta por Polge (Polge et al., 1949;
Lovelock & Polge, 1954), y posteriormente el efecto del dimetil sulfóxido (DMSO) (Lovelock,
1954; Lovelock & Bishop, 1959; Snedeker & Gaunya, 1970), una gran variedad de compuestos
potencialmente crioprotectores han sido investigados, si bien glicerol y DMSO siguen siendo
los más utilizados (Morris, 1980; Kartha, 1984). Debe tratarse de sustancias hidrosolubles y
presentar nula o baja toxicidad (Lovelock, 1954; Lovelock & Bishop, 1959). Por su modo de
acción, los crioprotectores pueden ser divididos en permeantes (que atraviesan la membrana
celular y pueden actuar intracelularmente) y los no permeantes (que no pueden cruzar la
membrana, y solo actúan extracelularmente).
Los crioprotectores permeantes, tales como glicerol, metanol, DMSO, etilenglicol y
propilenglicol, son sustancias de bajo peso molecular que penetran en la célula y actúan
protegiendo el citoplasma de la formación de hielo y sus estructuras internas de sus efectos
derivados (Benson, 2008). Estas sustancias disminuyen la temperatura de congelación
intracelular y aumentan la viscosidad de la solución, impidiendo la formación de cristales de
hielo (Merymn & Williams, 1980, 1985; Woods et al., 1999). Adicionalmente, son sustancias
que actúan como tampón osmótico durante el enfriamiento y calentamiento, contribuyendo a la
protección de las membranas celulares (Pegg, 1984). Los crioprotectores no permeantes son
sustancias frecuentemente de mayor peso molecular que, a pesar de no penetrar en el interior de
las células, tienen una acción protectora extracelular. Tal es el caso de la sacarosa (Benson,
2008). Se ha recomendado emplear una mezcla de sustancias crioprotectoras penetrantes y no
penetrantes, para aprovechar los efectos de ambos tipos de comportamientos (Fahy et al.,
1984).
El aumento de la concentración de solutos extracelulares desciende el potencial osmótico y,
para mantener la igualdad del potencial hídrico a ambos lados de la membrana, la
correspondiente cantidad de agua debe salir de las células, lo que da lugar a deshidratación
intracelular. La reducción de la cantidad de agua en el citoplasma se traduce en un aumento de
la concentración de los solutos intracelulares, con un efecto similar al de la penetración de
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8. DISCUSIÓN GENERAL/GENERAL DISCUSSION
agentes crioprotectores, en cuanto a aumento de la viscosidad celular, con el consiguiente
impedimento de la nucleación de hielo e inducción de la vitrificación (Benson, 2008).
Esta estrategia puede llevarse a cabo de manera aislada, por ejemplo, en los protocolos
donde se emplea la solución PSV3, que solo contiene sacarosa (Nishizawa et al., 1993;
Matsumoto et al., 1995; Makowska et al., 1999), o de manera combinada con crioprotectores
penetrantes. Tal es el caso en los protocolos donde se emplea PVS1 (Uragami et al., 1989,
Uragami, 1990), PVS2 (Sakai et al., 1990; Niwata, 1995; Sakai, 2003), y PVS4 (Sakai, 2000).
El caso del DMSO es especial, pues no solo atraviesa la membrana celular, sino que facilita
el que otras sustancias la atraviesen (Lovelock, 1954; Lovelock & Bishop, 1959). Esto no
siempre es deseable, en el contexto de la crioconservación, pues un efecto secundario de su uso
es la posibilidad de que alguna sustancia con capacidad mutagénica pueda atravesar la
membrana celular y nuclear con su ayuda, y causar alteraciones genómicas (Maclean & Hall,
1987, Chetverikova, 2011). El DMSO también podría interaccionar electrostáticamente con las
regiones hidrofílicas de los fosfolípidos de la membrana, evitando que su fluidez disminuyera a
temperaturas por debajo de 5ºC, lo que se ha relacionado con lesiones celulares provocadas por
frío (Kumamaru et al., 2010).
La viscosidad de las soluciones está directamente relacionada con la probabilidad de su
vitrificación. El empleo de diferentes crioprotectores (permeantes y no permeantes) en altas
concentraciones tiene como resultado tanto el aumento de la viscosidad como de la temperatura
de transición vítrea de las soluciones, como se ha visto en el estudio termodinámico recogido
en el Capítulo 6 (Towill & Jarret, 1992; Saragusty & Arav, 2011), características que aumentan
la probabilidad de vitrificación.
Algunos agentes crioprotectores, como el glicerol, tienen otro efecto adicional: la protección
directa de proteínas y membranas celulares frente a la deshidratación. Estos agentes, de tamaño
y características moleculares similares al agua, son capaces de sustituirla en las posiciones
donde, en condiciones de hidratación normales, se requerirían moléculas de agua para mantener
la conformación nativa (Charron et al., 2002; Mendes et al., 2004).
Los crioprotectores son esenciales para el éxito de la crioconservación de materiales
biológicos pero también son, de una manera u otra, prejudiciales para las células cuando son
utilizados en altas concentraciones. El uso combinado de diferentes crioprotectores para
conseguir este efecto permite reducir la concentración individual de cada uno, lo que disminuye
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8. DISCUSIÓN GENERAL/GENERAL DISCUSSION
la toxicidad global (Fahy, 1986). Los mismos procesos de añadir y eliminar las sustancias
crioprotectoras a los tejidos generan un estrés osmótico al material biológico y a las membranas
celulares que puede repercutir en su viabilidad.
Así, la reducción del contenido de agua (o el aumento de la concentración de solutos) es uno
de los principales factores determinantes del éxito de los procesos de crioconservación, como
se ha mostrado en los estudios presentados en los Capítulos 4 y 5. La presencia de hielo
intracelular, aun cuando la velocidad de enfriamiento empleada fue alta, en tejidos en que el
contenido acuoso era relativamente elevado, es evidenciada por las micrografías de cryo-SEM
presentadas en los mencionados capítulos, y confirmada por los experimentos de DSC
realizados en condiciones similares. La cantidad total de hielo y el tamaño de sus cristales en el
citoplasma son menores a medida que disminuye la cantidad de agua o, lo que es equivalente,
aumenta la concentración de solutos. Cuando este contenido de agua alcanza un determinado
umbral, la presencia de hielo no puede ser detectada por cryo-SEM o por DSC.
Otros dos factores, altamente interrelacionados, han sido destacados como de suma
importancia (Yavin & Arav, 2007): la velocidad de enfriamiento y calentamiento, y el volumen
de los especímenes.
Al respecto de la velocidad de cambio de temperatura, los procesos de crioconservación se
podrían dividir en aquellos que se llevan a cabo a velocidades relativamente lentas y los que
requieren velocidades lo más altas posibles (Sakai & Engelmann, 2007; Benson, 2008). En el
primer caso, se trata de procedimientos que aprovechan la formación de hielo extracelular para
extraer el agua intracelular (Rall et al., 1978; Mazur et al., 1981). Para evitar que, durante este
proceso, se forme hielo en el citoplasma, el enfriamiento debe ser inicialmente lento. Sin
embargo, como este proceso suele ir asociado a la adición de crioprotectores potencialmente
tóxicos, una velocidad excesivamente reducida daría lugar a una larga permanencia de estos
agentes en las células, a temperaturas a las que esta toxicidad puede ser más acusada (Muldrew
& McGann, 1994). La velocidad óptima de enfriamiento es específica para cada tipo de célula
y está directamente determinada por parámetros como el tamaño de las células, relación
superficie/volumen, permeabilidad de la membrana plasmática al agua y los crioprotectores,
grado de deshidratación celular y la relación entre la temperatura y la permeabilidad de la
membrana. Una vez alcanzado el grado de deshidratación requerido, en una segunda etapa el
proceso de enfriamiento se realiza a alta velocidad.
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8. DISCUSIÓN GENERAL/GENERAL DISCUSSION
Sin embargo, los métodos de crioconservación que han sido estudiados en este trabajo no
contemplan la formación de hielo extracelular. La crioconservación sin presencia de hielo fue
desarrollada inicialmente para células animales (Fahy, 1986) y posteriormente extendida a las
vegetales por Sakai (2004). Los métodos en los que se induce la vitrificación mediante
deshidratación celular y enfriamiento rápido son empleados ventajosamente en tejidos
heterogéneos complejos, para los que sería difícil o imposible encontrar velocidades de
enfriamiento óptimas para los distintos tipos de células (Meryman, 1966a; Benson, 2004,
2008). Se ha comprobado que tejidos de muy diversas especies vegetales toleran la
deshidratación, responden bien a los estreses osmóticos y soportan el enfriamiento rápido en
NL, con poca o ninguna pérdida de viabilidad (Yamada et al., 1991; Niino et al., 1992;
Matsumoto et al., 1994; Reinhoud, 1996; Senulka et al., 2007).
En los tres métodos que han sido considerados aquí (vitrificación-droplet -Capítulo 3 y 4-,
vitrificación -Capítulo 3- y encapsulación-deshidratación -Capítulo 5), la reducción del
contenido en agua es llevado a cabo mediante deshidratación osmótica mediante
crioprotectores o mediante evaporación por flujo de aire, previamente al inicio del
enfriamiento. Así, la velocidad óptima para este proceso sería la más rápida posible (Sakai &
Engelmann, 2007; Kaczmarczyk et al., 2011).
Es de destacar que en estos métodos, como se ha visto en el Capítulo 3, el enfriamiento y el
calentamiento son procesos “simétricos”, y tan imprescindible para la supervivencia es el
enfriamiento rápido como el calentamiento rápido (Meryman, 1966b). Tanto la fase de
deshidratación como la de rehidratación tienen lugar a temperatura ambiente y los procesos de
cambio de temperatura tienen un mayor grado de éxito cuanto más rápidamente ocurren
(Uchendu & Reed, 2008). Esto es debido a que, a pesar de la elevada deshidratación inducida y
de sus efectos sobre la temperatura de equilibrio de congelación, la movilidad molecular y la
temperatura de transición vítrea, aún existen zonas de temperatura que deben ser atravesadas
(tanto durante el proceso de enfriamiento como en el de calentamiento) en las cuales la
formación de hielo es posible. Así, un factor importante es la permanencia del sistema en esas
condiciones donde la probabilidad de formación de hielo aumenta con el tiempo, durante el
menor intervalo posible.
Los fenómenos de formación de hielo en condiciones de alta concentración de solutos y
elevada viscosidad y a temperaturas intermedias entre Tf y TG están aún bajo estudio y la
variación de la probabilidad de formación de hielo con la temperatura es poco conocida. Sin
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8. DISCUSIÓN GENERAL/GENERAL DISCUSSION
embargo, se considera que la fase de enfriamiento podría implicar un alto riesgo de nucleación
de hielo, a temperaturas más cercanas a TG, pero el crecimiento de estos cristales sería difícil en
esas condiciones. Sin embargo, sería en el proceso posterior de calentamiento donde estos
núcleos ya formados podrían, a temperaturas ya más cercanas a Tf, crecer, causando daños a las
células (Rall & Fahy, 1985; Sakai, 1997; Sakai & Engelmann, 2007).
Como se ha visto en el Capítulo 3, el procedimiento más habitual es el que emplea la
inmersión en nitrógeno líquido, para el enfriamiento, y en baños a temperaturas entre 20 y 40ºC
para el calentamiento, dando lugar a velocidades similares (pero de signo opuesto), pues los
gradientes térmicos que dan lugar a estas velocidades son equivalentes. Sin embargo, existen
una gran variedad de procedimientos para acelerar estos procesos. Por ejemplo, el factor
limitante de la velocidad de enfriamiento es el llamado efecto Leidenfrost, la formación de una
capa de nitrógeno gas, de baja conductividad térmica, y que reduce significativamente el
intercambio de calor con el nitrógeno líquido. Los procedimientos explorados para de evitar
este problema incluyen la inmersión en nitrógeno líquido en equilibrio con la fase sólida
(González-Benito et al., 2003), pero también el empleo de otros fluidos criogénicos que no
presentan este problema, tales como freón, o propano o etano líquido. La eliminación de la capa
gaseosa y la aceleración del intercambio térmico se han facilitado mediante técnicas de
inmersión forzada en el nitrógeno líquido (incluso mediante disparo de la muestra), y también
se recurre al peculiar diagrama de fase del agua para, aumentando la presión hidrostática hasta
2000 atmósferas, conseguir un enfriamiento acelerado. Sin embargo, estos procedimientos no
suelen ser empleados en la crioconservación rutinaria, pues implican complicaciones técnicas,
equipos o suministros costosos o no siempre accesibles, y en general solo se recurre a ellos en
determinadas tareas de experimentación científica, por ejemplo, en la obtención de muestras
vitrificadas para la observación por microscopía electrónica sin la formación de artefactos
debidos al hielo formado.
Si bien la velocidad de enfriamiento y calentamiento fue estudiada desde muy temprano
(Luyet & Gehenio, 1940; Mazur et al., 1957; Meryman & Kafig, 1955), la dedicación
experimental a esta tarea ha sido escasa teniendo en cuenta su importancia. Además la
complejidad de los efectos observados dio lugar a dificultades en la interpretación de la
relación velocidad-viabilidad. En ocasiones se ha considerado que las velocidades de cambio
térmico muy altas podrían dar lugar a daños en los especímenes, por la generación de
gradientes térmicos con bruscas variaciones de volumen. Sin embargo, los procesos que aquí
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8. DISCUSIÓN GENERAL/GENERAL DISCUSSION
son considerados tienen lugar en muestras de tamaño lo suficientemente reducido para que el
equilibrio térmico ocurra de manera rápida y los gradientes térmicos son minimizados. De
hecho no hay referencias de daños por esta causa en sistemas vegetales en crioconservación,
aun cuando se han recurrido a técnicas para aumentar la velocidad de enfriamiento, como es el
mencionado empleo de mezclas de nitrógeno líquido y sólido (González-Benito et al., 2003).
Esto nos lleva al tercer factor de importancia, el tamaño del espécimen a ser crioconservado.
El éxito de los procesos de crioconservación es mayor a menor tamaño de éste, como se puede
ver en los Capítulos 4, 5 y 6, por múltiples razones, algunas en relación con el proceso de
deshidratación y de tratamiento con crioprotectores. La difusión de éstos hacia el interior de las
células (y su salida posterior) es más rápida cuanto menor sea el tamaño de muestra. Y lo
mismo ocurre con los procesos de deshidratación y rehidratación, sea cuál sea el método
empleado. La relación superficie/volumen (que, variando la superficie de manera cuadrática y
el volumen, cúbica, es claramente, inversamente proporcional al diámetro equivalente) permite
racionalizar este comportamiento. Esto implica que las situaciones de posible daño fisiológico
ocasionadas por la presencia de crioprotectores potencialmente tóxicos o mutagénicos, de
estreses asociados a la deshidratación y a la alta concentración de solutos, pueden ser limitadas
a menores intervalos de tiempo. Hay que recordar que estos procesos de estrés y posible daño
estarían también detenidos en condiciones de vitrificación, con lo que el periodo en el que
pueden deteriorarse los sistemas a ser crioconservados incluye solo aquel del inicio y el final
del proceso, y no durante el almacenamiento, por largo que éste sea (Meryman, 1966; Sakai &
Engelmann, 2007; Kaczmarczyk et al., 2011).
El tamaño también influye en la velocidad de cambio térmico. De hecho, la manera más
eficiente de aumentar la velocidad de los procesos tanto de enfriamiento como de
calentamiento, es reducir el espécimen a tratar al volumen lo más pequeño posible (incluyendo
cualquier contenedor, encapsulado o solución circundante) (Arav, 1992; Arav et al., 2002;
Yavin & Arav, 2007). Como se ha podido observar en los Capítulos 3 y 5, aquellos
procedimientos donde los especímenes y sus contenedores tenían un tamaño global menor,
podían ser enfriados y calentados a mayores velocidades. La difusión de calor es un proceso
proporcional, entre otros factores, al espesor del material que debe ser atravesado por el flujo
térmico. Así, un el aumento del volumen del espécimen se traduce en el drástico incremento del
tiempo requerido para que su temperatura pueda ser alterada.
119
8. DISCUSIÓN GENERAL/GENERAL DISCUSSION
El tamaño de muestra, finalmente, tiene una relación directa con la probabilidad de
formación de hielo. Como se ha explicado, se trata de un proceso estocástico, en el que las
moléculas de agua se reorganizan para formar núcleos de hielo propulsadas por movimientos
Brownianos. La probabilidad de que este movimiento “al azar” dé lugar a un núcleo de hielo
que pueda generar un cristal es proporcional al número de moléculas de agua. Así, los
especímenes pequeños son menos proclives a la formación de hielo, en las mismas condiciones
que los mayores.
La crioconservación de organismos de gran tamaño, tales como relativamente grandes
órganos animales o animales completos, a parte de otros diversos obstáculos, se encuentra,
como uno de los principales impedimentos, con las dificultades derivadas del tamaño mucho
mayor de los especímenes, en comparación por ejemplo, con los estudiados en este trabajo, que
varían desde los 0,7 mg de ápices de menta deshidratados (Capítulo 4), a los 3,9 mg de los
igualmente deshidratados ápices de ajo (Capítulo 4), y a los 10 mg de cuentas de alginato,
incluyendo a ápices, completamente hidratadas (Capítulo 5). La gran diversidad de tipos
celulares a ser conservados y sus diversos requerimientos, así como la necesidad de alcanzar un
alto grado de recuperación en todos estos tipos celulares, se añaden a las mencionadas
dificultades (Meryman, 1966a).
Una de las cuestiones a tener en cuenta en el diseño de procedimientos para la
crioconservación es que los diversos tejidos y células de distintas especies y cultivares
presentan muy diversas respuestas frente a los estreses inducidos en estos procesos. Por una
parte, se procura diseccionar el material a crioconservar, reduciendo su tamaño al mínimo
estrictamente necesario para su posterior recuperación, en aras de trabajar con especímenes de
tamaño reducido, por las ventajas arriba indicadas. Sin embargo, esto implica desproteger estos
tejidos y exponerlos a fuertes estreses, agravados por los derivados de la manipulación de
grandes números de muestras, dilatados tiempos de espera y fluctuaciones de condiciones
ambientales. Como una solución de compromiso, se emplean el encapsulado mediante los
versátiles y completamente biocompatibles geles de alginato cálcico. Los efectos deletéreos de
las fluctuaciones incontroladas de contenido en agua, concentración de solutos y cambios
térmicos son apantallados por esta capa, básicamente formada por una red de polisacáridos
reteniendo agua.
En la actualidad, la crioconservación debe ser considerada como una técnica de gran
potencial para la conservación de recursos fitogenéticos. A pesar de ello, existe la necesidad de
120
8. DISCUSIÓN GENERAL/GENERAL DISCUSSION
profundizar en la investigación, tanto para mejorar los métodos existentes como para encontrar
otros nuevos, que se adapten a las necesidades de las diferentes especies que deben ser
conservadas.
121
9. CONCLUSIONES/CONCLUSIONS
9. CONCLUSIONS
- Mint shoot tip survival results suggest that the observed physiological response is
dependent upon the cryopreservation method and the cooling and warming rates associated.
This study demonstrates that faster thermal rates give rise to higher survival and recovery
percentages.
- Specimens of mint shoot tips at the final step of the droplet-vitrification cryopreservation
protocol become vitrified and no ice is formed, which is associated to the lack of damage in
the process and the resulting high viability. Meanwhile, shoot tips at previous steps of the
protocol show the formation of ice by both microscopic and calorimetric observations, which
is a determinant cause of damage leading to the observed null viability of these specimens.
The agreement between the results obtained by both cryo-SEM and DSC techniques is
relevant and the potential of its associated use is clearly shown.
conclude that the TG of commonly employed PVSs did not change with the wide
range of cooling and warming rates. The behavior of cryopreserved tissues might be, though,
different. Storage at very low temperature (LN, at -196ºC) would mean no risk due to
reorganization of the vitreous state. But higher temperatures, such as that of freezers or even
in LN vapor phase (at approx. -150ºC), might imply some reorganization after quench
cooling.
- Results obtained suggest the applicability of chitosan to the alginate beads employed in
cryopreservation, as the most important gel properties, such as its water interaction behaviour,
was found to be scarcely altered. The antimicrobial protection conferred by chitosan and their
9. CONCLUSIONES/CONCLUSIONS
antioxidant properties can be also of great interests, as often cultivation and growth after
cryopreservation can be impaired by microbial contamination or cellular oxidative damages.
10. REFERENCIAS/REFERENCES
10. REFERENCIAS
Ablett S, Darke AH, Izzard MJ, Lillford PJ. (1993) Studies of the glass transition in malto-
oligomers. In: The glassy state in foods. (JMV Blanshard & PJ Lillford). Nottingham
University Press. Nottingham, pp. 189-206.
Ait Barka E, Eullaffroy P, Clément C, Vernet G. (2004) Chitosan improves development, and
protects Vitis vinifera L. against Botrytis cinerea. Plant Cell Reports, 22:608-614.
Alberola N, Perez J, Tatibouet J, Vassoille R. (1983) Stability of the vitreous phase in water-
ethylene glycol mixtures studied by internal friction. The Journal of Physical Chemistry,
87:4264-4267.
Amborabé BE, Bonmort J, Fleurat-Lessard P, Roblin G. (2008) Early events induced by
chitosan on plant cells. Journal of Experimental Botany, 59:2317-2324.
Anbinder PS, Deladino L, Navarro AS, Amalvy JI, Martino MN. (2011) Yerba Mate Extract
Encapsulation with Alginate and Chitosan Systems: Interactions between Active
Compound Encapsulation Polymers. Journal of Encapsulation and Adsorption Sciences,
1:80-87.
Andres Y, Giraud L, Gerente C, Le-Cloiréc P. (2007) Antibacterial effects of chitosan
powder: Mechanisms of action. Environmental Technology, 28:1357-1363.
Angell CA. (1982) Supercooled water. In: Water-A comprehensive treatise. 7. (F Franks, ed.).
Plenum Press. New York, pp. 215-338.
Angell CA. (2000) Liquid fragility and the glass transition in water and aqueous solutions.
Chemical Reviews, 102:2627-2650.
Angell CA. (2004) Amorphous water. Annual Review of Physical Chemistry, 55:559-583.
Angell CA. (2008) Insights into phases of liquid water from study of its unusual glass-
forming properties. Science, 319:582-587.
Angell CA, Ngai KL, McKenna GB, McMillan PF, Martin SW. (2000) Relaxation in glass
forming liquids and amorphous solids. Journal of Applied Physics, 88:3113-3157.
Angell CA, Ogunl M, Slchlna WJ. (1982) Heat capacity of water at extremes of supercooling
and superheating. Journal of Physical Chemistry, 86:998-1002.
124
10. REFERENCIAS/REFERENCES
Angell CA, Sare JM, Sare EJ. (1978) Glass transition temperatures for simple molecular
liquids and their binary solutions. The Journal of Physical Chemistry, 82:2622-2629.
Angell CA, Shuppert J, Tucker JC. (1973) Anomalous properties of supercooled water. Heat
capacity, expansivity, and proton magnetic resonance chemical shift from 0 to -38º. The
Journal Physical Chemistry, 77: 3892-3899.
Angell CA, Smith DL. (1982) Test of the entropy basis of the Vogel-Tammann-Fulcher
equation. Dielectric relaxation of polyalcohols near Tg. The Journal of Physical Chemistry,
86:3845-3852.
Arav A. (1992) Vitrification of oocytes and embryos. In: Trends in embryo transfer. (A Lauria
& F Gandolfi, eds.). Portland Press. Cambridge, pp. 255-264.
Arav A, Yavin S, Zeron Y, Natan D, Dekel I, Gacitua H. (2002) New trends in gamete's
cryopreservation. Molecular and Cellular Endocrinology, 187:77-81.
Arvanitoyannis I, Blanshard JMV, Izzard MJ, Lillford PJ, Ablett S. (1993) Calorimetric study
of the glass transition occurring in aqueous glucose: fructose solutions. Journal of the
Science of Food and Agriculture, 63:177-188.
Arvy MP, Gallouin F. (2006) Especias, aromatizantes y condimentos. Ediciones Mundi-
Prensa. Madrid.
Ashmore SE. (1997) Status report on the development and application of in vitro techniques
for the conservation and use of plant genetic resources. International Plant Genetic
Resources Institute. Rome, pp. 11-18.
Bald WB. (1987) Quantitative Cryofixation. Hilger. Bristol, pp. 76-103.
Barber MS, Bertram RE, Ride JP. (1989) Chitin oligosaccharides elicit lignification in
wounded wheat leaves. Physiological and Molecular Plant Pathology, 34:3-12.
Benhamou N. (1992) Ultrastructural detection of -1,3-glucans in tobacco root tissues
infected by Phytophthora parasitica var. nicotianae using a gold-complexed tobacco -1,3-
glucanase. Physiological and Molecular Plant Pathology, 41:351-357.
125
10. REFERENCIAS/REFERENCES
Benhamou N, Kloepper JW, Tuzun S. (1998) Induction of resistance against Fusarium wilt of
tomato by combination of chitosan with an endophytic bacterial strain: ultrastructure and
cytochemistry of the host response. Planta, 204:153-168.
Benson EE. (1999) An introduction to plant conservation biotechnology. In: Plant
conservation biotechnology. (EE Benson, ed.). Taylor & Francis. London, pp. 3-10.
Benson EE. (2004) Cryoconserving algal and plant diversity: Historical perspectives and
future challenges. In: Life in the Frozen State. (B Fuller, N Lane & EE Benson, eds.). CRC
Press. London, pp. 299-328.
Benson EE. (2008) Cryopreservation theory. In: Plant cryopreservation: A practical guide.
(BM Reed, ed.). Springer. New York, pp. 15-32.
Benson EE, Reed BM, Brennan RM, Clacher KA, Ross DA. (1996) Use of thermal analysis
in the evaluation of cryopreservation protocols for Ribes nigrum L. germplasm.
CryoLetters, 17:347-362.
Berth G, Cölfen H, Dautzenberg H. (2002) Physicochemical and chemical characterisation of
chitosan in dilute aqueous solution. Progress in Colloid and Polymer Sciences, 119:50�–57.
Bhat S, Gupta SK, Tuli R, Khanuja SPS, Sharma S, Bagchi GD, Kumar A, Kumar S. (2001)
Photoregulation of adventitious and axillary shoot proliferation in menthol mint, Mentha
arvensis. Current Science, 80:878-881.
Bittelli M, Flury M, Campbell GS, Nichols EJ. (2011) Reduction of transpiration through
foliar application of chitosan. Agricultural and Forest Meteorology, 107:167-175.
Blanshard JM, Franks F. (1987) Ice crystallization and its control in frozen-food systems. In:
Food Structure and Behavior. (JMV Blanshard & PJ Lillford, eds.) Academic Press.
London, pp. 51-65.
Block W. (2003) Water status and thermal analysis of alginate beads used in cryopreservation
of plant germplasm. Cryobiology, 47:59-72.
Bouafia S, Jelti N, Lairy G, Blanc A, Bonnel E, Dereuddre J. (1996) Cryopreservation of
potato shoot tips by encapsulationdehydration. Potato Research, 39:69-78.
126
10. REFERENCIAS/REFERENCES
Brüning R, Samwer K. (1992) Glass transition on long time scales. Physical Review B,
46:318-322.
Buckley KA, Conway EJ, Ryan HC. (1958) Concerning the determination of total
intracellular concentrations by the cryoscopic method. Journal of Physiology, 143:236-245.
Cabrera JC, Messiaen J, Cambier P, van Cutsem P. (2006) Size, acetylation and concentration
of chitooligosaccharide elicitors determine the switch from defence involving PAL
activation to cell death and water peroxide production in Arabidopsis cell suspensions.
Physiologia Plantarum, 127:44-56.
Capaccioli S, Ngai KL. (2011) Resolving the controversy on the glass transition temperature
of water?. The Journal of Chemical Physics, 135:104504.
Carpenter JF, Dawson PE. (1991) Quantification of dimethyl sulfoxide in solutions and
tissues by high performance liquid chromatography. Cryobiology, 28:210-215.
Chambers HL. (1992) Mentha: genetic resources and the collection at USDA-ARS NCGR-
Corvallis. Lamiales News, 1:3-4.
Chang ZH, Baust JG. (1991) Physical aging of glassy state: DSC study of vitrified glycerol
systems. Cryobiology, 28:87-95.
Charron C, Kadri A, Robert M-C, Giegé R, Lorber B. (2002) Crystallization in the presence
of glycerol displaces water molecules in the structure of thaumatin. Acta
Crystallographica, D58:2060-2065.
Cheng X, Kaplan LA. (2003) Simultaneous analyses of neutral carbohydrates and amino
sugars in freshwaters with HPLC-PAD. Journal of Chromatographic Science, 41:434-438.
Chetverikova EP. (2011) Consequences of plant tissue cryopreservation (phenotype and
genome). Biophysics, 56:309-315.
Chua SP, Normah MN. (2011) Effect of preculture, PVS2 and vitamin C on survival of
recalcitrant Nephelium ramboutan-ake shoot tips after cryopreservation by vitrification.
Cryoletters, 32:506-515.
Chung YC, Wang HL, Chen YM, Li SL. (2003) Effect of abiotic factors on the antibacterial
activity of chitosan against waterborne pathogens. Bioresource Technology, 88:179-184.
127
10. REFERENCIAS/REFERENCES
Cole MD. (1992) The significance of the terpenoids in the Labiatae. In: Advances in labiate
science (RM Harley & T Reynolds, eds.). Royal Botanic Gardens. Kew, pp. 315-324.
Coviello T, Matricardi P, Marianecci C, Alhaique F. (2007) Polysaccharide hydrogels for
modified release formulations. Journal of Controlled Release, 119:5-24.
Craig S, Beaton CD. (1996) A simple cryo-SEM method for delicate plant tissues. Journal of
Microscopy, 182:102-105.
Cronquist A. (1981) An integrated system of classification of flowering plants. Columbia
University Press. New York.
Dam J, Schuck P. (2005) Sedimentation Velocity Analysis of Heterogeneous Protein-Protein
Interactions: Sedimentation Coefficient Distributions c (s) and Asymptotic Boundary
Profiles from Gilbert-Jenkins Theory. Biophysical Journal, 89:651-666.
Dangl JL, Jones JDG. (2001) Plant pathogens and integrated defence responses to infection.
Nature, 411:826-833.
Debenedetti PG, Stillinger FH. (2001) Supercooled liquids and the glass transition. Nature,
410:259-267.
Deladino L, Anbinder PS, Navarro AS, Martino MN. (2008) Encapsulation of natural
antioxidants extracted from Ilex paraguariensis. Carbohydrate Polymers, 71:126-134.
Dereuddre J, Scottez C, Arnaud Y, Duron M. (1990) Résistance d'apex caulinaires de
vitroplants de Poirier (Pyrus communis L. cv Beurré Hardy), enrobés dans l'alginate, à une
déshydratation puis à une congélation dans l'azote liquide: effet d'un endurcissement
préalable au froid. Comptes Rendus de l'Académie des Sciences, 10:317-323.
Draget KI. (2000) Alginates. In: Handbook of hydrocolloids (G Phillips & P Williams, eds.).
Woodhead. Cambridge, pp. 379-395.
Draget KI, Skjåk-Bræk G, Smidsrød O. (1997) Alginate based new materials. International
Journal of Biological Macromolecules, 21:47-55.
Draget KI, Taylor C. (2011) Chemical, physical and biological properties of alginates and
their biomedical implications. Food Hydrocolloids 25:251-256.
128
10. REFERENCIAS/REFERENCES
Dumet D, Block W, Worland R, Reed BM, Benson EE. (2000) Profiling cryopreservation
protocols for Ribes ciliatum using differential scanning calorimetry. CryoLetters, 21:367-
378.
Eaton P, Fernandes JC, Pereira E, Pintado ME, Xavier Malcata F. (2008) Atomic force
microscopy study of the antibacterial effects of chitosans on Escherichia coli and
Staphylococcus aureus. Ultramicroscopy, 108:1128-1134.
Ediger MD, Angel CA, Nagel SR. (1996) Supercooled liquids and glasses. Journal of
Physical Chemistry, 100:13200-13212.
El Ghaouth A, Arul J, Asselin A, Benhamou N. (1992) Antifungal activity of chitosan on
postharvest pathogens: induction of morphological and cytological alterations in Rhizopus
stolonifer. Mycological Research, 96:769-779.
El Ghaouth A, Arul J, Wilson C, Benhamou N. (1994) Ultrastructural and cytochemical
aspects of the effect of chitosan on decay of bell pepper fruit. Physiological and Molecular
Plant Pathology, 44:417-432.
El Hadrami A, Adam LR, El Hadrami I, Daayf F. (2010) Chitosan in Plant Protection. Marine
Drugs 8:968-987.
El Hassni M, El Hadrami A, Daayf F, Chérif M, Ait Barka E, El Hadrami I. (2004) Chitosan,
antifungal product against Fusarium oxysporum f. sp. albedinis and elicitor of defence
reactions in date palm roots. Phytopathologia Mediterranea, 43:195-204.
Elliott JAW, Prickett RC, Elmoazzen HY, Porter KR, McGann LE. (2007) A multisolute
osmotic virial equation for solutions of interest in biology. Journal of Physical Chemistry
B, 111:1775-1785.
Engelmann F. (1997) In vitro conservation methods. En Biotechnology and Plant Genetic
Resources: Conservation and Use. (JA Callow, BV Ford-Lloyd & HJ Newbury, eds.).
CAB International. Wellingford, pp. 119-162.
Engelmann F. (2004) Plant cryopreservation: progress and prospects. In vitro Cellular &
Developmental Biology-Plant, 40:427-433.
129
10. REFERENCIAS/REFERENCES
Engelmann F. (2008) Importance of cryopreservation for the conservation of plant genetic
resources. In: Cryopreservation of tropical plant germplasm (F Engelmann & H Takagi,
eds.). International Plant Genetic Resources Institute. Rome, pp. 8-20.
Engelmann F, González-Arnao MT, Wu Y, Escobar R. (2008) The development of
encapsulation dehydration. In: Plant Cryopreservation: A Practical Guide. (BM Reed, ed.).
Springer. New York.
Engelmann F, Takagi H. (2000) Cryopreservation of Tropical Plant Germplasm - Current
Research Progress and Applications. Tsukuba & International Plant Genetic Resources
Institute. Rome, p. 496.
Fabre J, Dereuddre J. (1990) Encapsulation-dehydration: A new approach to cryopreservation
of Solanum shoot- tips. CryoLetters, 11:413-426.
Fahn A. (1979) Secretory tissues in plants. Academic Press. London, pp. 158-222.
Fahy GM. (1986) Vitrification: a new approach to organ cryopreservation. Progress in
Clinical and Biological Research, 224:305-335.
Fahy, GM, MacFarlane DR, Angell CA, Meryman HT. (1984) Vitrification as an approach to
cryopreservation. Cryobiolology, 21:407-426.
Faoro F, Maffi D, Cantu D, Iriti M. (2008) Chemical-induced resistance against powdery
mildew in barley: the effects of chitosan and benzothiadiazole. BioControl, 53:387-401.
Flora Europaea (on line version) http://rbg-web2.rbge.org.uk/FE/fe.html (13/01/2013)
Franks F. (1972) Water, a comprehensive treatise. Plenum Press. New York.
Fujikawa S. (1980) Freeze-fracture and etching studies on membrane damage on human
erythrocytes caused by formation of intracellular ice. Cryobiology, 17:351-362.
Fujikawa S. (1981) The effect of various cooling rates on the membrane ultrastructure of
frozen human erythrocytes and its relation to the extent of haemolysis after thawing.
Journal of Cell Science, 49:369-382.
Fujita H. (1962) Mathematical Theory of Sedimentation Analysis. Academic Press. New
York.
130
10. REFERENCIAS/REFERENCES
Franks, F. (1986) Unfrozen water - yes - unfreezable water - hardly - bound water - certainly
not. CryoLetters, 7:207-207.
Fullerton GD, Keener CR, Cameron IL. (1994) Correction for solute/solvent interaction
extends accurate freezing point depression theory to high concentration range. Journal of
Biochemistry Biophysical Method, 29:217-235.
Gao C, Zhou GY, Xu Y, Hua TC. (2005) Glass transition and enthalpy relaxation of ethylene
glycol and its aqueous solution. Thermochimica Acta, 435:38-43.
George M, Abraham TE. (2006) Polyionic hydrocolloids for the intestinal delivery of protein
drugs: alginate and chitosan-a review. Journal of Controlled Release, 114:1-14.
González-Arnao MT, Engelmann F. (2006) Cryopreservation of plant germplasm using the
encapsulation-dehydration technique: review and case study on sugarcane. CryoLetters,
27:155-168.
González-Arnao MT, Moreira T, Urra C. (1996) Importance of pre growth with sucrose and
vitrification for the cryopreservation of sugarcane apices using encapsulation-dehydration.
CryoLetters, 17:141-148.
González-Arnao MT, Panta A, Roca WM, Escobar RH, Engelmann F. (2008) Development
and large scale application of cryopreservation techniques for shoot and somatic embryo
cultures of tropical crops. Plant Cell, Tissue and Organ Culture, 92:1-13.
González-Arnao MT, Urra C, Engelmann F, Ortiz R, Fe C. (1999) Cryopreservation of
encapsulated sugarcane apices: effect of storage temperature and storage duration.
CryoLetters, 20:347-352.
González-Benito ME, Clavero-Ramírez I, López-Aranda JM. (2004) Review. The use of
cryopreservation for germoplasm conservation of vegetatively propagated crops. Spanish
Journal of Agricultural Research, 2:341-351.
González-Benito ME, Mendoza-Condori VH, Molina-García AD. (2007) Cryopreservation of
in vitro shoot apices of Oxalis tuberosa Mol. CryoLetters, 28:23-32.
González-Benito ME, Núñez-Moreno Y, Martín C. (1998) A protocol to cryopreserve nodal
explants of Antirrhium microphyllum by encapsulation-dehydratation. CryoLetters,
19:225-230.
131
10. REFERENCIAS/REFERENCES
González-Benito, M.E., Salinas, P., Amigo, P. 2003. Effect of seed moisture content and
cooling rate in liquid nitrogen on legume seed germination and seedling vigour. Seed
Science and Technology, 31:423-434.
Graybill JR, Horton HF. (1969) Limited fertilization of steelhead trout eggs with
cryopreserved sperm. Journal of the Fisheries Board of Canada, 26:1400-1404.
Goy RC, Britto D, Assis OBG. (2009) A review of the antimicrobial activity of chitosan.
Polímeros, 19:241-247.
Guignon B, Torrecilla JS, Otero L, Ramos AM, Molina-García AD, Sanz PD. (2008) The
initial freezing temperature of foods at high pressure. Critical Reviews in Food Science and
Nutrition, 48:328-340.
Gupta R. (1991) Agrotechnology of medicinal plants. In: The medicinal plant industry. (ROB
Wijessekera, ed.). CRC Press. Boca Raton, pp. 43-57.
Halmagyi A, Deliu C, Coste A. (2005) Plant regrowth from potato shoot tips cryopreserved
by a combined vitrification-droplet method. CryoLetters, 26:313-322.
Halmagyi A, Deliu C, Isac V. (2010) Cryopreservation of Malus cultivars: comparison of two
droplet protocols. Science Horticulture, 124:387-392.
Harding K. (2004) Genetic integrity of cryopreserved plant cells: A review. CryoLetters,
18:217-230.
Harding SE, Schuck P, Abdelhameed AS, Adams G, Kök MS, Morris GA. (2011) Extended
Fujita Approach to the Molecular Weight Distribution of Polysaccharides and other
Polymeric Systems. Methods, 54:136-144.
Hashimoto T, Tasaki Y, Harada M, Okada T. (2011) Electrolyte-doped ice as a platform for
atto- to femtoliter reactor enabling zeptomol detection. Analitical Chemistry, 83:3950-
3956.
Henshaw GG, Stamp JA, Westcott JJ. (1980) Tissue culture and germoplasm storage. In:
Plant cell cultures: results and perspectives. (F Sala, R Parisi, R Cella & O Cifferi, eds.).
Elsevier. Amsterdam, pp. 277-282.
132
10. REFERENCIAS/REFERENCES
Hills BP, Cano C, Belton PS. (1991) Proton NMR relaxation studies of aqueous
polysaccharide systems. Macromolecules, 1:2944-2950.
Hills BP, Takacs SF, Belton PS. (1990) A new interpretation of proton NMR relaxation time
measurements of water in food. Food Chemistry, 37:95-111.
Hills BP, Tang H, Belton PS, Khaliq A, Harris RK. (1998) NMR Oxygen-17 studies of the
state of water in a saturated sucrose solution. Journal Molecular Liquids, 75:45-59.
Hirai D, Saki A. (1999) Cryopreservation of in vitro-grown axillary shoot-tip meristems of
mint (Mentha spicata L) by encapsulation vitrification. Plant Cell Reports, 19:150-155.
Hirano S, Nakahira T, Nakagawa M, Kim SK. (1999) The preparation and applications of
functional fibres from crab shell chitin. Journal of Biotechnology, 70:373-377.
Hubálek Z. (2003) Protectants used in the cryopreservation of microorganisms. Cryobiology,
46:205-229.
Hyland LL, Taraban MB, Hammouda B, Bruce YY. (2011) Mutually reinforced
multicomponent polysaccharide networks. Biopolymers, 95:840-851.
Iglesias HA, Chirife J. (1978) Delayed crystallization of amorphous sucrose in humidified
freeze dried model systems. Journal of Food Technology, 13:137-144.
International Board for Plant Genetic Resources. (1982) The design of seed storage facilities
for genetic conservation. IBPGR. Rome.
Iriti M, Picchi V, Rossoni M, Gomarasca S, Ludwig N, Garganoand M, Faoro F. (2009)
Chitosan antitranspirant activity is due to abscisic acid-dependent stomatal closure.
Environmental and Experimental Botany, 66:493-500.
Islam M, Dembele D, Keller ER. (2005) Influence of explant, temperature and different culture
vessels on in vitro culture for germplasm maintenance of four mint accessions. Plant Cell,
Tissue and Organ Culture, 81:123-130.
Johari GP. (2003) Water�’s T-endotherm, sub-T peak of glasses and T of water. The Journal of
Chemical Physics, 119:2935.
Johari GP. (2005) Water�’s size-dependent freezing to cubic ice. The Journal of Chemical
physics, 122:194504.
133
10. REFERENCIAS/REFERENCES
Johari GP, Astl G, Mayer E. (1990) Enthalpy relaxation of glassy water. Journal of Chemical
Physics, 92:809-810.
Jouve L, Hoffmann L, Hausman JF. (2004) Polyamine, carbohydrate, and proline content
changes during salt stress exposure of aspen (Populus tremula L.): involvement of
oxidation and osmoregulation metabolism. Plant Biology, 6:74-80.
Kaczmarczyk A, Rokka VM, Keller ERJ. (2011) Potato shoot tip cryopreservation. A review.
Potato Research, 54:45-79.
Kami D, Kasuga J, Arakawa K, Fujikawa S. (2008) Improved cryopreservation by diluted
vitrification solution with supercooling-facilitating flavonol glycoside. Cryobiology,
57:242-245.
Kartha KK. (1984) Culture of shoot meristems: Pea. In: Cell Culture and Somatic Cell
Genetics of Plants. Vol 1: Laboratory Procedures and their Applications. (IK Vasil, ed.).
Academic Press. Orlando, pp. 106-110.
Kartha KK, Leung NL, Mroginski LA. (1982) In vitro growth-responses and plant-
regeneration from cryopreserved meristems of cassava (Manihot esculenta Crantz),
Zeitschrift fur Pflanzenphysiologie, 107:133-140.
Kauzmann W. (1948) The nature of the glassy state and the behavior of liquids at low
temperatures. Chemistry Review, 43: 219-256.
Keller ERJ. (2002) Cryopreservation of Allium sativum L. (Garlic). In: Cryopreservation of
Plant Germplasm II. (LE Towil & PS Bajaj, eds.). Springer. Berlin, pp. 37-47.
Keller ERJ, Dreiling M. (2003) Potato cryopreservation in Germany - using the droplet
method for the establishment of a new large collection. Acta Horticulturae, 623:193-200.
Khor E, Shiong Loh C. (2005) Artificial seeds. In: Applications of Cell Immobilisation
Biotechnology. (V Nedovic & R Willaert, eds.). Springer. Amsterdam, pp. 527-537.
Kim HH, Lee JK, Yoon JW, Ji JJ, Nam SS, Hwang HS, Cho EG, Engelmann F. (2006a)
Cryopreservation of garlic bulbil primordia by the droplet-vitrification procedure.
CryoLetters, 27:143-153.
134
10. REFERENCIAS/REFERENCES
Kim HH, Yoon JW, Park YE, Cho EG, Sohn JK, Kim TS, Engelmann F. (2006b)
Cryopreservation of potato cultivated varieties and wild species: critical factors in droplet
vitrification. CryoLetters, 27:223-234.
Kim W-T, Chung H, Shin II-S, Yam KL, Chung D. (2008) Characterization of calcium
alginate and chitosan-treated calcium alginate gel beads entrapping allyl isothiocyanate.
Carbohydrate Polymers, 71:566-573
King AH. (1983) Brown seaweed extracts (Alginates). Food Hydrocolloids, 2:115-188.
Knorr D. (1984) Use of chitinous polymers in food. Food Technology, 38:85-94.
Kohl I, Bachmann L, Hallbrucker A, Mayer E, Loerting T. (2005a) Liquid-like relaxation in
hyperquenched water at <= 140 K. Physical Chemistry Chemical Physics, 7:3210-3220.
Kohl I, Bachmann L, Mayer E, Hallbrucker A, Loerting T. (2005b) Water Behaviour: Glass
transition in hyperquenched water?. Nature, 435:E1-E1.
Kong M, Chen XG, Xing K, Park HJ. (2010) Antimicrobial properties of chitosan and mode
of action: A state of the art review. International Journal of Food Microbiology, 144:51-63.
Krajewska B, Wydro P, Ja czyk A. (2011) Probing the modes of antibacterial activity of
chitosan. Effects of pH and molecular weight on chitosan interactions with membrane
lipids in Langmuir films. Biomacromolecules, 12:4144-52.
Kryszczuk A, Keller J, Grübe M, Zimnoch-Guzowska E. (2006) Cryopreservation of potato
(Solanum tuberosum L.) shoot tips using vitrification and droplet method. Food
Agriculture Environment, 4:196-200.
Kuchitsu K, Kosaka H, Shiga T, Shibuya N. (1995) EPR evidence for generation of hydroxyl
radical triggered by N-acetylchitooligosaccharide elicitor and a protein phosphatase
inhibitor in suspension-cultured rice cells. Protoplasma, 188:138-142.
Kulikov SN, Chirkov SN, Il�’ina AV, Lopatin SA, Varlamov VP. (2006) Effect of the
Molecular Weight of Chitosan on Its Antiviral Activity in Plants. Applied Biochemistry
and Microbiology, 42:200�–203.
135
10. REFERENCIAS/REFERENCES
Kumamaru T, Uemura Y, Inoue Y, Takemoto Y, Siddiqui SU, Ogawa M, Satoh H. (2010)
Vacuolar processing enzyme plays an essential role in the crystalline structure of glutelin
in rice seed. Plant and Cell Physiology, 51:38-46.
Langis R, Schnabel B, Earle ED, Steponkus PL. (1989) Cryopreservation of Brassica
campestris L. Cell suspensions by vitrification. Cryoletters, 10:421-428.
Lawrence BM. (1985) A review the world production of essential oils. Perfumer and Flavorist,
10:1-16.
Lee YC. (1996) Carbohydrate analyses with high-performance anion-exchange
chromatography. Journal of Chromatography A, 720:137-149.
Leindenfrost JG. (1966) On the Fixation of Water in Diverse Fire. International Journal of
Heat Mass Transfer, 9:1154-1166 (translation of the original 1756 publication).
Leprince O, Walters-Vertucci C. (1995) A calorimetric study of the glass transition behaviors
in axes of Phaseolus vulgaris L. seeds with relevance to storage stability. Plant Physiology,
109:1471-1481.
Leuba JL, Stössel P. (1986) Chitosan and other polyamines: Anti-fungal activity and
interaction with biological membranes. In: Chitin in Nature and Technology. (RAA
Muzzarelli, C Jeuniaux & GW Gooday, eds.). Plenum Press. New York, pp. 215-222.
Leunufna S, Keller ERJ. (2005) Cryopreservation of yams using vitrification modified by
including droplet method: effects of cold acclimation sucrose. CryoLetters, 26:93-102.
Levine H, Slade L. (1992) Glass transition in foods. In: Physical Chemistry of Foods. (HG
Schwartzberg & RW Hartel, eds.) Marcel Dekker. New York, pp. 83-221.
Levitt J. (1956) The hardiness of plants. Academic Press. New York, p. 278.
Levitt J. (1980) Responses of Plants to Environmental Stress. Chilling, Freezing and High
Temperature Stresses. 2nd Ed. Academic Press. New York.
Loewenfeld C, Back F. (1980) Guia de hierbas y especias. Ediciones Omega. Barcelona.
Lorenzi HE, Matos FJ de A. (2002) Plantas medicinais no Brasil/ Nativas e exóticas. Instituto
Plantarum. Nova Odessa.
136
10. REFERENCIAS/REFERENCES
Lovelock JE. (1954) The protective action of neutral solutes against haemolysis by freezing
and thawing. Biochemical Journal, 56:265.
Lovelock JE, Bishop MWH. (1959) Prevention of freezing damage to living cells by dimethyl
sulfoxide. Nature, 183:1394-1395.
Lovelock JE, Polge C. (1954) The immobilization of spermatozoa by freezing and thawing
and the protective action of glycerol. Biochemical Journal, 58:618.
Luyet BJ. (1965) Phase transitions encountered in the rapid freezing of aqueous solutions.
Annals of the New York Academy of Sciences, 12:502-521.
Luyet BJ, Gehenio PM. (1940) Life and death at low temperatures. Biodynamica Press.
Normandy.
McCartney SA, Sadtchenko V. (2013) Fast scanning calorimetry studies of the glass transition
in doped amorphous solid water: Evidence for the existence of a unique vicinal phase.
Journal of Chemical Physics, 138:084501.
McCown BH, Sellmer JC. (1987) General media and vessels suitable for woody plant culture.
In: Cell and tissue culture in forestry, General Principles and Biotechnology. (JM Bonga &
DZ Durzan, eds.). Martinus Nijhoff Publishers. Dordrecht, pp. 4-16.
Maclean N, Hall BK. (1987) Cell commitment and differentiation. Cambridge University
Press. New York.
Makowska Z, Keller J, Engelmann F. (1999) Cryopreservation of apices isolated from garlic
(Allium sativum L.) bulbils and cloves. Cryoletters, 20:175-182.
Martín C, Cervera MT, González-Benito ME. (2011) Genetic stability analysis of
chrysanthemum (Chrysanthemum x morifolium Ramat) after different stages of an
encapsulation�–dehydration cryopreservation protocol. Journal of Plant Physiology,
168:158-166.
Martins MBG. (1998) Biometria tecidual e identificação dos tricomas secretores presentes em
Mentha spicata L. e em Mentha spicatata x suaveolens (Lamiaceae). Revista Hispeci e
Lema, 3:43-48.
137
10. REFERENCIAS/REFERENCES
Masuda T, Kitahara K, Aikawa Y, Arai S. (2001) Determination of carbohydrates by HPLC-
ECD with a novel stationary phase prepared from polystyrene-based resin and tertiary
amines. Journal of the American Chemical Society, 17:895-898.
Matos FJA. (1998) Farmacias vivas: sistemas de utilização de plantas medicinais projetado para
pequenas comunidades. Universidade Federal do Ceará. Fortaleza.
Matsumoto T, Saki A, Nako Y. (1998) A novel preculturing for enhancing the survival of in
vitro-grown meristems of wasabi (Wasabia japonica) cooled to -196°C by vitrification.
CryoLetters, 19:27-36.
Matsumoto T, Sakai A, Takahashi C, Yamada K. (1995) Cryopreservation in vitro grown
apical meristems of wasabi (Wasabi japonica) by encapsulation-vitrification method.
CryoLetters, 16:189-206.
Matsumoto T, Sakai A, Yamada K. (1994) Cryopreservation of in vitro grown apical
meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant-
regeneration. Plant Cell Reports, 13:442-446.
Mazur P. (1963) Kinetics of water loss from cells at subzero temperatures and the likelihood
of intracellular freezing. Journal of General Physiology, 47:347-369.
Mazur P. (1970) Cryobiology: the freezing of biological systems. Science. New York.
Mazur P. (1984) Freezing of living cells: mechanisms and implications. American Journal of
Physiology-Cell Physiology, 247:125-142.
Mazur P. (2004) Principles of cryopreservation. In: Life in the frozen state (B Fuller, N Lane
& E Benson, eds.). CRC Press. London, pp. 3-66.
Mazur P, Pinn IL, Kleinhans FW. (2007) The temperature of intracellular ice formation in
mouse oocytes vs. the unfrozen fraction at that temperature. Cryobiology, 54:223-233.
Mazur P, Rall WF, Leibo SP. (1984) Kinetics of water loss and the likelihood of intracellular
freezing in mouse ova. Influence of the method of calculating the temperature dependence
of water permeability. Cell Biophysics, 6:197-213.
138
10. REFERENCIAS/REFERENCES
Mazur P, Rall WF, Rigopoulos N. (1981) Relative contributions of the fraction of unfrozen
water and of salt concentration to the survival of slowly frozen human erythrocytes.
Biophysical Journal, 36:653-675.
Mazur P, Rhian MA, Mahlandt BG. (1957) Survival of Pasteurella tularensis in gelatin-saline
after cooling and warming at subzero temperatures. Archives of Biochemistry and
Biophysics, 71:31-51.
Mendes MA, Souza BM, Marques MR, Palma MS. (2004) The effect of glycerol on signal
suppression during electrospray ionization analysis of proteins. Spectroscopy, 18:339-345.
Meryman HT. (1966a) Foreword. In Cryobiology. Academic Press, London.
Meryman HT. (1966b) Review of Biological Freezing. In Cryobiology. Academic Press.
London, pp. 1-114.
Meryman HT, Kafig E. (1955) The freezing and thawing of whole blood. Proceedings of the
Society for Experimental Biology and Medicine, 90:587-589.
Meryman HT, Williams RJ. (1985) Basic principles of freezing injury to plant cells: natural
tolerance and approaches to cryopreservation. In: Cryopreservation of plant cells and
organs (LA Withers, ed.). CRC Press. Boca Raton, pp. 13-47.
Meryman HT, Williams RJ, Withers LA, Williams JT. (1980) Mechanisms of freezing injury
and natural tolerance and the principles of artificial cryoprotection. In: Crop genetic
resources. The conservation of difficult material (LA Withers & JT Williams, eds.).
International Union of Biological Sciences. Paris, pp. 5-37.
Meste ML, Champion D, Roudaut G, Blond G, Simatos D. (2002) Glass transition and food
technology: A critical appraisal. Journal of Food Science, 67:2444-2458.
Minami E, Kuchitsu K, He DY, Kouchi H, Midoh N, Ohtsuki Y, Shibuya N. (1996) Two
novel genes rapidly and transiently activated in suspension-cultured rice cells by treatment
with N-acetylchitoheptaose, a biotic elicitor for phytoalexin production. Plant and Cell
Physiology, 37:563-567.
Mishima O, Stanley HE. (1998) The relationship between liquid, supercooled and glassy
water. Nature, 396:329-335.
139
10. REFERENCIAS/REFERENCES
Morris GJ. (1980) Plant cells. In: Plant cells and low temperature preservation in medicine
and biology. (MJ Ashwood & J Farrant, eds.). Pitman Medical. Kent, pp. 253-284.
Morris GA, Castile J, Smith A, Adams GG, Harding SE. (2009) Macromolecular
conformation of chitosan in dilute solution: A new global hydrodynamic approach.
Carbohydrate Polymers, 76:616�–621.
Morris GA, Kok MS, Harding SE, Adams GG. (2010) Polysaccharide drug delivery systems
based on pectin and chitosan. Biotechnology and Genetic Engineering Reviews, 27:257-
284.
Mounib MS, Hwang PC, Idler DR. (1968) Cryogenic preservation of Atlantic cod (Gadus
morhua) sperm. Journal of the Fisheries Board of Canada, 25:2623-2632.
Moynihan CT, Easteal AJ, Bolt MA, Tucker J. (1976) Dependence of the fictive temperature
of glass on cooling rate. Journal of the American Ceramic Society, 59:12-16.
Muldrew K, Acker JP, Elliott AW, McGann LE. (2004) The water to ice transition:
implications for living cells. In: Life in the Frozen State (B Fuller, N Lane & EE Benson,
eds.), CRC Press, London, pp. 67-108.
Muldrew K, McGann LE. (1994) The osmotic rupture hypothesis of intracellular freezing
injury. Biophysical journal, 66:532-541.
Munsi PS. (1992) Nitrogen and phosphorus nutrition response in Japanese mint cultivation.
Acta Horticulturae, 306:436-443.
Murashige T, Skoog F. (1962) A revised medium for rapid bioassays with tobacco tissue
cultures. Physiologia Plantarum, 15:473-497.
Murthy SSN. (1997) Study of the phase behaviour of the supercooled aqueous solutions of
dimethyl sulfoxide, ethylene glycol and methanol. Journal Physical Chemistry, 101:6043-
6049.
Muthusamy J, Staines HJ, Benson EE, Mansor M, Krishnapillay B. (2005) Investigating the
use of fractional replication and taguchi techniques in cryopreservation: a case study using
orthodox seeds of a tropical rainforest tree species. Biodiversity and Conservation,
14:3169-3185.
140
10. REFERENCIAS/REFERENCES
Muzzarelli RAA, Tarsi R, Filippini O, Giovanetti E, Biagini G, Varaldo PE. (1990)
Atimicrobial properties of N-carboxybutyl chitosan. Antimicrobial Agents Chemotherapy,
34:2019-2023.
Nadarajan J, Mansor M, Krishnapillay B, Staines HJ, Benson E, Harding K. (2008)
Applications of differential scanning calorimetry in developing cryopreservation strategies
for Parkia speciosa, a tropical tree producing recalcitrant seeds. CryoLetters, 29:95-110.
Niino T, Sakai A, Yakuwa H, Nojiri K. (1992) Cryopreservation of in vitro-grown shoot tips
of apple and pear by vitrification. Plant Cell, Tissues and Organ Culture, 28:261-266.
Nishizawa S, Sakai A, Amano Y, Matsuzawa T. (1993) Cryopreservation of asparagus
(Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration
by vitrification. Plant Science, 1:67-73.
Niwata E. (1995) Cryopreservation of apical meristems of garlic (Allium sativum L.) and high
subsequent plant regeneration. CryoLetters, 16:102-107.
No HK, Lee KS, Kim ID, Park MJ, Meyers SP. (2003) Chitosan treatment effects yield,
ascorbic acid content, and hardness of soybean sprouts. Journal of Food Science, 68:680-
685.
No HK, Young Park N, Ho Lee S, Meyers SP. (2002) Antibacterial activity of chitosan and
chitosan oligomers with different molecular weights. International Journal of Food
Microbiology, 74:65-72.
Obbard R, Iliescu D, Cullen D, Chang J, Baker I. (2003) SEM/EDS comparison of polar and
seasonal temperate ice. Microscopy Research and Technology, 62:49-61.
Ohnoa H, Igarashib M, Hondota T. (2005) Salt inclusions in polar ice core: location and
chemical form of water-soluble impurities. Earth and Planetary Science Letters, 232:171-
178.
Otero L, Molina-García AD, Sanz PD. (2002) Some interrelated thermophysical properties of
liquid water and ice. I. A user-friendly modelling review for food high-pressure
processing. Critical Reviews in Food Science and Nutrition, 42:339-352.
141
10. REFERENCIAS/REFERENCES
Palma-Guerrero J, Jansson HB, Salinas J, Lopez-Llorca LV. (2008) Effect of chitosan on
hyphal growth and spore germination of plant pathogenic and biocontrol fungi. Journal of
Applied Microbiology, 104:541-553.
Panis B, Piette B, Swennen R. (2005) Droplet vitrification of apical meristems: a
cryopreservation protocol applicable to all Musaceae. Plant Science, 168:45-55.
Panis B, Lombardi M. (2006) Status of cryopreservation technologies in plants (food and
forest crops). In: The role of biotechnology in exploring and protecting agricultural genetic
resources (J Ruane & A Sonnino, eds.). FAO. Rome, pp. 61-78.
Pasparakis G, Bouropoulos N. (2006) Swelling studies and in vitro release of verapamil from
calcium alginate and calcium alginate-chitosan beads. International Journal of
Pharmaceutics, 323:34-42.
Patel TR, Harding SE, Ebringerova A, Deszczynski M, Hromadkova Z, Togola A, Paulsen
BS, Morris GA, Rowe AJ. (2007) Weak self-association in a carbohydrate system.
Biophysical Journal, 93:741-749.
Paulet F, Engelmann F, Glaszmann JC. (1993) Cryopreservation of apices of in vitro plantlets
of sugarcane (Saccharum sp. Hybrids) using encapsulation-dehydration. Plant Cell
Reports, 12:525-529.
Pearce RS. (1988) Extracellular ice and cell shape in frost-stressed cereal leaves: a low-
temperature scanning-electron-microscopy study. Planta, 175:313-324.
Pegg DE. (1984) Red cell volume in glycerol/sodium chloride/water mixtures. Cryobiology,
2:234-239.
Pennycooke JC, Towill LE. (2000) Cryopreservation of shoot tips from in vitro plants of
sweet potato [Ipomoea batatas (L.) Lam.] by vitrification. Plant Cell Reports, 19:733-737.
Phunchindawan M, Hirata K, Sakai A, Miyamoto K. (1997) Cryopreservation of encapsulated
shoot primordia induced in horseradish (Armoracia rusticana) hairy root cultures. Plant
Cell Reports, 16:469-73.
Piccaglia R, Dellacecca V, Marotti M, Giovanelli E. (1993) Agronomic factors affecting the
yields and the essential oil composition of peppermint (Mentha x piperita L.). Acta
Horticulture, 344:29-40.
142
10. REFERENCIAS/REFERENCES
Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at
low temperatures. Nature, 164:666.
Pritchard HW, Dickie JB. (2003) Predicting Seed Longevity: the use and abuse of seed
viability equations. In: Seed Conservation, Turning Science into Practice. (RD Smith, JB
Dicke, SH Linington, HW Pritchard & RJ Probert, eds.). Royal Botanic Gardens. Kew, pp.
655-721.
Rabea EI, El Badawy MT, Stevens CV, Smagghe G, Steurbaut W. (2003) Chitosan as
antimicrobial agent: Applications and mode of action. Biomacromolecules, 4:1457-1465.
Rai MK, Asthana P, Singh SK, Jaiswal VS. (2009) The encapsulation technology in fruit
plants-A review. Biotechnology Advances, 27:671-79.
Rajasekaran K. (1996) Regeneration of plants from cryopreserved embryogenic cell
suspension and callus cultures of cotton (Gossypium hirsutum L.). Plant Cell Reports,
15:859-864.
Rajendran SK, Basu SK. (2009) Alginate-Chitosan Particulate System for Sustained Release
of Nimodipine. Tropical Journal of Pharmaceutical Research, 8:433-440.
Rall WF, Fahy GM. (1985) Ice-free cryopreservation of mouse embryos at -196 °C by
vitrification. Nature, 24:387-402.
Rall WF, Mazur P, Souzu H. (1978) Physical-Chemical Basis of the Protection of Slowly
Frozen Human Erythrocytes by Glycerol. Biophysical Journal, 23:101-120.
Ralston G. (1993) Introduction to Analytical Ultracentrifugation. Beckman Instruments.
Fullerton.
Ramløv H, Wharton DA, Wilson PW. (1996) Recrystallization in a Freezing Tolerant
Antarctic Nematode, Panagrolaimus davidi, and an Alpine Weta, Hemideina maori
(Orthoptera; Stenopelmatidae). Cryobiology, 33:607-613.
Rao PS, Suprasanna P, Ganapathi TR, Bapat VA. (1998) Synthetic seeds: concepts, methods
and application. In: Plant tissue culture and molecular biology. (PV Srivastava, ed.).
Narosa Publishing House. New Delhi, pp. 607-619.
143
10. REFERENCIAS/REFERENCES
Ravi-Kumar MNV. (2000) A review of chitin and chitosan applications. Reactivity and
Functionality of Polymers, 46:1-27.
Reddy MV, Arul J, Angers P, Couture L. (1999) Chitosan treatment of wheat seeds induces
resistance to Fusarium graminearun and improves seed quality. Journal of Agricultural
Food Chemistry, 47:1208-1216.
Reed BM. (1988) Cold acclimation as a method to improve survival of cryopreserved Rubus
meristems. CryoLetters, 9:166-171.
Reed BM. (2008) Plant cryopreservation: A practical guide. Springer. New York.
Reinhoud PJ. (1996) Cryopreservation of tobacco suspension cells by vitrification. Ph.D.
Thesis. Rijks University. Leiden.
Rinaudo M. (2006) Chitin and chitosan: Properties and applications. Progress in Polymer
Science, 31:603-632.
Robinson W. (1931) Free and bound water determinations by the heat of fusion of ice method.
Journal Biological Chemistry, 92:699-709.
Romanazzi G, Nigro F, Ippolito A, Di Venere D, Salerno M. (2002) Effects of pre-and
postharvest chitosan treatments to control storage grey mold of table grapes. Journal of
Food Science, 67:1862-1867.
Roos YH. (1995) Glass transition: related physicochemical changes in foods. Food
Technology, 49:97-102.
Roos YH, Karel M. (1991) Applying state diagrams to food processing and development.
Food Technology, 45:66-68.
Ruan SL, Xue QZ. (2002) Effects of chitosan coating on seed germination and salt-tolerance
of seedlings in hybrid rice (Oryza sativa L.). Acta Agronomica Sinica, 28:803-808.
Sacandé M, Buitink J, Hoekstra FA. (2000) A study of water relations in neem (Azadirachta
indica) seed that is characterized by complex storage behavior. Journal Experimental
Botany, 51:635-643.
144
10. REFERENCIAS/REFERENCES
Sakai A. (1997) Potentially valuable cryogenic procedures for cryopreservation of cultured
plant meristems. In: Conservation of Plant Genetic Resources in Vitro. (MK Lazdan & EC
Cocking, eds.). Science Publishers. New York, pp. 53-66.
Sakai A. (2000) Development of cryopreservation techniques. In: Cryopreservation of tropical
plant germplasm. (F Engelmann & H Takagi, eds.). International Plant Genetic Resources
Institute. Rome, pp. 1-7.
Sakai A. (2003) Proceedings of the International Workshop on Cryopreservation of Bio-
Genetic Resources. International Technical Cooperation Center, Rural Development
Administration. Suwon, pp. 3-18.
Sakai A. (2004) Plant cryopreservation. In: Life in the Frozen State (B Fuller, N Lane & EE
Benson, eds.). CRC Press. London, pp. 329-345.
Sakai A, Engelmann F. (2007) Vitrification, encapsulation-vitrification and droplet-
vitrification: a review. CryoLetters, 28:151-172.
Sakai A, Kobayashi S, Oiyama I. (1990) Cryopreservation of nucellar cell of navel orange
(Citrus sinensis Obs. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports, 9:30-33.
Sakai A, Kobayashi S, Oiyama I. (1991) Cryopreservation of nucellar cells of navel orange
(Citrus sinensis Osb.) by a simple freezing method. Plant Science, 74:243-248.
Sakai A, Matsumoto T, Hirai D, Niino T. (2000) Newly developed encapsulation�–dehydration
protocol for plant cryopreservation. CryoLetters, 21:53-62.
Sakai A, Ötsuka K, Yoshida S. (1968) Mechanism of survival in plant cells at super-low
temperatures by rapid cooling and rewarming. Cryobiology, 4:165-173.
Sakar D, Naik PS. (1998) Cryopreservation of shoot tips of tetraploid potato (Solanum
tuberosum L.) clones by vitrification. Annals of Botany, 82:455-461.
Salinas J. (2002) Molecular mechanisms of signal transduction in cold acclimation. In:
Frontiers in Molecular Biology. (BD Hames & DM Glover, eds.). Oxford University Press,
Oxford, pp. 116-139.
145
10. REFERENCIAS/REFERENCES
Sanchéz-Dominguéz D, Bautista-Baños S, Ocampo PC. (2007) Efecto del quitosano en el
desarrollo y morfologia de Alternaria alternata (FR.) Keissl. Anales de Biologia, 29:23-
32.
Sant R, Panis B, Taylor M, Tyagi A. (2008) Cryopreservation of shoot-tips by droplet
vitrification applicable to all taro (Colocasia esculenta var. esculenta) accessions. Plant
Cell, Tissues and Organ Culture, 92:107-111.
Saragusty J, Arav A. (2010) Current progress in oocyte and embryo cryopreservation by slow
freezing and vitrification. Reproduction, 141:1-19.
Schäfer-Menhur A, Schumacher HM, Mix-Wagner G. (1997) Cryopreservation of potato
cultivars - design of a method for routine application in genebanks. Acta Horticulturae,
447:477-482.
Schilcher H. (1988) Quality requirements and quality standards for medicinal, aromatic and
spices plants. Acta Horticulture, 249:33-44.
Schiller M, von der Heydt H, März F, Schmidt P. (2002) Quantification of sugars and organic
acids in hygroscopic pharmaceutical herbal dry extracts. Journal Chromatography A,
968:101-111.
Scottez C, Chevreau E, Godard N, Arnaud Y, Duron M, Dereuddre J. (1992)
Cryopreservation of cold-acclimated shoot tips of pear in vitro cultures after encapsulation-
dehydration. Cryobiology, 29:691-700.
Senaratna T. (1992) Artificial seeds. Biotechnology Advances, 10:379-392.
Snedeker WH, Gaunya WS. (1970) Dimethyl sulfoxide as a cryoprotective agent for freezing
bovine semen. Journal of Animal Science, 30:953-956.
Senula A, Keller JER, Sanduijav T, Yohannen T. (2007) Cryopreservation of cold-acclimated
mint (Mentha spp.) shoot tips using a simple vitrification protocol. CryoLetters, 28:1-12.
Shibli RA. (2000) Cryopreservation of black iris (Iris nigricans) somatic embryos by
encapsulation-dehydration. CryoLetters, 21:39-46.
146
10. REFERENCIAS/REFERENCES
Shibli RA, Shatnawi MA, Subaih WS, Ajlouni MM. (2006) In vitro conservation and
cryopreservation of plant genetic resources: a review. World Journal of Agricultural
Sciences, 2:372-382.
Shibli RA, Smith MAL, Shatnawi M. (1999) Pigment recovery from encapsulated-dehydrated
Vaccinium pahalae cryopreserved cells. Plant Cell, Tissue and Organ Culture, 55:119-123.
Simões CMO, Spitzer V. (2007) Óleos voláteis. In: Farmacognosia: da planta ao medicamento.
UFRGS. Porto Alegre, pp. 467-496.
Singh J. (1979) Freezing of protoplasts isolated from cold-hardened and non-hardened winter
rye. Plant Science Letters, 16:195-201.
Sinha VR, Kumria R. (2001) Polysaccharides in colon-specific drug delivery. International
Journal of Pharmaceutics, 224:19-38.
Slade L, Levine H. (1995) Glass Transitions and Water-Food Structure Interactions.
Advances in Food and Nutrition Research, 38:103-269.
Slade L, Levine H, Reid DS. (1991) Beyond water activity: recent advances based on an
alternative approach to the assessment of food quality and safety. Critical Reviews in Food
Science and Nutrition, 30:115-360.
Song YS, Adler D, Xu F, Kayaalp E, Nureddin A, Anchan RM, Demirci U. (2010)
Vitrification and levitation of a liquid droplet on liquid nitrogen. Proceedings of the
National Academic of Sciences of the United States, 107:4596-4600.
Sperling LH. (1986) Introduction to Physical Polymer Science. Interscience. New York, pp.
224-295.
Steponkus PL, Langis R, Fujikawa S. (1992) In: Advances in Low Temperature Biology. (PL
Steponkus, ed.). JAI Press. Hamptomill, pp. 1-16.
Steponkus PL, Uemura M, Webb MS. (1993) A contrast of the cryostability of the plasma
membrane of winter rye and spring oat-two species that widely differ in their freezing
tolerance and plasma membrane lipid composition. In: Advances in Low-temperature
Biology. (PL Steponkus, ed.), Vol. 2. JAI Press. London. pp. 211�–312.
147
10. REFERENCIAS/REFERENCES
Steponkus PL, Webb MS. (1992) Freeze-induced dehydration and membrane destabilization
in plants. Water and Life. Springer-Verlag. Berlin, pp. 338-362.
Sun WQ. (1999) State and Phase Transition Behaviors of Quercus rubra Seed Axes and
Cotyledonary Tissues: Relevance to the Desiccation Sensitivity and Cryopreservation of
Recalcitrant Seeds. Cryobiology, 38:372-385.
Sun T, Yao Q, Zhou D, Mao F. (2008) Antioxidant activity of N-carboxymethyl chitosan
oligosaccharides. Bioorganic & Medicinal Chemistry Letters, 18:5774-5776.
Sun T, Zhou D, Xie J, Mao F. (2006) Preparation of chitosan oligomers and their antioxidant
activity. Chemistry of Materials Science, 225:451-456.
Suzuki M, Ishikawa M, Okuda H, Noda K, Kishimoto T, Nakamura T, Ogiwara I, Shimura I,
Akihama T. (2006) Physiological changes in gentian axillary buds during two-step
preculturing with sucrose that conferred high levels of tolerance to desiccation and
cryopreservation. Annals of Botany, 97:1073-1081.
Swan TW, O�’Hare D, Gill RA, Lynch PT. (1999) Influence of preculture conditions on the
post-thaw recovery of suspension cultures of Jerusalem artichoke (Helianthus tuberosus
L.). CryoLetters, 20:325-336.
Swenson J, Teixeira J. (2010) The glass transition and relaxation behavior of bulk water and a
possible relation to confined water. Journal of Chemical Physics, 132:014508.
TA Instruments. Determination of Polymer Crystallinity by DSC. Thermal Analysis
Application Brief. No. TA123
http://www.tainstruments.com/main.aspx?n=2&id=181&main_id=338&siteid=11
(10/01/2013)
Tahtamouni RW, Shibli RA. (1999) Preservation at low temperature and cryopreservation in
wild pear (Pyrus syriaca). Advances in Horticultural Science, 13:156-160.
Takeda K, Murata K, Yamashita S. (1998) Thermodynamic investigation of glass transition in
binary polyalcohols. Journal of Non-Crystaline Solids, z213:273-279.
Teixeira AS, González-Benito ME, Molina-García AD. (2012) Tissue and cytoplasm
vitrification in cryopreservation monitored by low temperature scanning electron
148
10. REFERENCIAS/REFERENCES
microscopy (cryo-SEM). In: Current microscopy contributions to advances in science and
technology. (A Méndez-Vilas, ed.). Formatex. Badajoz, pp. 872-879.
Teixeira AS, Gonzalez-Benito ME, Molina-García AD. (2013) Glassy state and
cryopreservation of mint shoot tips. Biotechnology Progress, 29:707-717.
Terbojevich M, Muzzarelli RAA. (2000) Chitosan. In: Handbook of hydrocolloids. (G
Phillips & P Williams, eds.). Woodhead. Cambridge, pp. 367-378.
Thomashow MF. (1998) Role of Cold-Responsive Genes in Plant Freezing Tolerance. Plant
Physiology, 118:1-7.
Touchell DH & Dixon KW. (1994) Cryopreservation for seedbanking of Australian species.
Annals of Botany, 74:541-546.
Towill LE. (1990) Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell
Reports, 9:178-180.
Towill LE. (1996) Vitrification as a method to cryopreserve shoot tips. In: Plant tissue culture
concepts and laboratory exercises. (RS Trigiano & DJ Gray, eds.). CRC Press. Boca Raton,
pp. 297-304.
Towill LE. (2002) Cryopreservation of Mentha (mint). In: Cryopreservation of Plant
Germplasm II. Springer. Heidelberg, pp. 151-163.
Towill LE, Bonnart R. (2003) Cracking in a vitrification solution during cooling or warming
does not affect growth of cryopreserved mint shoot tips. CryoLetters, 24:341-346.
Towill LE, Jarret RL. (1992) Cryopreservation of sweet potato (Ipomoea batatas (L.) Lam.)
shoot-tipsby vitrification. Plan Cell Reports, 11:175-178.
Ubbink J, Kruger J. (2006) Physical approaches for the delivery of active ingredients in foods.
Trends in Food Science and Technology, 17:244-254.
Uchendu EE, Reed BM. (2008) A comparative study of three cryopreservation protocols for
effective storage of in vitro-grown mint (Mentha spp.). CryoLetters, 29:181-188.
Uhrich KE, Cannizzaro SM, Langer RS, Shakesheff KM. (1999) Polymeric systems for
controlled drug release. Chemical Reviews, 99:3181-3189.
149
10. REFERENCIAS/REFERENCES
Uragami A. (1990) Cryopreservation by vitrification of cultured cells and somatic embryos
from mesophyll tissue of asparagus. Acta Horticulturae, 271:109-116.
Uragami A, Sakai A, Nagai M, Takahashi T. (1989) Survival of cultured cells and somatic
embryos of Asparagus officinalis cryopreserved by vitrification. Plant Cell Reports, 8:418-
421.
Umrath W. (1983) Calculation of the freeze-drying time for electron-microscopical
preparations. Mikroskopie, 40:9-34.
Velikov V, Borick S, Angell CA. (2001) The glass transition of water, based on
hyperquenching experiments. Science, 294:2335-2338.
Verdonck E, Schaap K, Thomas L. (1999) A discussion of the principles and applications of
modulated temperature DSC (MTDSC). International Journal of Pharmaceutics, 192:3-20.
Vertucci CW. (1989) The effects of low water contents on physiological activities of seeds.
Physiology Plantarum, 77:172-176.
Vertucci CW. (1990) Calorimetric studies of the state of water in seed tissues. Biophysical
Journal, 58:1463-1471.
Vold IMN. (2004) Periodate oxidised chitosans: Structure and solution properties. Ph.D.
Thesis. Norwegian University of Science and Technology. Trondheim.
Volk GM, Caspersen A, Walters C. (2007) Shoot tip structural and biophysical responses to
plant vitrification solution 2 (PVS2) exposure. Cryobiology, 55:358.
Volk GM, Harris JL, Rotindo KE. (2006) Survival of mint shoot tips after exposure to
cryoprotectant solution components. Cryobiology, 52:305-308.
Volk GM, Walters C. (2006) Plant vitrification solution 2 lowers water content and alters
freezing behavior in shoot tips during cryoprotection. Cryobiology, 52:48-61.
Vujovic T, Sylvestre I, Ruzic D, Engelmann F. (2011) Droplet-vitrification of apical shoot
tips of Rubus fruticosus L. and Prunus cerasifera Ehrh. Scientia Horticulturae, 130:222-
228.
Walstra P. (2002) Physical Chemistry of Foods. CRC Press. New York.
150
10. REFERENCIAS/REFERENCES
Walters C, Wheeler L, Stanwood PC. (2004) Longevity of cryogenically stored seeds.
Cryobiology, 48:229-244.
Wang N, Reed BM. (2003). Development, detection, and elimination of Verticillium dahliae in
mint shoot cultures. Hortscience, 38:67-70.
Wesley-Smith J, Vertucci CW, Berjak P, Pammenter NW, Crane J. (1992) Cryopreservation
of Desiccation-Sensitive Axes of Camellia sinensis in Relation to Dehydration, Freezing
Rate and the Thermal Properties of Tissue Water. Journal of Plant Physiology, 140:596-
604.
Wesley-Smith J, Walters C, Pammenter NW, Berjak P. (2001) Interactions among water
content, rapid (nonequilibrium) cooling to -196ºC, and survival of embryonic axes of
Aesculus hippocastanum L. seeds. CryoLetters, 42:196-206.
Wharton DA. (2011) Cold tolerance of New Zealand alpine insects. Journal of Insect
Physiology, 57:1090-1095.
Wiggins PM. (1990) Role of water in some biological processes. Microbiology Reviews, 54:
432-449.
Wolf AV, Brown MG, Prentiss PG. (1985) Concentrative properties of aqueous solutions. In:
Handbook of Chemistry and Physics. (RC Weast, ed.) CRC Press. Boca Raton, pp. 219-
271.
Wolfe J, Bryant G, Koster KL. (2002) What is �‘unfreezable water�’, how unfreezable is it and
how much is there? CryoLetters. 23:157-166.
Woods EJ, Zieger MA, Gao DY, Critser JK. (1999) Equations for obtaining melting points for
the ternary system ethylene glycol/sodium chloride/water and their application to
cryopreservation. Cryobiology, 38:403-407.
Xie W, Xu P, Liu Q. (2011) Antioxidant activity of water-soluble chitosan derivatives.
Bioorganic & Medicinal Chemistry Letters, 11:1699-1701.
Yamada T, Sakai A, Matsumura T, Higuchi S. (1991) Cryopreservation of apical meristems
of white clover (Trifolium repens L.) by vitrification. Plant Science, 78:81-87.
151
10. REFERENCIAS/REFERENCES
Yavin S, Arav A. (2007) Measurement of essential physical properties of vitrification
solutions. Theriogenology, 67:81-89.
Yue Y, Angell CA. (2004) Clarifying the glass-transition behaviour of water by comparison
with hyperquenched inorganic glasses. Nature, 427:717-720.
Yue Y, Angell CA. (2005) Water behaviour: Glass transition in hyperquenched water?
(reply). Nature, 435:E1-E2.
Zame nik J, Bilav ík A, Faltus M. (2007) Importance, involvement and detection of glassy
state in plant meristematic tissue for cryopreservation. Cryobiology, 5:324-378.
Záme ník J, Faltus M, Bilav ík A, Kotková R. (2012) Comparison of Cryopreservation
Methods of Vegetatively Propagated Crops Based on Thermal Analysis. In: Current
Frontiers in Cryopreservation. (I Katkov, ed.). InTech. New York, pp. 335-358.
Zhou YG, Yang YD, Qi YG, Zhang ZM, Wang XJ, Hu XJ. (2002) Effects of chitosan on
some physiological activity in germinating seed of peanut. Journal Peanut Science, 31:22-
25.
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11. ANEXOS/ANNEXES
153
11. ANNEXES
Annex I. COMPOSITION OF VITRIFICATION SOLUTIONS
Figure annex 1. Molecular structures of the components of the vitrification solutions.
11. ANEXOS/ANNEXES
154
Table 11.1. Molar composition of plant vitrification solutions. mol of each species in the PVS Chemical species
Molecular weight (g mol-1) PVS1 PVS2 PVS2 mod PVS3 PVS3 mod
Sorbitol 182.17 0.050 - - - - EG 62.07 0.209 0.242 0.266 - - DMSO 78.13 0.077 0.192 0.211 - 0.064 PG 76.09 0.171 - - - - Glycerol 92.09 - 0.326 0.411 0.5439 0.543 Sucrose 342.3 - 0.400 0.400 0.146 0.146 Water 18.02 3.557 2.236 1.737 1.587 1.337
Table 11.2. Water content on relation to total mass and molar relations for PVS.
water mass/total mass mol water/mol total mol water/mol solutes 0.609 0.875 7.018 0.354 0.659 1.928 0.270 0.574 1.349 0.222 0.697 2.303 0.187 0.640 1.776
11. ANEXOS/ANNEXES
155
Annex II. PUBLICATIONS
Tissue and cytoplasm vitrification in cryopreservation monitored by low temperature scanning electron microscopy (cryo-SEM) A.S. Teixeira1, M.E. González -Benito2 and A.D. Molina-García1 1ICTAN-CSIC, José Antonio Novais 10, Madrid, 28040, Spain 2Dpto. de Biología Vegetal, E.U.I.T. Agrícola, Universidad Politécnica de Madrid, Ciudad Universitaria 28040, Madrid,
Spain
Cryopreservation of cells and tissues frequently relies on inducing cytoplasm vitrification for storage with almost complete stoppage of both chemical reactions and physical processes and without ice crystal biological damage. The most frequently employed technique used for monitoring ice formation and vitrification in these systems is differential scanning calorimetry (DSC). This technique, which was used in this study to observe ice formation in cryoprotecting agent solutions and mint shoot tips, is fairly sensitive for detecting ice formation, but not so for directly observing glass transition. Besides, the possibility of coincident thermal phenomena obscuring the small glass transition signature in DSC and the lack of any spatial resolution, make valuable the information obtained from other sources. Low temperature scanning electron microscopy (cryo-SEM) is able to show ice crystals formed in the cooling process employed to introduce samples into the microscope, very similar to the cooling step of cryopreservation protocols. The images from samples that, after DSC evidence, have no ice crystals and are vitrified, appear completely unetched in cryo-SEM micrographs. Consequently, cryo-SEM is proposed as a suitable method to ascertain cells and tissues effective vitrification in cryopreservation.
Keywords glass transition; ice dynamics; DSC; mint tips
1. Introduction Cryopreservation comprises a family of procedures allowing the long-term storage of viable biological material at low temperature. Currently many different tissue and cell types (of human, animal, plant or microorganisms origin) are cryopreserved [1-3]. Low temperature storage is employed to avoid deterioration or viability loss occurring at room temperature. But the cooling and warming processes involved are also likely to irreversibly damage cells and tissues. Ice crystal formation is known to cause cell death and loss of viability of stored material [4], so the procedures applied are designed to avoid it. Ice formation requires an initial nucleation step, energetically disfavourable, that involves the reorganization of a large number of water molecules driven by stochastic Brownian movement. Only when nucleii have reached a certain size, ice crystal growth becomes energetically favourable, even at temperatures well below the equilibrium freezing point [5]. The protocols developed and the general strategy followed differs greatly, depending on the material type, tissue, and even concrete species. Nevertheless, most protocols aim at reducing the intracellular water content (either by air or osmotic dehydration, or extracted by the extracellular formation of ice), increasing at the same time the intracellular solute concentration. The result is an increase of the cytoplasmic microviscosity, slowing the water reorganization process required for ice nucleation. Viscosity, which is also increasing steeply as the system temperature decreases, can reach such high values that movement is virtually stopped. In this situation, denominated glassy or vitreous state, most chemical reactions and physical processes are considered to be detained, and ice formation is deemed as impossible [6, 7]. Plant cryopreservation is often performed by means of two step procedures in which first, the water content is reduced (increasing, so, both solute concentration and cytoplasm viscosity) and then temperature is quickly reduced, to cross the glassy state thermal border, the glass transition temperature, TG. Although there exist many other fluids with heat transfer and cryogenic properties more favourable than liquid nitrogen (LN), because cryopreservation is, after all, a set of standard preparative procedures, quick plunging into nitrogen is usually applied, as there exist a LN widespread supply system, at reasonable cost. Besides, the glass transition of many cryopreserved specimens often lies within the interval -120 to -90ºC, and the temperature of boiling LN (-196ºC) provides a large enough temperature difference to ensure a fast cooling drive, as well as a safe storage ambient, well below TG [5, 8]. Droplet-vitrification protocol is widely applied to different plant species. In it, in order to increase heat transfer rates, the tissue portion to be preserved is included into a small drop of vitrification solution (after a set of previous treatments, causing dehydration but also inducing natural defence mechanisms, useful to protect cells towards both dehydration and low temperature). Several drops of this solution (very concentrated in sugars and related low-molecular weight cryoprotecting compounds) are placed into a small strip of aluminium foil, which is then immersed directly, either into LN for cooling, or, at the recovery stage, into room-temperature or warm culture medium. This protocol is particularly successful when applied to mint shoot tips, and the reported viability reached is high [7, 9].
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The transition between liquid and glassy state, not a proper phase transition, can be detected by monitoring a number of properties ultimately related to molecular mobility [10], such as volumetric or mechanical ones. In the context of cryopreservation, the glass transition is most frequently studied by means of differential scanning calorimetry (DSC). This technique allows to detect even small amounts of water freezing (due to its high phase change enthalpy) and also to directly observe glass transition as a step in the heat capacity (CP) baseline. DSC is, however, not a very sensitive technique for this purpose, as the CP change observed is often very small. Sensitive equipment, yielding a stable baseline, is required, and as the step span depends on the amount of vitrified material, in many occasions its observation is problematic [11]. Low temperature scanning electron microscopy (cryo-SEM) is a user-friendly microscopic technique, allowing an easy, quick and artefact free observation of biological materials. The main draw-back of electron microscopy applications to biological materials is their high water content and its incompatibility with the high vacuum required. Cryo-SEM allows the observation of these materials without tedious dehydration procedures, often causing loss or alteration of structural features in specimens. This technique is particularly useful for the observation of the effect of the cryopreservation processes, as the cooling into LN step can be considered equivalent to the final stage of the cryopreservation protocol. Contrast in this technique is low for high water content biological material, poor in high atomic weight atoms. So, an etching step is included, in which a temperature rise allows the partial sublimation of ice, creating depressions that are visible after gold covering. We are considering in this chapter the employment of cryo-SEM as a tool to visualize glassy state in cells and tissues submitted to cryopreservation processes. Mint shoot tips treated following the droplet-vitrification protocol were used as a test material, and the steps of the protocol with decreasing water content were compared to show examples of specimens with intracellular ice and vitrified.
2. Materials and Methods
2.1 Plant Vitrifying Solution 2
Plant Vitrifying Solution 2 (PVS2) was prepared as 30% w/v glycerol, 15% w/v ethylene glycol, 15% w/v dimethyl sulfoxide and 0.4 M sucrose in plant growth medium salts [6]. For Some DSC and Cryo-SEM experiments, PVS2 was diluted with milli-Q deionized water to 25%, 50% and 75%.
2.2 Plant material pre-culture and mint shoot tips extraction
A scheme of the cryopreservation protocol showing the stages in which the specimens were observed is shown in Fig 1. Shoot tips were extracted from Mentha x piperita in vitro plants, maintained by monthly subculture on MS medium [12] with 3% sucrose. Incubation took place at constant temperature (25°C) with a photoperiod of 16 h, and an irradiance of 50 µlmol m-2 s-1 from fluorescent tubes. Shoots were cut into one-node segments, transferred to fresh medium and incubated at alternating temperatures of 25°C (day) and -1°C (dark), always with 16 h photo- and thermoperiod, 50 µlmol m-2 s-1irradiance, provided by fluorescent tubes. After 3-weeks of culture under these conditions, shoot tips (1-2 mm) were isolated from the axillary buds.
2.3 Mint shoot tips dehydration steps
Shoot tips were pre-cultured overnight in a Petri dish with 2 mL of liquid MS medium containing 0.3 M sucrose, over filter paper, at 25°C (observation stage a). Thereafter, the explants were transferred to a Petri dish with 2 mL of loading
Figure 1. Scheme of the steps of the droplet-vitrification cryopreservation protocol employed, see text for more details on media and solutions employed.
a: preculture
Overnight, in MS medium with 0.3 M sucrose
b: loading c: dehydration
20 min incubation in 2M glycerol, 0.4 M
20 min incubation in PVS2 solution
LN
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solution (2 M glycerol + 0.4 M sucrose), over filter paper, for 20 min, at room temperature (observation stage b). Finally, they were dehydrated in 2.0 mL PVS2, in a Petri dish, on filter paper, for 20 min at 0ºC (observation stage c).
2.4 Low temperature scanning electron microscopy Cryo-SEM observations were performed using a Zeiss DSN 960 scanning microscope equipped with a Cryotrans CT-1500 cold plate (Oxford, UK). Cryo-SEM allows sample observations without the need of prior chemical fixing or drying processes. Three shoot tips in the same stage of the cryopreservation protocol (see above) were fitted on a special bracket with their axes vertically aligned, and this piece was introduced in LN under low pressure, physically fixing tissues for microscopic observation. Then, the holder was introduced in the pre-chamber of the Cryotrans cold plate, at -180 ºC, and specimens were fractured perpendicularly to their axis, to obtain suitable observation surfaces. Finally, samples were inserted in the microscope, etched for three min at -90ºC, gold-covered, and observed at -150/-160ºC under secondary electron mode. Drops of PVS2 solution and 50% water dilution were deposited on the microscope sample holder and cooled in LN at reduced pressure. They were not fractured, but directly etched for three min at -90ºC, gold-covered, and observed at -150/-160ºC under secondary electron mode.
2.5 Differential scanning calorimetry
DSC experiments were performed with a TA-1000 instrument (TA Instruments, New Castle, DE, USA) using a scanning rate of 10ºC min-1. Five shoot tips in the same stage of the cryopreservation protocol were introduced in a DSC pan, which was sealed and weighed. 10 µl samples of PVS2 and water dilutions were also enclosed in DSC pans. Both sample types were submitted to cooling scans from room temperature to -85ºC, short (1 min) equilibration periods at this temperature, followed by warming scans, back to room temperature. Later, pans with tips were punctured and dried in an oven at 85ºC for 72 hours, when they showed constant weight. Thermograms were analysed using the standard procedures provided in the Universal Analysis Program (TA Instruments). Ice thawing thermal events (more precise than freezing events) were considered. The routine produced the temperature corresponding to the event onset and that to its peak, the former corresponding to the equilibrium freezing temperature. The process associated enthalpy, proportional to the event area, was also obtained.
3. Results and Discussion
3.1 Plant Vitrifying Solution 2 DSC studies PVS2 solution was employed as a model of a simple vitrifying system. Its composition was adjusted to enable tissue dehydration and protection towards low temperature effects [6]. Some of its components are membrane-penetrating, while some others are not. Its water content is 40,3 % and it is known to vitrify at -112ºC [13]. Figure 2 shows heating DSC thermograms obtained with pure PVS2 solution and several of its water dilutions. The pure PVS2 solution shows no ice formation events either at the cooling (not shown) or heating steps. The diluted mixtures show proof of ice formation. The samples of 25% and 50% PVS2 concentration show melting events corresponding to ice formed in the cooling step. The 75% PVS2 samples had the water mobility so reduced that ice was not formed in the cooling step. However, some ice was formed and quickly melted, during the heating step.
3.2 Plant vitrifying solution 2 cryo-SEM observation
Figure 3 shows the surface of a drop of PVS2 solution and its 50% water dilution deposited on the microscope sample holder without fracture. The clear lumps on the drop surface are volatile substances deposits originated by the lack of a clean surface generated by freeze-fracture. The pure PVS2 drop shows no darker regions that could be associated to ice crystal formation and later sublimation during the etching process. Meanwhile, the 50% PVS2 dilution shows dark holes interpreted as ice crystals formed in the cooling step and later evacuated by sublimation during etching. The pure PVS2 solution is known to be in glassy state at the -150ºC of the microscope stage [13]. It is shown to form no ice upon cooling, after the presented DSC results. Its 50% dilution however, is forming ice crystals, after these DSC measurements, and this is in good agreement with the microscopic observations, where the dark holes that can be observed after etching would correspond to the ice crystals formed. We conclude that pure, vitrified PVS2 solution undergoes no sublimation and no etching-enhanced contrast. Vitrified water has an amorphous structure [14,15]. Interactions among water molecules and other solution components are strong, though disordered, which may impair cooperative phenomena and may have an important role in phase change kinetics. The null or very slow sublimation of vitreous water has been previously reported [16, 17].
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Specimens in the stages a (preculture), b (loading) and c (dehydrated) of the droplet-vitrification protocol were studied, after Fig. 1. Figure 4 shows their typical re-warming DSC thermograms. Table 1 shows the thermal parameters derived from these DSC experiments: Tf(onset) and Tf(peak), respectively, the onset temperature of the melting endotherm (corresponding to the equilibrium freezing temperature) and its peak temperature. The freezing enthalpy ( H) is also showed.
Table 1. Thermal parameters of the thawing events obtained from mint tips DSC thermograms.
Tf(onset) (ºC) Tf(peak) (ºC) H (J g-1)a -5.9 ± 1.1 -1.35 ± 0.34 292 ± 16 b -15.5 ± 0.1 -7.27 ± 0.21 188 ± 14 c - - -
3.3 Mint tips DSC studies Specimens both in steps a and b of the cryopreservation protocol undergo freezing (not shown) and melting events in the respective cooling and re-warming scans. Freezing events at cooling scans take place at the nucleation temperature, shifted to lower values by a considerable supercooling degree and are not appropriate to obtain the equilibrium freezing temperature, especially in a situation of high solute concentration and consequent low molecular mobility, favouring supercooling. Both Tf(onset) and Tf(peak) decrease from a to b, which is in good agreement with a significant increase in the solute concentration with the second incubation step. The amount of frozen water (proportional to the freezing enthalpy) is consequently reduced also, from a to b, as less water is available for ice formation when the solute concentration increases. Thermograms corresponding to specimens in step c show a flat baseline, with no freezing or thawing event in either cooling or warming DSC scans, spanning from room temperature -85 ºC (only warming scans from -50 to 15 ºC are shown.
3.4 Mint tips cryo-SEM studies Figure 5 shows typical cryo-SEM micrographs of mint shoot tips cooled in LN in the microscope cryo-unit, as described. Specimens in both a and b protocol steps showed a clearly visible tissular and cellular structure, with some organelles and vacuolar membrane elements present, among a dark ice crystal mosaic surrounded by a clearer supercooled cryo-concentrated solution matrix. Specimen a shows larger ice crystals while the alteration of cellular
Figure 2. Typical DSC thermograms corresponding to re-warming processes of PVS2 solution and its 25%, 50% and 75% water dilutions. Scanning rate was 10 ºC min-1.
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structures by ice is more evident than in b. Meanwhile, specimen c shows a grey continuous mass masking all structural elements in the field, not affected by the etching procedure applied, and ascribed to vitrified solution.
The combined results of DSC and cryo-SEM would be in good agreement, implying that part of the water contained in the system for specimens on steps a and b becomes frozen when cooled under -85ºC (with less amount of ice formation in the latter) while specimens in step c would avoid ice formation, presumably getting vitrified in the cooling process, as they showed no ice trace, either microscopically or calorimetrically. This is also in good agreement with the literature data for viability as a result of applying the droplet-vitrification method to several systems, including mint apices [6]. DSC is the most frequently employed technique used for monitoring ice formation and glass transition in cryopreserved systems. While the high value of the phase change enthalpy for ice melting ensures the detection of even very small amounts of ice, glass transition has a much smaller reflection on DSC thermograms. Besides, glass
a b
Figure 3. Cryo-SEM micrographs of pure PVS2 solution (a) and 50% water dilution (b). The bar corresponds to 10 µm.
a
b c
-8 -6 -4 -2 0 2 4 6
-60 -50 -40 -30 -20 -10 0 10 20 Temperature (ºC)
Hea
t flo
w (
W g
-1 )
1
Figure 4. Typical DSC thermograms corresponding to re-warming processes of mint shoot tips in different stages of the droplet cryopreservation protocol: a, preculture; b, loading, and c, dehydration. Each experiment was performed with five shoot tips. Scanning rate was 10 ºC min-1. (See Materials and Methods and Figure 1 for more details).
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
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transitions, showed in thermograms as a small baseline displacement, are much less energetic than freezing of thawing processes and are often difficult to be observed in low-mass samples, such as those here considered (total sample mass for each DSC experiment for c-step specimens, about 3.5 mg; total mass of water in these samples, about 2.5 mg). Besides, many other associated phenomena, causing a stronger distortion on the heat capacity baseline, can take place at the same time [11]. A common example is lipid melting which, being very wide processes can cover underlying glass transition taking place of the aqueous phase [18]. A further difficulty is the global character of the information obtained by calorimetric methods. Although many different types of cells and tissues could be present in cryopreserved specimens, the information on ice formation and/or vitrification given by DSC refers to an average over the whole specimen. The main interest lies, however, in the particular class of cells and tissues that may have been identified as essential for survival. In these cases, the possible use of a tool such as cryo-SEM could be very valuable. Tissues or cells with ice crystal marks or vitrified could be readily discriminated, no matter their size or water content. Other phenomena contributing to the energetic state of the sample, such as lipid melting or other unrelated phase transition, are not interfering, and besides, each particular type of cell can be identified and its glassy state directly checked. It must be noted that the cryo-SEM technique is directly reporting the lack of sublimated ice during etching [16, 17]. Ice formation is completely impaired in vitrified solutions, but in regions over TG but close to it (at least up to about 20ºC over TG), although ice has the physical possibility to be formed, its occurrence is very unlikely in a short time period, as the molecular mobility is already very low. So, the cryo-SEM methods would not allow discrimination between these two possible states, both of them not permitting ice formation, at least for short time scales.
4. Conclusions Low temperature scanning electron microscopy is useful to monitor ice formation in cells and tissues, in conditions very close to that of cryopreservation protocols. The information obtained from this technique is in perfect agreement with the ice formation and vitrification data obtained by differential scanning calorimetry. The combined used of these two techniques yielding complementary information is considered as very promising for further studies concerning the mechanism of both cryopreservation and glass formation and evolution.
Acknowledgements Work funded from project “CRYODYMINT” (AGL2010-21989-C02-02), Spanish “Plan Nacional I+D+i 2008-2011”. A.S.T. supported by the CSIC JAE-Pre program, partially funded from the European Social Fund. The expert technical work of F. Pinto is acknowledged.
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
© 2012 FORMATEX 877
a
b
c
Figure 5. Cryo-SEM micrographs of mint shoot tips in different steps of the droplet cryopreservation protocol: preculture, a; loading, b; dehydrated c. The bar corresponds to 10 µm.
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
© 2012 FORMATEX 878
References [1] Towill LE. Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell Rep. 1990;9:178-180. [2] Mazur P, Pinn IL, Kleinhans FW. The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at
that temperature. Cryobiology. 2007;54:223-233. [3] Nadarajan J, Mansor M, Krishnapillay B, Staines HJ, Benson E, Harding K. Applications of differential scanning calorimetry
in developing cryopreservation strategies for Parkia speciosa, a tropical tree producing recalcitrant seeds. Cryoletters. 2008;29: 95-110.
[4] Mazur P, Seki S, Pinn IL, Kleinhans FW, Edashige K. Extra- and intracellular ice formation in mouse oocytes. Cryobiology. 2005;51:29-53.
[5] Zamecnik J, Bilavcik A, Faltus M. Importance, involvement and detection of glassy state in plant meristematic tissue for cryopreservation. Cryobiology. 2007;55:324-378.
[6] Sakai A, Engelmann F. Vitrification, encapsulation-vitrification and droplet-vitrification: a review. CryoLetters. 2007;28:151-172.
[7] Senula A, Joachim Keller ER, Sanduijav T, Yohannen T. Cryopreservation of cold-acclimated mint (Mentha SPP.) shoot tips using a simple vitrification protocol. Cryoletters 2007;28:1-12.
[8] Reed BM (ed.). Plant Cryopreservation: A Practical Guide. New York, NY: Springer; 2008. [9] Volk GM, Walters C. Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during
cryoprotection. Cryobiology. 2006;52:48–61. [10] Kauzmann W. The nature of the glassy state and the behavior of liquids at low temperatures. Chem. Rev. 1948;43:219–256. [11] Verdonck E, Schaap K, Thomas, L. A discussion of the principles and applications of Modulated Temperature DSC
(MTDSC). Int. J. Pharm.1999;192:3–20. [12] Murashige T, Skoog F. A revised medium for rapid bioassays with tobacco tissue cultures. Physiol. Plantarum.1962;15:473-
497. [13] Teixeira AS, Faltus M, Záme ník J, Kotková R, González-Benito ME, Molina-García AD. Invariance of the Glass Transition
Temperature of Plant Vitrification Solutions with Cooling Rate. Cryobiology. 2012;65: in press. [14] Mishima O, Stanley HE. The relationship between liquid, supercooled and glassy water. Nature,1998; 396:329-
335. [15] Angell CA. Amorphous water. Annual review of physical chemistry,2004;55 (1):559-583. [16] Umrath W. Calculation of the freeze-drying time for electron-microscopical preparations. Mikroskopie. 1983;40:9-34. [17] Franks F. Metastable water at subzero temperatures. J. Microsc. Oxford. 1986;141(Part 3):243-249. [18] Teixeira AS, Ballesteros D, Molina-García AD, Walters C. Interactions between water and triacylglycerols may explain faster
aging rates in stored germplasm at low temperature. Cryobiology. 2012;65: in press.
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
© 2012 FORMATEX 879
Glassy State and Cryopreservation of Mint Shoot Tips
Aline S. TeixeiraICTAN, Instituto de Ciencia y Tecnolog!ıa de Alimentos y Nutrici!on, CSIC, Madrid 28040, Spain
M. Elena Gonz!alez-BenitoDpto. de Biolog!ıa Vegetal, E.U.I.T. Agr!ıcola, Universidad Polit!ecnica de Madrid, Ciudad Universitaria, Madrid 28040, Spain
Antonio D. Molina-Garc!ıaICTAN, Instituto de Ciencia y Tecnolog!ıa de Alimentos y Nutrici!on, CSIC, Madrid 28040, Spain
DOI 10.1002/btpr.1711Published online March 27, 2013 in Wiley Online Library (wileyonlinelibrary.com)
Vitrification refers to the physical process by which a liquid supercools to very low tem-peratures and finally solidifies into a metastable glass, without undergoing crystallization ata practical cooling rate. Thus, vitrification is an effective freeze-avoidance mechanism andliving tissue cryopreservation is, in most cases, relying on it. As a glass is exceedingly vis-cous and stops all chemical reactions that require molecular diffusion, its formation leads tometabolic inactivity and stability over time. To investigate glassy state in cryopreservedplant material, mint shoot tips were submitted to the different stages of a frequently usedcryopreservation protocol (droplet-vitrification) and evaluated for water content reductionand sucrose content, as determined by ion chromatography, frozen water fraction and glasstransitions occurrence by differential scanning calorimetry, and investigated by low-tempera-ture scanning electron microscopy, as a way to ascertain if their cellular content was vitri-fied. Results show how tissues at intermediate treatment steps develop ice crystals duringliquid nitrogen cooling, while specimens whose treatment was completed become vitrified,with no evidence of ice formation. The agreement between calorimetric and microscopicobservations was perfect. Besides finding a higher sucrose concentration in tissues at themore advanced protocol steps, this level was also higher in plants precultured at 25/21!Cthan in plants cultivated at 25!C. VC 2013 American Institute of Chemical Engineers Biotech-nol. Prog., 29:707–717, 2013Keywords: ice crystal, dehydration, droplet-vitrification, DSC, cryo-SEM
Introduction
Biologically material, most frequently that being used forplant propagation (i.e., germplasm), can be stored at lowtemperature for long periods. Plant germplasm, particularlyseeds, pollen, or reproductive tissues, is often kept at liquidnitrogen (LN) temperature to avoid deterioration.1 Cryopre-servation is currently an important tool for germplasm long-term storage, requiring minimal space and maintenance, andthe number of species and cultivars cryopreserved quicklyincreases.2 Low-temperature storage of living tissues andcells must avoid ice crystal formation (especially intracellu-lar) during the whole process (cooling, storage, and finalrewarming), as ice results lethal for cells, impairing speci-mens viability.3,4
Most techniques used include an initial step designed toreduce the tissues water content (with the limit of avoidingdehydration damage) and increase the cytoplasm internal vis-cosity. This step may include the incorporation of extrinsicviscosity enhancing substances (such as sugars, glycerol, orethylene glycol) and the induction of cold adaptation and
defense mechanisms by the plant cells. A second step con-sists of quick cooling by plunging into LN to avoid ice for-mation. The chances of ice crystallization are limited byboth a decrease in the amount and mobility of intracellularwater and an increase in cooling rates, hindering the rear-rangement of water molecules into ice, a necessary previousnucleation step. Cooling to LN temperature places the sys-tem well into the glassy state. Once vitrified, despite sometime-depending phenomena still taking place, relatively largemolecular reorganizations, as those required for ice forma-tion, are not possible and specimens can be stored for hypo-thetically indefinite periods, without ice being formed.5 Athird step consists of the rewarming process, which must beperformed quickly enough to avoid ice nucleation whilecrossing by the temperature region comprised between theglass transition temperature (TG) and the equilibrium freez-ing point (Tf).
6–8 Progressive dehydration and rapid coolinghave been found to avoid the formation and growth of lethalintracellular ice in plant cells.9
Many apparently independent physical properties undergolarge changes associated to the glass transition within similartemperature ranges. For example, the TG values determinedby calorimetry, thermal expansivity, or viscosity are oftennearly identical.10 This is a result of all these properties
Correspondence concerning this article should be addressed to A. S.Teixeira at [email protected]
VC 2013 American Institute of Chemical Engineers 707
being parallelly influenced by changes in molecular mobilityoccurring during the glass transition.
The initial works on mint cryopreservation by vitrificationincluded gradual dehydration using a mixture of ethyleneglycol, dimethyl sulfoxide (DMSO), and polyethylene gly-col.1 A successful method among those usually used forplant germplasm cryopreservation is that named droplet-vitri-fication.11 It is derived from the droplet-freezing technique12
developed for preserving shoot tips, which are placed indroplets of cryoprotective medium and cooled slowly in aprogrammable freezer. In the droplet-vitrification method,the specimen is initially incubated in solutions with increas-ing concentration of cryoprotecting and dehydrating agentsfor different periods. Then it is treated and included in asmall drop of cryoprotecting agents solution (containing highconcentrations of glycerol, ethylene glycol, DMSO, and su-crose13) and deposited on a small strip of aluminum foil.Finally, the strip with the droplets is quickly plunged intoLN and then stored in a cryovial in a LN container. Warm-ing is carried out similarly, plunging the strip plus dropletsinto warm liquid medium containing 1.2 M sucrose and, af-ter 20 min unloading, shoot tips are retrieved and placed onrecovery medium. The results of this method, in terms of vi-ability, have been good for a variety of systems.2,11
The main advantage of this technique is enabling veryhigh cooling and warming rates because of the small volumeof medium in which the explants are placed.2 Many proto-cols involve exposure of plant shoot apices to PVS2 at0!C.13,14 To prevent cellular damage, cryoprotectant solu-tions both dehydrate and penetrate cells to stabilize proteinsand membranes. However, the mode of action of each com-ponent may differ with cell type, species, temperature, andother solution components.15,16 A particular component ofthe cryoprotectant solutions used, DMSO, enhances chemicalpenetration into cells and, in spite of its own cytotoxicity—for which testing the treatment duration is essential—it hasbeen found to promote survival of cryoexposed cells.8,17,18
The interplay between exogenous and endogenous carbohy-drates plays an important role on plant resistance to stresses.For example, a two-step preculture treatment of gentian budswith increasing sucrose concentrations produced a raise ofabscisic acid, followed by an increase of endogenous sucroseconcentration, all of which resulting in a higher dehydrationtolerance.19 Various high-performance liquid chromatography(HPLC) techniques have been developed to facilitate carbohy-drate analyses in an effort to better understand their role inbiochemical processes. High-performance ion chromatogra-phy (IC) coupled with pulsed amperometric detection (PAD)is an efficient method to quantify carbohydrates in naturalsamples and food products.20–24
Differential scanning calorimetry (DSC) is a well-knowntechnique used for studying thermal properties of thermallydriven processes. It has been used for long in cryopreserva-tion studies, as it yields interesting information on freezingand vitrification processes.5,8,25–27 Water freezing latent heatis large, so calorimetry is a sensitive method to monitor iceformation or thawing, even in small quantities. Besides theamount of ice formed, after knowledge of the system’s watercontent, the ratio of frozen–unfrozen water may also beobtained. DSC also yields Tf, and its reduction speaks ofincreasing solute concentrations. TG is also acquired, if scansare performed at sufficiently low temperature, although DSCis not particularly sensitive for this purpose.
Scanning electron microscopy (SEM) is a powerful anduser-friendly microscopic technique, able to provide a wealthof structural information on a wide range of samples. Whencombined with a cryo-stage (cryo-SEM), it is especiallyinteresting to study the microstructure of biological systems,not altered in sample preparation.28 Low-pressure LN frozensamples preserve most structural relations present in theoriginal sample, with little or no artifacts. Cryo-SEM allowssample observation without need for prior chemical fixing ordrying. Ice crystals formed are small and not altering tissuestructures. Ice formed inside cells can be observed, afterfracture in the cryo-stage and a suitable etching process, asblack “void” regions surrounded by gray ridges formed byhighly cryo-concentrated solution either frozen as euthectic,unfrozen, or even vitrified.
In this work, these three powerful techniques, DSC, cryo-SEM, and HPLC, have been applied to follow the behaviortoward LN cooling of specimens of plant germplasm—mint(Menta 3 piperitha) shoot tips—in different steps of thedroplet-vitrification protocol. To date, no publication pro-vides data obtained during this cryopreservation protocol,but only of its final outcome. Actually, very little physicalinformation exists on actual cryopreserved or vitrified plantspecimens. Besides, the combined use of information result-ing from these physicochemical techniques allows us to bet-ter understand the phenomena taking place between thenormal and the vitrified states of plant tissues and back torestore its viability as germplasm. Interesting relationsamong sucrose and cryoprotectant content in cells and itsfreezing and vitrification behavior are found, as well as withthe other factors ruling over the physical control of ice for-mation: water content and cooling rate. The data presentedhere are expected to help to design improved cryopreserva-tion procedures, involving less damaging cryoprotectantsand/or suitable for problematic species.
Materials and Methods
Plant material preculture and shoot tips extraction
Shoot tips were extracted from in vitro shoots of Mentha3 piperita. In vitro plants were monthly subcultured on me-dium MS29 with 3% sucrose and incubated at constant tem-perature (25!C) with a photoperiod of 16 h and an irradianceof 50 mmol m22 s21 from fluorescent tubes. One-node seg-ments were obtained from these shoots, transferred to freshmedium, and incubated at alternating temperatures of 25!C(day) and 21!C (night), always with 16 h photoperiod andthermoperiod, 50 mmol m22 s21 irradiance, provided by fluo-rescent tubes (stage a, see Figure 1). After 3 weeks of cul-ture under these conditions, shoot tips (1–2 mm) wereexcised from axillary buds.
Incubation and dehydration of shoot tips
A protocol based on that of Senula et al.11 was followed.Five excised shoot tips were precultured overnight with 2mL of liquid MS medium containing 0.3 M sucrose at 25!Cover filter paper (stage b, see Figure 1). Thereafter, theexplants were transferred to a Petri dish with 2 mL of load-ing solution (2 M glycerol1 0.4 M sucrose) over filter paper,for 20 min, at room temperature (stage c). Finally, they wereosmotically dehydrated in 2 mL PVS2 (plant vitrification so-lution 2: 30% w/v glycerol, 15% w/v ethylene glycol, 15%w/v DMSO, and 0.4 M sucrose in plant growth medium13)
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in a Petri dish, on filter paper, for 30 min at 0!C (stage d).Immersion in LN was carried out in the cryo-SEM or insidethe aluminum pans of DSC, depending on the experiment. Aschematic of these steps can be seen in Figure 1.
Plant recovery and viability
Treated and control shoot tips were warmed from LN tem-perature by plunging into warm liquid medium with 1.2 Msucrose for 20 min. After the unloading of cryoprotectants,shoot tips were cultured on regrowth medium, composed ofsolid MS medium supplemented with 0.5 mg L21 BAP1 0.3M sucrose, cultivated in the dark for 24 h and finally placedinto the culture room at 22–25!C and 16 h illumination. Forcontrol samples (2LN), all steps were carried out, includingthe incubation in PVS2 for 30 min. After that the explantswere rinsed in 1.2 M sucrose solution for 20 min and thenplaced on the regrowth medium.
Survival and regrowth were calculated as rates over thetotal number of shoot tips used. Survival was defined includ-ing all forms of visible viability (evidence of green struc-tures or callus) observed 1–2 weeks after rewarming.Regrowth was defined as the formation of small plantlets, 4weeks after rewarming.
Low-temperature SEM
Low temperature SEM (cryo-SEM) observations were per-formed with a Zeiss DSN 960 scanning microscope equippedwith a Cryotrans CT-1500 cold plate (Oxford, UK). Threeshoot tips in the same stage of the cryopreservation protocolwere fitted on a special bracket with their axes verticallyaligned. The geometry of sample and sample holder requiredoptimization for both freezing and fracturing procedures:these steps require stable mechanical mounting of the sampleon a support. This piece was plunged into LN under lowpressure, physically fixing tissues for microscopic observa-tion. This cooling step was considered equivalent to thecooling process taking place in the actual cryopreservationprocess by LN immersion. The holder was then inserted inthe prechamber of the Cryotrans cold plate, at 2180!C, andspecimens were fractured perpendicularly to their axis toobtain a suitable observation surface (a cartoon of the tipsbefore introduction in the microscope and after fracture canbe seen in Figure 2). Later, samples were inserted in themicroscope and etching (partial ice sublimation induced toprovide contrast) was performed for 3 minutes at 290!C.Once etched and recooled, the sample was retrieved into thecryopreparation chamber and coated with high-purity Au,which acts as a conductive contact for electrical charge. The
etched and coated sample was reinstated into the cooledSEM stage and observed using an accelerating voltage of 15kV at 2150/2160!C.
Dry matter and water content determination
Six sets of five tips were used for determining the drymater content (dm) in shoot tips at the different stages of thecryopreservation protocol. It resulted from weighing afteroven drying ("85!C, 72 h) and was expressed as relative tothe total tip mass. The water content (Wc) of shoot tips couldbe calculated for stages a and b, directly as the differencebetween its total mass and dm. It was expressed as relativeto the total shoot tip mass (Wc(s)) or the dry mass (Wc(dm)).
Differential scanning calorimetry
DSC experiments were performed with a Mettler-ToledoDSC 30 instrument (Mettler-Toledo, Griefsen, Switzerland).Cooling was performed using the calorimeter control (10!Cmin21), or for higher rates, by quenching the samples in LN(see below). The response of shoot tips apices to each stepin the mint protocol was evaluated. Five shoot tips in thesame stage of the cryopreservation protocol were introducedin a DSC pan, which was sealed and weighed. Shoot tipshad been sequentially treated with MS medium, loading so-lution, and PVS2. Samples were submitted to a cooling scanfrom room temperature to 2150!C and a short 5 min equili-bration at this temperature, followed by a warming scan (at
Figure 1. Scheme of the steps of the droplet cryopreservation protocol used.
See text for more details on media and solutions used.
Figure 2. Cartoon showing the three mint tips on the micros-copy holder before insertion in the microscope (A)and after equatorial freeze fracture (B).
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10!C min21), back to room temperature. Calorimetric datawere collected from two replicates per treatment.
For the quenching method shoot tips, sealed into a DSCpan, were quickly plunged into LN. Pans were subsequentlycold-loaded into a prechilled DSC (2150!C) for analysis.
Later, pans were punctured and dried in an oven at 85!C,for 72 h, when they showed constant weight. Thermogramswere analyzed using the standard procedures provided in theMettler-Toledo STARe software. Ice thawing thermal events(more precise than freezing events) were used for extractionof Tf. The routine produced the temperature corresponding tothe event onset, Tf(onset), corresponding to the equilibriumfreezing temperature and that to the peak, Tf(peak).
The equilibrium freezing temperature can be related to thewater activity (aw) of the system to its osmolality (p) and toits solute content.30,31 For comparative purposes, a “sucroseequivalent” concentration (Suceq) and a “sodium chlorideequivalent” (NaCleq) were determined from Tf, assuming thatthe only solute present was sucrose or sodium chloride,respectively. Tf(peak) data were used for the determination ofthese parameters, as it was considered to better represent thebehavior of the whole phase change. Equation (1)30 was fol-lowed for aw determination:
ln aw 5DHf w# $Aw Tf2Tf%# $= TfTf%# $; (1)
where DHf(w) is the pure water freezing enthalpy (specific,per water gram), Aw is the water molecular weight, and Tf*is the equilibrium freezing point of pure water. The osmolal-ity was calculated using Eq. 231:
p5DTf=1:86; (2)
where DTf is the freezing point depression, which for waterat atmospheric pressure, being the pure substance freezingpoint 0!C, equals to Tf with a positive sign.
Suceq and NaCleq could not be calculated from Raoult’slaw, using analytical equations,32 and only valid for morediluted solutions. Experimental data from literature30,33 andcalorimetric determinations performed on concentrated su-crose solutions (data not shown) were used to relate thesehypothetic solute concentrations with the observed cryo-scopic temperature decrease.
The process-associated enthalpy, phase change enthalpyDHf, proportional to the event area, was also obtained. Itwas expressed as relative to either the total shoot tip mass(DHf(s)), its dry mass (DHf(dm)), or its water content (DHf(w))for allowing easier data comparison.
Water fractions: frozen water (Wf) and unfrozen water(Wu) were calculated by comparison of DHf and the purewater freezing enthalpy (DHf(w)5 333.4 J g21 at 0!C), to-gether with the total water contents previously determined,using Eq. (3):
Wf5 DHf=DHf w# $! "
;Wu5Wc2Wf : (3)
Specific enthalpy values (per sample gram) were alwaysused after the corresponding oven dry pan weighs. Enthalpyvalues measured at temperatures different from the corre-sponding phase change equilibrium temperature, 0!C, areunderevaluated, as a result of the difference between waterand ice heat capacities (Cpw and Cph, respectively). So, thevalue of DHf(w) was corrected using Eq. (4), before applyingEq. (3). Single freezing temperatures, Tf(peak), were used, andCpw and Cpi data were obtained from a data calculation
routine produced in this laboratory,34 after interpolationwhen required, and other sources.35,36
DHf corr5DHf2#Tf
0
CpWdT2#0
Tf
Cp jdT
$ %(4)
Frozen (Wf) and unfrozen (Wu) water contents wereexpressed as relative to the total shoot tip mass (Wf(s) andWu(s)), its dry mass (Wf(dm) and Wu(dm)), or its total watercontent, when available, (Wf(w) and Wu(w)).
The glass transition can be observed as a small step in theheat capacity thermogram baseline. The corresponding heatcapacity increment is very small and, in our experiments,only could be appreciated by using 30 shoot tips in the sameDSC pan.
For comparative purposes, calorimetric experiments wereperformed with samples of the cryoprotectant solutions usedin each stage of the droplet protocols (i.e., liquid MS me-dium with 0.3 M sucrose, loading solution, and PVS2).Experiments were performed in the same conditions thanthose carried out with shoot tip specimens.
Sucrose content assessment in shoot tips
Specimens were analyzed after each stage of the cryopre-servation procedure for its sucrose content. Five shoot tipswere weighed (average fresh weight 3.06 0.1 3 1023 g)and mortar-ground into a fine powder in LN. As grindingproceeded, the vessel was slowly warmed. Immediately, 1.5mL Milli-Q water was added and the mixture was filteredthrough a syringe-driven filter (Millex-GS, 0.22 lm).
The determination of sucrose was carried out by an IonChromatography 817 Bioscan (Metrohm, Herisau, Switzer-land) system equipped with a Metrosep Carb 1–250 columnand a PAD with a gold electrode. A three-step PAD protocolwas used with the following time intervals (ms) and poten-tials (mV): t1, 400/E1510.05 (detection); t2, 200/E2510.75 (cleaning); and t3, 400/E3520.15 (regenera-tion). Samples (1.5 mL) were injected using an autosampler(model, 838 Advanced Sample Processor, Metrohm, Herisau,Switzerland) and the flow rate through the column was 1 mLmin21 and the eluent was 100 mM NaOH.
Identification of sucrose was performed by comparisonwith the retention time of the pure standard sucrose (Merck)and the data were acquired using IC Net 2.3 software. Su-crose content was expressed as ppm and the data were themean of three replicates. Results (Suc) are expressed as ppmof the total tip mass.
Results and Discussion
Detection of glassy state and ice by cryo-SEM
Two different observation approaches were compared: sec-ondary electron and backscattered electron image. Basically,the same information was obtained with both; however, thecellular structures could be better appreciated using backscat-tered electrons, and this was used in the observations andmicrographs presented here.
Figure 3 shows the micrographs showing shoot tips cooledin LN in the microscope cryounit as described in the Materi-als and Methods section, in different moments of the dropletcryopreservation protocol: control (A); stage a, cold treat-ment (B); stage b, preculture (C, D); stage c, loading (E, F);and stage d, dehydration (G, H). Micrographs C, E, and G
710 Biotechnol. Prog., 2013, Vol. 29, No. 3
show lower magnification images of the shoot tips axialplane. The concentric cell distribution in the equatoriallyfractured tips could be appreciated. There were no noticeabledifferences among central or more external cells, indicatingthat the fracture was close to the apical dome, so effectivehomogeneity for temperature and solute and water concentra-tions could be assumed for tip cells.
Ice crystals formed in the more concentrated solutionswere smaller because of the limitations to crystal growthcaused by the higher solution viscosity. On the other hand,conversion of water into ice during cooling increased theconcentration of solutes in the more concentrated regions.The etching procedure applied gave rise to a contrast in the
SEM micrograph, removing part of the ice formed by subli-mation and leaving the darker regions separated by sector or“ridges” of freeze-concentrated solution that can be appreci-ated in micrographs A–F. This behavior allowed visualiza-tion of individual crystals. Sublimation is only taking placeat an appreciable rate out of pure ice crystals, whereas waterin glassy state has a sublimation rate almost negligible.37,38
So, when solutions become vitrified, they cannot be etchedin this way. Micrographs G and H show a complete lack ofthe structural details revealed by etching.
Determination of preculture effect by cryo-SEM
Figures 3A,B show, respectively, typical cryo-SEM micro-graphs after 3 weeks of culture at 25!C (control apices) or at25/21!C. Widespread cellular disruption occurred in controlapices, as it can be observed in micrograph 3A, where icecan be seen as dark areas inside the cell’s cytoplasm andvacuole; meanwhile, clearer parts would correspond to thesupercooled cryoconcentrated solution matrix. Cold-treatedshoot tips (3B) showed the formation of smaller ice crystals,mostly in the vacuole space, which indicates a higher solutecytoplasmic concentration with respect to control apices. Theobserved size crystal reduction is understood because of anincrease in the amount of endogenous cryoprotecting agentsdeveloped by the plant during the cold hardening procedure,confirming the positive influence of tips cold hardeningbefore cryopreservation observed by other workers.11
Observed plant recovery and viability
Both survival and recovery reached a 96% value for speci-mens treated with the complete droplet cryopreservation pro-tocol (i.e., quenched in LN after stage d, dehydration inPVS2). Meanwhile, survival and recovery of previous stages,a to c, were 0% for all cases. The control samples (2LN)showed a 100% survival and recovery.
Analysis of the droplet-vitrification protocol stepsby cryo-SEM
Micrographs of specimens in the stages b (preculture) (C,D), c (loading) (E, F), and d (dehydration) (G, H) of thedroplet-vitrification protocol can be seen in Figure 3. Speci-mens in both b and c stages showed a clearly visible tissularand cellular structure, with some organelles and vacuolarmembrane elements present. Specimens in stage b showedlarger ice crystals and a higher degree of alteration of cellu-lar structures by ice than those in stage c. Tips treated withalternating temperatures (see previous section) had a similaraspect to those in step b (preculture). The ice crystal sizedifference could be due to concentration differences amongsamples, as the crystals formed in the more diluted solutioncan grow to a larger final size, whereas those occurring inconcentrated solutions, where molecular mobility isimpaired, would be smaller.
During the treatment in the loading solution, the cells ofspecimens in stage c of the protocol accumulated exogenousglycerol and other solutes of endogenous origin. These cellswere clearly partly plasmolyzed as a result of the osmoticdehydration imposed by the loading treatment, which wasrelated to the progressive acquisition of tolerance towardfreezing during the protocol.
PVS2, the cryopreservation solution used in the last step,affected cellular freezing properties.8 It has components that
Figure 3. Cryo-SEM micrographs of shoot tips cooled in LN inthe microscope cryounit (see Materials and Methods).
Control (A); stage a, cold treatment (B); stage b, preculture (Cand D); stage c, loading (E and F); and stage d, dehydration (Gand H). The bar corresponds to 10 lm (A, B, D, F, and H) or 50lm (C, E, and G).
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are penetrating (dimethyl sulfoxide, glycerol, and ethyleneglycol) and nonpenetrating (sucrose) for the cellular mem-brane of most cell types.39 This complex solution served todehydrate shoot tips and to change the behavior of the waterremaining within shoot tips.
Micrographs of specimens in the stage d of the protocol,after osmotic dehydration in PVS2 solution, presumably witha lower water content and more concentrated in solutesinduced by permeation from the cryopreservation solution,showed a gray continuous, little detailed surface, indicatingvitrification (Figures 3G,H).
Changes in the dry mass and water content during thedroplet-vitrification protocol
The dry mass and water content of shoot tips, derivedfrom its weight difference after drying, are shown in Table1. For step a and b, where water was the only volatile sub-stance (overseeing very small quantities of organic com-pounds), Wc could be obtained by this procedure and ispresented in Table 1, expressed as related to the total samplemass Wc(s) or to the dry mass Wc(dm). The water content oftips undertook a small decrease with the first steps of thecryopreservation protocol, associated to sucrose incubationin stage b. Water content for stages c and d could not bederived from oven-drying data, as the amount of volatilesubstances apart from water was high. Nevertheless, it couldbe assumed that there was a large decrease in the water con-tent associated to step c, as inferred from the increase in dm,which was in good agreement with the reported microscopicobservations showing a smaller ice crystal size in these sam-ples and with the sucrose content data (see below).
Stage d, following the same arguments, would show thecomplex result of water osmotically driven out of the cell bythe strong potential of the very concentrated PVS2 solution,rich in the nonpenetrating sucrose, entrance of the penetratingagents DMSO, ethylene glycol, and glycerol, and water reten-tion inside the cell by the increased internal presence of thesecryoprotectors,8,39–42 notwithstanding the increasing presenceof endogenous cryoprotecting (and often water binding)agents, as a result of the plant response to the induced stress.
Measurement of ice heat of fusion using DSC
Figure 4 shows typical rewarming DSC thermograms forthe same specimens types (at stages a–d) previously cooled
at a rate of 10!C min21. Table 1 shows the thermal parame-ters derived from DSC experiments, Tf(onset) and Tf(peak),respectively, the onset temperature of the melting endotherm(corresponding to the equilibrium freezing temperature) andits peak temperature. The freezing enthalpy (DH) is alsoincluded. The information that both enthalpy and equilibriumfreezing temperature can provide is of the highest interest, asit yields information on the water content within cells and,in a context where it is difficult to know the detailed cyto-plasm composition and interacting effects of endogenous andexogenous solutes, to draw conclusions on the availability ofthis water, to form ice, to permit diffusion-driven processesand even to keep the stability of protein and membranes.Specimens both in stages b and c of the cryopreservationprotocol showed freezing (not shown) and melting events inthe respective cooling and rewarming scans.
The equilibrium freezing temperature is a good raw datato determine accurately solution characteristics, such aswater activity and frozen and unfrozen water.32 However,
Table 1. Compositional and Thermal Parameters of the Thawing Events Obtained from DSC Thermograms for Mint Shoot Tips Specimens atDifferent Stages (a–d) of the Droplet Cryopreservation Protocol
Cold Treatment(Stage a)
Preculture(Stage b)
Loading(Stage c)
Dehydration(Stage d)
dm (gdm gsample21) 0.1176 0.009 0.1516 0.009 0.3446 0.049 0.2526 0.013
Wc(s) (gwater gsample21) 0.8836 0.009 0.8496 0.009 – –
Wc(dm) (gwater gdm21) 7.566 0.65 5.646 0.39 – –
Tf(onset) (!C) 22.966 0.06 24.96 1.8 27.936 0.79 –
Tf(peak) (!C) 20.486 0.08 20.586 0.21 25.56 1.1 –
DHf(s) (J gsample21) 2416 16 209.856 0.12 93.06 7.2 –
DHf(dm) (J gdm21) 1,8006 650 1,3946 83 2741 60 –
DHf(w) (J gw21) 2726 14 247.16 2.5 – –
Wf(s) (gwater gsample21) 0.7246 0.47 0.6326 0.000 0.2906 0.023 0
Wf(dm) (gwater gdm21) 6.206 0.88 4.206 0.25 0.866 0.19 0
Wf(w) (gwater gwater21) 0.8206 0.045 0.7446 0.007 – 0
Wu(s) (gwater gsample21) 0.1596 0.038 0.2176 0.009 – –
Wu(dm) (gwater gdm21) 1.346 0.22 1.446 0.14 – –
Wu(w) (gwater gwater21) 0.1806 0.045 0.2566 0.007 – –
aw 0.99546 0.001 0.99446 0.002 0.94706 0.001 –
p (Osm) 0.266 0.04 0.316 0.12 2.976 0.60 –
Figure 4. Typical DSC thermograms corresponding torewarming processes of shoot tips in differentstages of the droplet cryopreservation protocol:cold treatment (stage a), preculture (stage b), load-ing (stage c), and dehydration (stage d).
Each experiment was performed with five shoot tips. Theinsert shows an expanded thermogram section which allowsthe glass transition, obtained with 30 tips, to be appreciated.Scanning rate was 10!C min21 (See Materials and Methodsand Figure 1 for more details). The ordinates scale of thethermograms baseline is arbitrary.
712 Biotechnol. Prog., 2013, Vol. 29, No. 3
the precision yielded by DSC for melting temperatures, espe-cially in complex systems, such as natural tissues, is limited.The onset temperature is considered usually as the best esti-mation of the equilibrium freezing point. But, in complexsolutions, compartmentalized in different isolated pools, withmarkedly different composition and water contents (cyto-plasm, vacuoles, intercellular space, different cells, and tis-sues) the onset temperature would rather correspond to themelting of the most concentrated pool, which would thaw atthe lowest temperature.
We have considered that, for these sample types, the peaktemperature was giving a better estimation of the averagemelting of the specimens studied. Moreover, the DSC-meas-ured temperature was affected by the thermal differences (aswell as the compositional ones) within the sample, not negli-gible at a relatively fast scanning speed, as 10!C min21.
The temperatures determined were decreasing steeply withthe progress of the cryopreservation protocol, which spokeabout the increase in the “number” of solute molecule con-centration. The last stage showed no thawing event, andthere would be no frozen water at all in these conditions.From Tf, the aqueous solution parameters aw and p werederived and shown in Table 1.
The thawing enthalpy is, in practice, a much more accurateparameter than freezing enthalpy, depending less on kinetic,geometric, or compartmentalization issues. The phase changeenthalpy, which is presented in Table 1 as relative to eitherthe total sample mass, DHf(s), the dry mass, DHf(dm), or thetotal water content, DHf(w), decreased as the protocol pro-gresses. After correction for the different values of the purewater melting enthalpy at the actual phase change temperaturefor each of these processes, the amount of thawed water ineach process could be calculated by simple comparison withthis quantity. The resulting frozen water content, presented inTable 1 relative to either the total sample mass, Wf(s), the drymass, Wf(dm), or the total water content, Wf(w), again markedlydecreased as the protocol progressed. The last step could alsobe comprised in this trend, as Wf would equal to zero.
The unfrozen water fraction (derived from the frozenwater fraction) can help to visualize the changes undergoingin the cryopreserved tissues, and is also shown in Table 1relative to the total sample mass, Wu(s), the dry mass, Wu(dm),or the total water content, Wu(w). Traditionally, frozen waterwas understood and often named as free water, whereas theunfrozen fraction was considered as bound water.43 Thesedenominations are much criticized nowadays,44 as the differ-ences found in the actual binding of these two water frac-tions are very small, if any at all.45–50
For comparative purposes, Figure 5 shows typical calori-metric thermograms obtained during rewarming for the cryo-protective solutions used (at stages b–d), in the sameconditions than those applied in the experiments of Figure 4.DHf(s) for stage b (liquid MS medium with 0.3 M sucrose)was 2286 2 J g sample
21, whereas for stage c (loading solu-tion) it was 1386 2 J g sample
21. Stage d (PVS2) showed nomelting exotherm. DHf(w) for stage b amounted to 2506 3 Jgw
21. The Tf(onset) were 23.97!C6 0.2!C for stage b and24.68!C6 0.3!C for stage c.
Effects of quench-cooling on heat of fusion of ice
In Figure 6, thermograms of the rewarming DSC stage ofmint shoot tips cooled by quenching are compared with
those at a rate of 10!C min21 in specimens of step a. It canbe seen that, in the quench cooled sample, the thawing tem-perature decreased and the enthalpy increased slightly. Theseresults, although consistent in all repeats and for experimentsin different conditions (data not shown), were unexpected,and of difficult explanation. A possibility is that the fastcooling might trap solute molecules within the ice phase,which would later contribute to an average effective meltingat lower temperatures.51–53
Nevertheless, this behavior, although may be further fromequilibrium than those observed in the slow cooling, wouldcorrespond more closely to the real events taking place incryopreservation practice.
Detection of glass transition by DSC
Specimens at the last stage of the cryopreservation proto-col did not show any freezing event, in any case, either atthe cooling (not shown) or melting runs of DSC studies.Nevertheless, in many experiments, the step in the heatcapacity baseline showing the actual glass transition in DSC
Figure 5. Typical DSC thermograms corresponding torewarming processes of the cryopreservation solu-tions used in different stages of the droplet cryo-preservation protocol: preculture (stage b): liquidMS medium containing 0.3 M sucrose, loading(stage c): loading solution, and dehydration (staged): PVS2.
The insert shows an expanded thermogram section whichallows the glass transition to be appreciated. Scanning ratewas 10!C min21 (See Materials and Methods and Figure 1for more details). The ordinates scale of the thermogramsbaseline is arbitrary.
Figure 6. DSC scans showing warming at 10!C min21 forshoot tips treated at the preculture stage (b) of thedroplet cryopreservation protocol.
Cooling rate was 10!C min21 or quenching (direct immersionof the DSC pan in LN).
Biotechnol. Prog., 2013, Vol. 29, No. 3 713
thermograms could not be appreciated. Cooling rates of10!C min21, controlled by the calorimeter itself, or exter-nally driven, by quick introduction into LN (quenching)were tested, but the glass transition could only be observed(for both cooling rates) for samples containing 30 shoot tips.Calorimetry is often claimed to be a rather insensitivemethod to observe the glass transition,54 and this is espe-cially applicable here, as the dehydrated tips have a verysmall weight (total average sample mass for each DSCexperiment—five tips—for d-stage specimens was "3.5 mgand the total mass of water in these samples was 2.5 mg).
As shown in the insert of Figure 5, PVS2 solution is alsoshowing a glass transition in these conditions, centered at2120!C.
Changes in sucrose content during the droplet-vitrificationprotocol
The actual composition of the cellular contents in cryopre-servation conditions is critical for the formation of ice or thevitrification of the system. Most studies rather equate thiscomposition (or the physical behavior of the cytoplasm) tothat of the cryopreservation solutions applied, which is notnecessarily correct. A simple extraction procedure and a gen-eral chromatographic method for small carbohydrate mole-cule quantification calibrated for sucrose were applied, as afirst approach to work out this composition. The sucrose con-tent data obtained are presented in Table 2, expressed asrelated to the total sample mass, Suc(s), or to the dry mass,Suc(dm), reflecting the increase in sucrose content within tis-sues, not only as a result of the actual increase of the contentof this compound but also of the decrease in that of water.
There was a strong increase in its level between stages band c (Table 2). Meanwhile, the sucrose incubation of thepreculture leading to stage b only implied a moderateincrease (about 10%) of the internal sucrose level; the load-ing with glycerol gave rise to a dramatic increase of thisconcentration (sevenfold). The stage d, dehydration, afterPVS2 treatment underwent a further increase, but muchmore moderate. This could be interpreted as a reflection ofthe actual cellular water content, and was in good agreementwith the data of water contents and its frozen and unfrozenfraction shown in Table 1.
Other sources for changes in sucrose levels, different fromthe alteration in the water content, were possible, such as theactual penetration of sucrose, which could be facilitated bythe presence of DMSO in the PVS2 solution, an agentknown for promoting the penetration of other solutes throughmembranes.2
Equivalent solute concentrations (isoosmotic) can beobtained from the freezing equilibrium temperatures obtainedby DSC.55 The observed decrease in this temperature can berelated by Raoult’s law to the molal concentration of the
solution. In the case of tissues, it would rather be an averageof the different solution pools in the different cell types andsubcellular compartments of the specimens studied. The ana-lytical equations are only valid for solutions much morediluted than those considered here, and their applicationwould overestimate the solvent concentration required toinduce the observed cryoscopic decrease. The Tf(peak)obtained was, so, compared with tabulated data sets, comple-mented when necessary with experimental DSC determina-tions for concentrated solutions (data not shown). Equivalentsucrose concentration, Suceq, and equivalent sodium chlorideconcentrations, SaCleq, were calculated and presented in Ta-ble 2. These coarse estimations could not describe the com-plex composition of cellular fluids. Nevertheless, as theobserved decreases in Tf were caused by the increased num-ber of molecules in solution, the amount of small molecularweight compounds (either dissociated as NaCl or not, as su-crose) would have a more determinant role than the presenceof larger macromolecules and other specific plant-generatedsubstances.
When comparing the chromatographic sucrose contentwith these isoosmotic solute estimated concentrations, itcould be seen that the total solute content of cellular com-partments must be much higher than the sucrose amountreported. Especially, a high increase in the estimated soluteconcentrations could be appreciated after incubation with theloading solution, may be reflecting the entrance of significantamounts of glycerol in cells, apart from the already com-mented dehydration. It must be noted, when comparing Ta-ble 2 parameters, that although the sucrose contentchromatographically determined refers to the total or drysample mass of the shoot tips, the estimated sucrose and so-dium chloride contents, as originated from the cryoscopicdecrease, refer to the solution mass (only considering solutesand solvent).
General discussion
The combined results of DSC and cryo-SEM are in goodagreement, implying that part of the water contained in thesystem for specimens on steps a and b becomes frozen whencooled in LN (with less amount of ice formation in the lat-ter), while specimens in step c would avoid ice formation,presumably getting vitrified in the cooling process, as theyshowed no ice trace, either microscopically or calorimetri-cally. This is also in good agreement with our data for via-bility and recovery and those of literature, resulting ofapplying the droplet method to several systems, includingmint apices.11,56–64
The different experimental approaches used agree in areduction of the cellular water content with the cryopreserva-tion protocol progress. The major dehydration would takeplace during the loading solution incubation (stage c, showingmuch lower water content than b). This agrees well with the
Table 2. Solute Content of Mint Shoot Tips at the Different Stages of the Droplet Cryopreservation Protocol
Cold Treatment(stage a)
Preculture(stage b)
Loading(stage c)
Dehydration(stage d)
Suc(s) (ppmtotal sample weight) 5,9006 600 6,5006 300 46,9006 700 53,0006 4,000Suc(dm) (g gdm
21) 0.0596 0.006 0.0446 0.002 0.1376 0.002 0.2096 0.015Suceq (ppm)* 8,800 10,800 470,000 –
NaCleq (ppm)* 8,000 10,000 91,000 –
*These concentrations, as derived from the freezing point depression, refer to the solution mass (mass of solutes plus mass of solvent), not takinginto consideration any nondissolved substances.
714 Biotechnol. Prog., 2013, Vol. 29, No. 3
dry weight evolution, as well as the calorimetric frozen watercontent and the observed decrease of the equilibrium freezingtemperature. The decrease in the size of ice crystals observedby cryo-SEM and the increases in the sucrose contentobtained by ionic chromatography would confirm this result.
Regarding the last and most interesting stage d, there isless information available, as on one hand, the presence oflarge quantities of volatile substances precludes the measure-ment of a reliable water content by differential weighing,and on the other hand, there were no freezing eventsobserved that could yield information about water fractionsor cryoscopic decrease. Nevertheless, the sucrose contentdata could imply a further dehydration degree over stage c,unless a significant amount of the sucrose contained in PVS2solution could have penetrated cells facilitated by DMSO.The evidences of vitrification derived from the cryo-SEMobservations and the glass transition detected in DSC (aswell as the lack of freezing or thawing events at any scan-ning rate) justify a higher cytoplasm viscosity in stage dthan in stage c specimens, evidently not vitrified. This vis-cosity increase could be originated either by a further watercontent reduction or by the entrance in cells of the viscosityenhancing PVS2 components (or both).
The incubation with glycerol, a known penetratingagent,8,39–42 has been reported to promote water exit fromcells, driven by the osmotic gradient, but also a substitutionof part of this water by glycerol inside cells.39 The compari-son of total shoot tips specimen and dry mass (Table 1) withwater content data from other workers,8 whose mint shoottip protocol is not equivalent but similar enough, allows tocalculate that for stage c specimens, at a water content of"1.8 gwater gdm
21, and the amount of glycerol possibleincorporated to shoot tips would be small (about 0.1 gwatergdm
21). Although in this case most of the effect of the proto-col step would be, then, simple osmotic dehydration, thesame type of calculation applied to stage d yields, for awater content of "0.7 gwater gdm
21, an entrance of extrinsiccompounds corresponding to nearly 60% of the final speci-men mass (about 2.3 gwater gdm
21).
A general reflection on these data may acknowledge thenatural variability of the properties determined here. In spiteof the efforts to prepare equivalent start point materials andto treat them in a similar way, differences in size, permeabil-ity, microscopic scale damage, and all short of small compo-sitional and microstructural differences would exist in a waynot easy to monitor. Comparison with other similar experi-ments (data not shown), it can be observed that this variabil-ity would have a larger impact on the equilibrium freezingtemperature data, while the enthalpy and associated waterfractions would be more constant. And, an absolute observa-tion is that for all repeats, sets of experiments, and evencooling rates applied, the final stage of the cryopreservationprotocol is never producing calorimetrically detectable for-mation of ice. This is in good agreement with the viabilityresults found for this protocol,11 viability that is incompati-ble with the formation of ice.
The calorimetric properties (melting enthalpies and equilib-rium freezing and glass transition temperatures) of thecryoprotecting solutions are not too different of those of thespecimens treated with them. In addition to some differencesbeing evident, this calorimetric similarity cannot be takingas a proof of equal composition and/or behavior in otherrespects.
The probabilities of ice formation in specimens under TGand over TG but at temperatures not too elevated (roughly,no more than 20!C over TG) are similar, if a short frametime is considered, such as those corresponding to cooling orwarming processes in cryopreservation. But, this may be notso for longer time periods, such as those corresponding tostorage. Although storage is normally carried out below2150!C, well under these systems TG, it must be remem-bered that, when over TG, at long storage periods, ice forma-tion is always a real possibility.38,65
A further consideration can be derived from the lack ofice observations in DSC at the last stage of the cryopreserva-tion protocol, when the cooling is carried out at 10!C min21,a rather slow speed. This, generally observed for this sam-ples and this protocol (data not shown), would be indicativeof a possible excessive amount of cryoprotecting agents,although, for the biological material used here, we find thatthere is no appreciable damage, at least in terms of viabilityand recovery. Cooling at 10!C min21 would mean that thesamples are in the ice formation window (roughly, withoutconsidering the differences in ice formation probabilities fordifferent temperature regions, between 220 and 2110!C)for about 9–10 min. Quench cooling would mean a perma-nence in this ice formation possible temperature window foronly a few seconds (data not shown). Considering only theeffects of the cryopreservation protocol in the formation ofice (not valuating here the possible membrane or proteinprotection toward low temperature or dehydration, for exam-ple) a reduction in the concentration of the vitrification solu-tion used could produce specimens that may be ice-formation prone at low cooling rates but would not form iceat quenching rates, which are those actually used in practicalcryopreservation. This observation should be taken into con-sideration, especially when the effect of some of the cryo-protecting components of these solutions, such as DMSO, iscriticized for its possible mutagenic or cytotoxic effects.66
Conclusions
Specimens of mint shoot tips at the final step of the drop-let-vitrification cryopreservation protocol become vitrifiedand no ice is formed, which is associated to the lack of dam-age in the process and the resulting high viability. Mean-while, shoot tips at previous steps of the protocol show theformation of ice by both microscopic and calorimetric obser-vations, which is a possible cause of damage leading to theobserved low viability of these specimens. The agreementbetween both cryo-SEM and DSC techniques is good andthe potential of its associated use is clearly shown.
Acknowledgments
This work has been carried out thanks to project“CRYODYMINT” (AGL2010-21989-C02-02) of the SpanishMinistry of Science and Innovation. A.S. Teixeira was sup-ported by the CSIC, within the JAE-Pre program, partiallyfunded from the European Social Fund. The expert technicalwork of F. Pinto is also acknowledged.
Literature Cited
1. Towill LE. Cryopreservation of isolated mint shoot tips by vitri-fication. Plant Cell Rep. 1990;9:178–180.
2. Sakai A, Engelmann F. Vitrification, encapsulation-vitrificationand droplet-vitrification: a review. CryoLetters. 2007;28:151–172.
Biotechnol. Prog., 2013, Vol. 29, No. 3 715
3. Muthusamy J, Staines HJ, Benson EE, Mansor M, KrishnapillayB. Investigating the use of fractional replication and taguchitechniques in cryopreservation: a case study using orthodoxseeds of a tropical rainforest tree species. Biodivers Conserv.2005;14:3169–3185.
4. Nadarajan J, Mansor M, Krishnapillay B, Staines HJ, Benson E,Harding K. Applications of differential scanning calorimetry indeveloping cryopreservation strategies for Parkia speciosa, atropical tree producing recalcitrant seeds. CryoLetters.2008;29:95–110.
5. Zamecnik J, Bilavcik A, Faltus M. Importance, involvement anddetection of glassy state in plant meristematic tissue for cryopre-servation. Cryobiology. 2007;5:324–378.
6. Niino T, Sakai A, Yakuwa H, Nojiri K. Cryopreservation of invitro-grown shoot tips of apple and pear by vitrification. PlantCell Tissue Organ Cult. 1992;28:261–266.
7. Reinhoud PJ. Cryopreservation of tobacco suspension cells byvitrification. Doctoral Paper, Rijks University, Leiden, TheNetherlands, 1996.
8. Volk GM, Walters C. Plant vitrification solution 2 lowers watercontent and alters freezing behavior in shoot tips during cryo-protection. Cryobiology. 2006;52:48–61.
9. Wesley-Smith J, Walters C, Pammenter NW, Berjak P. Interac-tions among water content, rapid (nonequilibrium) cooling to2196!C, and survival of embryonic axes of Aesculus hippocas-tanum L. seeds. CryoLetters. 2001;42:196–206.
10. Kauzmann W. The nature of the glassy state and the behaviorof liquids at low temperatures. Chem Rev. 1948;43:219–256.
11. Senula A, Joachim Keller ER, Sanduijav T, Yohannen T. Cryo-preservation of cold-acclimated mint (Mentha spp.) shoot tipsusing a simple vitrification protocol. CryoLetters. 2007;28:1–12.
12. Kartha KK, Leung NL, Mroginski LA. In vitro growth-responses and plant-regeneration from cryopreserved meristemsof cassava (Manihot esculenta Crantz), Z Pflanzenphysiol.1982;107:133–140.
13. Sakai A, Kobayashi S, Oiyama I. Cryopreservation of nucellarcell of navel orange (Citrus sinensis Obs. var. brasiliensisTanaka) by vitrification. Plant Cell Rep. 1990;9:30–33.
14. Reed BM. Plant Cryopreservation: A Practical Guide. NewYork: Springer; 2008.
15. Dereuddre J, Scottez C, Arnaud Y, Duron M. R!esistance d’apexcaulinaires de vitroplants de Poirier (Pyrus communis L. cvBeurr!e Hardy), enrob!es dans l’alginate, "a une d!eshydratationpuis "a une cong!elation dans l’azote liquide: effet d’un endur-cissement pr!ealable au froid. C R Acad Sci. 1990;10:317–323.
16. Jouve L, Hoffmann L, Hausman JF. Polyamine, carbohydrate,and proline content changes during salt stress exposure of aspen(Populus tremula L.): involvement of oxidation and osmoregula-tion metabolism. Plant Biol. 2004;6:74–80.
17. Fahy GM, MacFarlane DR, Angell CA, Meryman HT. Vitrifica-tion as an approach to cryopreservation. Cryobiology.1984;21:407–426.
18. Benson EE. An introduction to plant conservation biotechnol-ogy. In: Benson EE, editor. Plant Conservation Biotechnology.London: Taylor & Francis; 1999:3–10.
19. Suzuki M, Ishikawa M, Okuda H, Noda K, Kishimoto T, Naka-mura T, Ogiwara I, Shimura I, Akihama T. Physiologicalchanges in gentian axillary buds during two-step preculturingwith sucrose that conferred high levels of tolerance to desicca-tion and cryopreservation. Ann Bot. 2006;97:1073–1081.
20. Carpenter JF, Dawson PE. Quantification of dimethyl sulfoxidein solutions and tissues by high performance liquid chromatog-raphy. Cryobiology. 1991;28:210–215.
21. Cheng X, Kaplan LA. Simultaneous analyses of neutral carbo-hydrates and amino sugars in freshwaters with HPLC-PAD. JChromatogr Sci. 2003;41:434–438.
22. Lee YC. Carbohydrate analyses with high-performance anion-exchange chromatography. J Chromatogr A. 1996;720:137–149.
23. Masuda T, Kitahara K, Aikawa Y, Arai S. Determination of car-bohydrates by HPLC-ECD with a novel stationary phase pre-pared from polystyrene-based resin and tertiary amines. J AmChem Soc. 2001;17:895–898.
24. Schiller M, von der Heydt H, M#arz F, Schmidt P. Quantificationof sugars and organic acids in hygroscopic pharmaceuticalherbal dry extracts. J Chromatogr A. 2002;968:101–111.
25. Matsumoto T, Sakai A, Yamada K. Cryopreservation of in-vitrogrown apical meristems of wasabi (Wasabia japonica) by vitrifi-cation and subsequent high plant-regeneration. Plant Cell Rep.1994;13:442–446.
26. Yamada T, Sakai A, Matsumura T, Higuchi S. Cryopreservationof apical meristems of white clover (Trifolium repens L.) by vit-rification. Plant Sci. 1991;78:81–87.
27. Gonz!alez-Benito ME, Mendoza-Condori VH, Molina-Garc!ıaAD. Cryopreservation of in vitro shoot apices of Oxalis tuber-osa Mol. CryoLetters. 2007;28:23–32.
28. Craig S, Beaton CD. A simple cryo-SEM method for delicateplant tissues. J Microsc. 1996;182:102–105.
29. Murashige T, Skoog F. A revised medium for rapid bioassayswith tobacco tissue cultures. Physiol Plantarum. 1962;15:473–497.
30. Fullerton GD, Keener CR, Cameron IL. Correction for solute/solvent interaction extends accurate freezing point depressiontheory to high concentration range. J Biochem Biophys Methods.1994;29:217–235.
31. Elliott JAW, Prickett RC, Elmoazzen HY, Porter KR, McGannLE. A multisolute osmotic virial equation for solutions of inter-est in biology. J Phys Chem B. 2007;111:1775–1785.
32. Guignon B, Torrecilla JS, Otero L, Ramos AM, Molina-Garc!ıaAD, Sanz PD. The initial freezing temperature of foods at highpressure. Crit Rev Food Sci Nutr. 2008;48:328–340.
33. Wolf AV, Brown MG, Prentiss PG. Concentrative properties ofaqueous solutions. In: Weast RC, editor. Handbook of Chemis-try and Physics. Boca Raton: CRC Press; 1985:D-219–D-271.
34. Otero L, Molina-Garc!ıa AD, Sanz PD. Some interrelated ther-mophysical properties of liquid water and ice. I. A user-friendlymodelling review for food high-pressure processing. Crit RevFood Sci Nutr. 2002;42:339–352.
35. Angell CA, Shuppert J, Tucker JC. Anomalous properties ofsupercooled water. Heat capacity, expansivity, and proton mag-netic resonance chemical shift from 0 to – 38!. J Phys Chem.1973;77:3892–3899.
36. Angell CA, Ogunl M, Slchlna WJ. Heat capacity of water atextremes of supercooling and superheating. J Phys Chem.1982;86:998–1002.
37. Umrath W. Calculation of the freeze-drying time for electron-microscopical preparations. Mikroskopie. 1983;40:9–34.
38. Franks F. Metastable water at subzero temperatures. J Microsc.1986;141:243–249.
39. Benson EE. Cryopreservation theory. In: Reed BM, editor. PlantCryopreservation: A Practical Guide. New York: Springer;2008:15–32.
40. Sch#afer-Menhur A, Schumacher HM, Mix-Wagner G. Cryopre-servation of potato cultivars—design of a method for routineapplication in genebanks. Acta Hort. 1997;447:477–482.
41. Hub!alek Z. Protectants used in the cryopreservation of microor-ganisms. Cryobiology. 2003;46:205–229.
42. Panis B, Lombardi M. Status of cryopreservation technologiesin plants (food and forest crops). In: Ruane J, Sonnino A, edi-tors. The Role of Biotechnology in Exploring and ProtectingAgricultural Genetic Resources. Rome: FAO; 2006:61–78.
43. Robinson W. Free and bound water determinations by the heatof fusion of ice method. J Biol Chem. 1931;92:699–709.
44. Franks F. Unfrozen water - yes - unfreezable water - hardly -bound water - certainly not. CryoLetters. 1986;7:207.
45. Hills BP, Takacs SF, Belton PS. A new interpretation of protonNMR relaxation time measurements of water in food. FoodChem. 1990;37:95–111.
46. Wiggins PM. Role of water in some biological processes.Microbiol Rev. 1990;54:432–449.
47. Hills BP, Cano C, Belton PS. Proton NMR relaxation studies ofaqueous polysaccharide systems. Macromolecules. 1991;1:2944–2950.
48. Slade L, Levine H, Reid DS. Beyond water activity: recentadvances based on an alternative approach to the assessment offood quality and safety. Crit Rev Food Sci Nutr. 1991;30:115–360.
716 Biotechnol. Prog., 2013, Vol. 29, No. 3
49. Hills BP, Tang H, Belton PS, Khaliq A, Harris RK. NMR Oxy-gen-17 studies of the state of water in a saturated sucrose solu-tion. J Mol Liq. 1998;75:45–59.
50. Wolfe J, Bryant G, Koster KL. What is ‘unfreezable water’,how unfreezable is it and how much is there? CryoLetters.2002;23:157–166.
51. Obbard R, Iliescu D, Cullen D, Chang J, Baker I. SEM/EDScomparison of polar and seasonal temperate ice. Microsc ResTech. 2003;62:49–61.
52. Ohnoa H, Igarashib M, Hondota T. Salt inclusions in polar icecore: location and chemical form of water-soluble impurities.Earth Planet Sci Lett. 2005;232:171–178.
53. Hashimoto T, Tasaki Y, Harada M, Okada T. Electrolyte-dopedice as a platform for atto- to femtoliter reactor enabling zepto-mol detection. Anal Chem. 2011;83:3950–3956.
54. Verdonck E, Schaap K, Thomas L. A discussion of the princi-ples and applications of modulated temperature DSC (MTDSC).Int J Pharm. 1999;192:3–20.
55. Buckley KA, Conway EJ, Ryan HC. Concerning the determina-tion of total intracellular concentrations by the cryoscopicmethod. J Physiol. 1958;143:236–245.
56. Keller ERJ, Dreiling M. Potato cryopreservation in Germany—using the droplet method for the establishment of a new largecollection. Acta Hort. 2003;623:193–200.
57. Halmagyi A, Deliu C, Coste A. Plant regrowth from potatoshoot tips cryopreserved by a combined vitrification-dropletmethod. CryoLetters. 2005;26:313–322.
58. Leunufna S, Keller ERJ. Cryopreservation of yams using vitrifi-cation modified by including droplet method: effects of coldacclimation and sucrose. CryoLetters. 2005;26:93–102.
59. Panis B, Piette B, Swennen R. Droplet vitrification of apicalmeristems: a cryopreservation protocol applicable to all Musa-ceae. Plant Sci. 2005;168:45–55.
60. Kim HH, Lee JK, Yoon JW, Ji JJ, Nam SS, Hwang HS, ChoEG, Engelmann F. Cryopreservation of garlic bulbil primordiaby the droplet-vitrification procedure. CryoLetters. 2006;27:143–153.
61. Kryszczuk A, Keller J, Gr#ube M, Zimnoch-Guzowska E. Cryo-preservation of potato (Solanum tuberosum L.) shoot tips usingvitrification and droplet method. Food Agric Environ.2006;4:196–200.
62. Sant R, Panis B, Taylor M, Tyagi A. Cryopreservation of shoot-tips by droplet vitrification applicable to all taro (Colocasiaesculenta var. esculenta) accessions. Plant Cell Tissue OrganCult. 2008;92:107–111.
63. Halmagyi A, Deliu C, Isac V. Cryopreservation of Malus culti-vars: comparison of two droplet protocols. Sci Hortic.2010;124:387–392.
64. Vujovic T, Sylvestre I, Ruzic D, Engelmann F. Droplet-vitrifica-tion of apical shoot tips of Rubus fruticosus L. and Prunus cera-sifera Ehrh. Sci Hortic. 2011;130:222–228.
65. Angell CA. Supercooled water. In: Franks F, editor. Water—AComprehensive Treatise, Vol. 7. New York: Plenum Press;1982:215–338.
66. Harding K. Genetic integrity of cryopreserved plant cells: areview. CryoLetters. 2004;18:217–230.
Manuscript received Oct. 26, 2012, and revision received Jan. 11,2013.
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