in vitro techniques as supportive tools for breeding and

1

Development of in vitro techniques as supportive

tools for breeding and mass clonal propagation of

Leucocoryne spp. (Amaryllidaceae)

Alejandro Félix Altamira Bravo

2016

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Pontificia Universidad Católica de Chile Facultad de Agronomía e Ingeniería Forestal

Development of in vitro techniques as supportive tools for breeding and mass clonal propagation of Leucocoryne spp.

(Amaryllidaceae)

Alejandro Félix Altamira Bravo

Thesis to obtain the degree of

Doctor

Ciencias de la Agricultura

Santiago, Chile, December 2016

3

Thesis presented as part of the requirements for the degree of Doctor en Ciencias de la Agricultura, approved by the

Thesis Committee

_____________________ Gloria Montenegro, Advisor

__________________ Dr. Patricio Arce Johnson

___________________ Dr. Levi Mansur

Santiago, December 2016

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In memory of my grandmother Adela, with whom I spent many days learning to appreciate and propagate plants during my childhood.

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This work was supported by CONICYT doctoral grant number 21110937

and FIA Project PYT-2012-0079.

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ACKOWLEDGEMENTS

I would like to thank all those who were members of my committee, Professors

Gloria Montenegro, Patricio Arce, Levi Mansur and Eduardo Olate for all their

valuable contributions in the development of this thesis.

I would also like to thank Professor Marlene Gebauer for her infinite support and

for allowing me to finish this thesis in her laboratory.

I also thank the ex-members of the “Laboratorio de cultivo in vitro y Ornamentales”

with whom I shared during the development of this thesis, especially Nicole Arenas

and Gonzalo Gutierrez for their friendship and support.

Thanks to my parents Sergio and Mónica and my sisters Camila and Josefa for

their love and unconditional support.

And especially I want to thank my partner Mario for all these years of friendship,

company, support and challenges that we have overcome together.

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CONTENTS

CHAPTER 1.

General Introduction ........................................................................... 8

CHAPTER 2.

An efficient clonal micropropagation protocol for Leucocoryne, a

geophyte genus endemic of Chile .................................................... 26

CHAPTER 3.

An efficient in vitro production system for Leucocoryne spp. plants

initiated from seeds .......................................................................... 68

CHAPTER 4.

Conclusions ...................................................................................... 92

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CHAPTER 1

GENERAL INTRODUCTION

9

1. Description of Leucocoryne genus.

Leucocoryne genus belongs to the Amaryllidaceae family and is endemic to Chile.

There are 17 described taxons in the genus (15 species and 2 subspecies),

however its taxonomic classification is still confusing due to its high morphological

variability and the occurrence of natural hybrids. Consequently some authors

mention up to 45 species as part of the genus (Muñoz and Moreira 2000;

Riedemannn and Aldunate 2001; Mansur 2002; Zoellner 2002; Mansur and

Cisternas 2005; Zuloaga et al. 2009; Olate and Schiappacasse 2013). The natural

life cycle of Leucocoryne can vary depending on the species but, in general, the

plant takes three years from seed to floral bulb size (Riedemannn and Aldunate

2001; Mansur 2002).

In general, this genus presents high adaptation to dry climates and is widely

distributed along Chile, from lat 20°S in deserted zones until lat 41°S in more rainy

temperate zones, with populations ranging from mild coastal habitats to more

colder mountainous areas where the soil is cover with snow for several months, at

altitudes of 2.800 m.a.s.l. in the Andes mountains. However, most of the

Leucocoryne populations grow in coastal areas in Central Chile (lat 30°-35°S), in

dry climates that show a marked rainy season from May to August and a 70 mm

average annual rainfall (Zoellner 1972; Kim et al. 1998; Mansur et al. 2004; Olate

and Schiappacasse 2013).

Plants of this genus are considered geophytes due the presence of an

underground storage structure, which in the case of Leucocoryne corresponds to a

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tunicated bulb. These bulbs have a spherical or oval shape, reaching 1.5 to 2.5 cm

in diameter, covered by brown dry membranes and having up to twenty fleshy

scales on the inside. Bulbs have a basal plate from which adventitious roots

develop (Hartmann et al. 1997; Zoellner 2002).

Leaves have a flaccid, glabrous and semi-fleshy appearance. From the bulbs a

single floral scape grow whose height can vary between 30 and 80 cm. Its apical

inflorescence it is an umbel that can have from 3 - 4 to 8 - 12 flowers depending on

the species, although 5 - 15 flowers have also been described. Flowers are perfect,

with the stigma and stamens attached to the floral tube, with 2.5 - 6 cm in diameter,

six tepals 1.4 - 2 cm in length exhibiting colors from white, blue-sky, purple, violet

and others (Zoellner 1972; Mansur 2002; Schiappacasse et al. 2002).

The androecium is composed of three or six fertile stamens and three fleshy

staminodes (infertile stamens) 6 mm in length, with color varying between white,

yellow, greenish or two-colored. The gynoecium has a cylindrical superior ovary,

with a short style and a capitate stigma. Fruit corresponds to a tricarpellary

dehiscent capsule, and each carpel has several seeds. In general, 15 - 40 seeds

per fruit are produced, which are small with sizes of 0.1 - 0.2 cm in diameter

(Muñoz and Moreira 2000; Mansur 2002; Schiappacasse et al. 2002; Zoellner

2002; Verdugo 2013).

Besides the diversity in shape and colors, it has been genetically verified the

existence of diversity in terms of chromosomal number, which can vary from 2n =

10 in Leucocoryne purpurea to 2n = 18 in Leucocoryne coquimbensis, with hybrids

showing 2n = 14, 2n = 20 y 2n = 22 (Mansur 2002; Araneda et al. 2004; Salas and

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Mansur 2004). Also, it has been reported six Leucocoryne species as self-

incompatible, which would explain the high genetic variability (Mansur 2002;

Mansur et al. 2004).

2. Advances in Leucocoryne domestication and commercial use.

The Leucocoryne genus is known in Chile by the common name "Huilli" and

internationally as "Glory-of-the-Sun". Because the high phenotypic variability of

flowers (colors, shapes and aroma) and also because its long vase life,

Leucocoryne has become a great alternative as an ornamental plant, either as cut

flower, potted or garden plant (Bridgen 2000; Mansur 2002; Olate and

Schiappacasse 2013; De la Cuadra et al. 2016).

The ornamental value of the different species has led to several studies and

domestication efforts and even incipient commercialization of them in countries like

Japan, Netherlands, Israel, New Zealand and USA (Bridgen 2000; Lancaster et al.

2000; Walton et al. 2008; Olate and Schiappacasse 2013; De la Cuadra et al.

2016). This interest is similar to the one occurred with Alstroemeria genus, which

has been used by companies in other countries, for selecting, breeding and

commercialization with high economic success, using Chilean germplasm. These

are examples of importance of identifying, selecting, propagating, breeding and

registration of plant material belonging to Chilean native genetic resources as

Leucocoryne, which could also lead to economic benefits and improve the

conservation of endangered populations (Jorquera et al. 2007).

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In order to take advantage of the commercial potential and also to conserve

Leucocoryne, which has already been affected by antrophic intervention of their

environment, conservation programs have been developed in Chile. Sustainable

germplasm banks, genetic and agronomic studies for future uses in landscaping,

and also breeding programs for releasing new cut-flower cultivars have been

developed by researchers at the Pontificia Universidad Católica de Valparaíso. To

date, three cut-flower cultivars ('Elena', 'Gabriela' and 'Paulina') have been

patented (Verdugo and Teixeira 2006). Nevertheless, an efficient mass

propagation system is still a pending challenge for successfully commercialization

of these new cultivars (Bridgen 2000; Mansur 2002; Araneda et al. 2004; Olate and

Schiappacasse 2013).

3. Propagation techniques

3.1 Seed propagation

Seed propagation of Leucocoryne naturally occurs when seeds are released from

its dehiscent capsule sometime during the spring. Seeds stay on the soil surface

during all summer and part of the autumn until the next rainy season (autumn and

winter), with temperatures ranging 10 to 15 °C (Mansur 2002; De la Cuadra et al.

2016). Once germination occurs, plants develop a single leaf and after 45 days a

small bulb starts to grow. This bulb can weight 0.06 g in average after 100 days

since germination, and then it enters in dormancy until the next season. During the

next autumn these small bulbs could produce shoots even in absence of water,

13

using for that their reserves getting then ready for the winter rains. During this

second growing season two leaves will appear and the plant will stay active for 100

- 120 days producing more bulb weight increase until it becomes dormant again

until next rainy season. Only during the third or fourth growing season the bulbs will

reach 0.3 g in weight and the flowering process will happen and new seeds will be

produced (Riedemannn and Aldunate 2001; Mansur 2002; De la Cuadra and

Mansur 2004).

Germination studies have been conducted on L. coquimbensis, L. ixioides and L.

purpurea, which have determined that water imbibition for one day and subsequent

cold stratification at 7°C for 7 weeks results in higher germination rates (over 90%)

compared to non-stratified seeds (Salazar 2001; Schiappacasse et al. 2002).

Similar studies have also achieved high germination rates by imbibition of

Leucocoryne seeds for 96 h and subsequent culture at 10 - 15°C, without a

stratification period, higher than germination observed at 20°C and no germination

at 25°C (Jara et al. 2006; De la Cuadra et al. 2016).

Due to the high genetic variability of Leucocoryne and self-incompatibility in some

of its species, seed propagation techniques have great disadvantages to maintain

interesting traits and massive propagation of new registered cultivars. Despite this,

seed propagation is still used as a valuable technique in breeding programs to

obtain new plant segregants (Mansur 2002; Mansur et al. 2004; De la Cuadra et al.

2016).

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3.2 Vegetative propagation

Vegetative propagation consists in obtaining new plants from other plant organs

like stems, leaves, roots, bulbs, corms and rhizomes. The main advantage related

to this techniques is that new developed plants are genetically identical to the

propagated plant (Schiappacasse et al. 2002; Kumar et al. 2010). In Leucocoryne,

as in other geophyte genus, natural vegetative propagation occurs in the second or

third year of growth, producing 1 - 2 lateral bulblets every one or two years

depending on the genotype. In some species and in particular environmental

conditions the plants can produce even more deep bulbs called "droppers"

(Riedemannn and Aldunate 2001; Mansur 2002; Schiappacasse et al. 2002).

In some tunicate bulb species artificial propagation techniques allow to increase

natural multiplication rates. These techniques are known as bulb-cutting methods

and they consist essentially in to generate a mechanical damage to the basal plate

or to the entire bulb. "Scooping" is one of these techniques that involve the

complete elimination of the basal plate, the apical meristem and the base of the

scales (Fig. 1b). In the "scoring" method, deep cuts to the base of the bulb are

done in order to damage its basal plate and the apical meristem (Fig. 1c). "Coring"

is other of these methods, and consists in the removal of a cylindrical section of the

basal plate, including the apical meristem (Fig. 1d). In other species it is used the

method called "sectioning", where the entire bulb is cut in two or more vertical

sections in order to produce adventitious bulblets (Fig. 1e) (Hartmann et al. 1997;

Schiappacasse et al. 2002; Olate and Bridgen 2005).

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Fig. 1 Artificial propagation techniques used in some bulb species. a) Intact bulb, b) Scooping, c) Scoring, d) Coring, and e) "Sectioning". Adapted from Kumar et al. (2010).

Studies in Leucocoryne coquimbensis have determined that sectioning a bulb in

equal sections could produce up to 2.2 bulbs of different sizes, while in

Leucocoryne ixiodes up to 5 new bulbs have developed from a singular initial bulb

(Schiappacasse et al. 2002). Nevertheless, in a similar study, L. ixioides

multiplication rates of intact bulbs were compared to bulbs multiplied using

scooping and scoring, founding no significant differences between the natural and

artificial methods (Salazar 2001). These results do not coincide with the successful

results observed in Hyacinthus and Scilla species, where scooping techniques

produce much higher multiplication rates than natural propagation, and it is used

for commercial production (Hartmann et al. 1997).

3.3 In vitro propagation

Micropropagation, or in vitro propagation, corresponds to the culture of explants

under aseptic conditions, onto a nutritive medium, under controlled temperature,

humidity and luminosity conditions, to produce high number of clonal plants in a

16

reduced period of time (Roca and Mroginski 1991; Hartmann et al. 1997; Kyte et al.

2013).

In vitro shoot growth can be achieved using direct organogenesis from axillary or

adventitious buds, or using indirect organogenesis from cells, cell suspensions or

calluses. However, indirect organogenesis has the disadvantage of increasing the

risk of producing genetically non true-to-type plants (somaclonal variation) and thus

affecting the homogeneity of the plants produced (George et al. 2008).

Micropropagation has several stages to obtain new plants. Stage 0 includes the

selection of the genotype and the initiation explant. Stage I corresponds to the

disinfection and establishment of the explant in aseptic culture conditions. Once

the explants are stabilized in vitro conditions, mass plant multiplication could be

performed (Stage II). Stage III of the micropropagation process is where the in vitro

rooting is achieved. Finally, once the plants are fully developed, plants are

transferred to ex vitro conditions to be culture into soil or potting soil in order to

start their acclimatization (Stage IV). Duration and conditions for each of these

stages must be adapted to different plant genotypes (George et al. 2008; Bach and

Sochacki 2012; Kyte et al. 2013).

In contrast with other ornamental geophyte species, only a few studies of in vitro

culture have been done in Leucocoryne sp. One of the main barriers hindering a

wider use of these techniques in geophyte species, is the high contamination

occurring during the in vitro initiation of bulbs, corms, rhizomes and tubers,

because their permanent contact with soil and high level of microorganisms, as

fungi and bacteria (Pedersen and Brandt 1992; Slabbert et al. 1993; Kritzinger et

17

al. 1998; Smith et al. 1999; Chang et al. 2003; Sochacki and Orlikowska 2005;

Paredes et al. 2014). Previous works in Leucocoryne species have reported

contamination rates between 42% and 100% during the in vitro initiation of bulbs

(Fuentevilla 2004). In order to surpass this problem, previous studies involving

Leucocoryne sp. Bulbs have used seeds as initial explants with the consequent

problem of uncertain genotypic homogeneity of the plants used (Ham 2002;

Briones 2003; Escobar et al. 2008). In one of these studies different concentrations

of MS (Murashige and Skoog 1962) were tested as germination media for L.

purpurea seeds (i.e. 12.5%, 25%, 37.5%, 50%, 75% and 100%) resulting in higher

germination rates when seeds were cultured onto 75% MS or lower (Ham 2002).

Escobar et al. (2008) studied indirect organogenesis in L. purpurea by using MS

medium supplemented with 30 g·L-1 sucrose and 0.6% agar, pH 5.7 and a

combination of different plant growth regulators: 2,4-Dichlorophenoxyacetic (2,4-

D), 6-bencyladenine (BA), 1-Phenyl-3-(1-2-3-thiadiazol-5-yl)-urea (TDZ), α-

naphthaleneacetic acid (ANA), 6-y-y-(dimethylallyamino)-purine (2iP) and 4-

amino-3,5,6-trichloropicolinic acid (Picloram). Callus proliferation and shoot

formation was achieved. Then, after seven months of culture, 3 - 4 bulbs/explant

were obtained when subculture was done in absence of plant growth regulators.

Also, in previous studies (Olate and Bridgen 2005; Olate 2006), using MS medium

supplemented with 30 g·L-1 sucrose and 0.7% agar and pH adjusted to 5.7, it was

observed that ligh/darkness and temperature treatments does not affect bulb

production in L. purpurea, L. coquimbensis and L. ixioides. In the same study, in

vitro bulb-cutting methods were also tested. An average of almost 7 bulbs/explants

18

were obtained using a scoring method in L. coquimbensis bulbs, and over 8

bulbs/explant were produced by sectioning the bulbs in four, contrasting with

scarce occurrence of new bulblets on intact bulbs.

4 Hypothesis and objectives.

In terms of commercial potential and native germplasm concerns there is an

obvious need to develop asexual propagation techniques to assist either plant

breeding and conservation programs of Leucocoryne spp. One of the techniques

that provide several advantages over sexual propagation is the clonal

micropropagation. This system could avoid the long life cycle from seed to

flowering bulb and the low vegetative propagation rates that naturally occur in

these species. In this sense, hybrids or interesting clonal lines would benefit from

an in vitro propagation system, using direct organogenesis for example, to

propagate reliable and large populations without altering the genetic and

phenotypic characteristics of the original germplasm. Therefore, future studies

regarding the in vitro multiplication of Leucocoryne spp. should focus on increasing

clonal multiplication rates and on the optimal environmental conditions for the

growth and development of the explants.

In this context, we formulated the following hypothesis:

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4.1 Hypothesis.

Adjustment and optimization of the culture media, plant growth regulators, explant

type and growing temperature produces a more effective initiation process and

higher multiplication rates during the in vitro propagation of Leucocoryne species,

thus improving breeding and conservation outcomes.

4.2 General objective.

To determine in vitro media culture, propagation techniques and

environmental conditions to increase the growth and propagation rate

of Leucocoryne species to support breeding and conservation programs.

4.3 Specific objectives.

1. To determine the effect of culture media and system on in vitro bulb propagation rates of Leucocoryne sp.

2. To establish the effect of plant growth regulators on in vitro bulb propagation of Leucocoryne sp. bulbs

3. To evaluate the effect of bulb-cutting methods on in vitro bulb propagation rates of Leucocoryne sp.

4. To determine the effect of culture temperature on in vitro seed germination of Leucocoryne sp.

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CHAPTER 2

AN EFFICIENT CLONAL MICROPROPAGATION PROTOCOL FOR

Leucocoryne, A GEOPHYTE GENUS ENDEMIC TO CHILE

Alejandro Altamira1, Eduardo Olate1, Marlene Gebauer1, Levi Mansur2 and Gloria

Montenegro1

1Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería

Forestal, Pontificia Universidad Católica de Chile. Vicuña Mackenna 4860, Macul,

Santiago, Chile.

2Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Avda. San

Francisco s/n, La Palma, Quillota, Chile

This chapter was sent to Plant Cell, Tissue and Organ Culture (PCTOC) - Journal of Plant Biotechnology – Springer (December 2016)

27

An efficient clonal micropropagation protocol for Leucocoryne, a geophyte

genus endemic to Chile

Alejandro Altamira1*, Eduardo Olate1, Marlene Gebauer1, Levi Mansur2 and Gloria

Montenegro1.

1 Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal,

Pontificia Universidad Católica de Chile. Vicuña Mackenna 4860, Macul, Santiago, Chile.

2 Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Avda. San

Francisco s/n, La Palma, casilla 4-D, Quillota, Chile

*Corresponding author:

Name: Alejandro Altamira

Address: Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería


Santiago, Chile

Telephone: +56 9 90915256

E-Mail: [email protected]

Keywords: Amaryllidaceae, bulb, plant tissue culture, cytokinin, culture media, cut flower.

28

Abstract

Leucocoryne (Amaryllidaceae) is a geophyte genus endemic to Chile with

exceptional characteristics to use it as cut flower, pot or garden plant. The

objective of this work is to develop and improve an in vitro protocol for mass clonal

propagation to assist breeding and conservation efforts. We evaluated different in

vitro culture systems (agar, cotton), nutrient media (MS, LS, B5), BAP

concentrations (0.5, 1.0, 1.5 and 2.0 mgL-1) and different type of explants (intact

bulb, scoring and sectioning) in four Leucocoryne genotypes (Leucocoryne

purpurea, L. vittata, L. ixioides and L. sp. Pichicuy). Using an improved disinfection

process it was possible to initiate bulbs in vitro with a low contamination rate of 7%.

High bulb sprouting percentages were achieved, with values between 80 – 100%.

There was a higher rate of bulb multiplication and higher shoot production when

BAP was added to the growing media. Bulb-cutting methods resulted in the highest

multiplication rates reported to date, obtaining 11-16 bulblets per sectioned bulb,

depending on the genotype used.

Abbreviations: BAP - N6-benzylaminopurine; IAA - indole-3-acetic acid; MS –

Murashige and Skoog medium (1962); LS - Linsmaier and Skoog medium (1965);

B5 – Gamborg et al. medium (1968); Benomyl - Methyl-1-(butylcarbamoyl)-2-

benzimidazole carbamate; Captan - N-(triclorometiltio) ciclohex-4-eno-1,2-

dicarboximida.

29

Introduction

Leucocoryne is a geophyte genus endemic to Chile that belongs to

Amaryllidaceae. It is commonly known as "Huilli" or "Glory-Of-The-Sun" (Mansur

2002; Zoellner 2002; Sassone et al. 2014).

These plants present exceptional characteristics to be used as cut flowers, potted

or landscape plants due to their long life in vase and wide variety of shapes,

designs and colors (Mansur 2002; De la Cuadra et al. 2016).

There are 17 taxa (15 species and 2 subspecies) in the genus. However,

classification of Leucocoryne can be very confusing due to its high variability in

morphology and the occurrence of natural hybrids (Zoellner 1972; Muñoz and

Moreira 2000; Mansur and Cisternas 2005; Olate and Schiappacasse 2013;

Sassone et al. 2014; Jara-Arancio et al. 2014). Plants develop a small tunicate

bulb 1.5-2.5 cm in diameter, thin leaves and a 1-3 floral scape 30-80 cm in height

arranged in a terminal umbel. Its colorful flowers are actinomorphic and trimerous

(6 tepals) varying in quantity from 3-4 to 8-12 depending on the species colors

ranging from white to sky-blue, to purple and violet, forming different patterns and

designs depending on the species. A distinctive characteristic of Leucocoryne

flowers is the presence of three prominent staminodes (sterile stamens) that

protrude from the central part of the corolla (Mansur 2002; Schiappacasse et al.

2002; Zoellner 2002; Olate and Bridgen 2005; Hoffmann et al. 2015).

30

Although Leucocoryne plants have been cultivated in European gardens since the

19th century (Muñoz and Moreira 2000), in the 1990’s the ornamental industry

started to show interest to produce these species commercially, in order to satisfy

the constant demand for new species and cultivars with new shapes and colors.

The latter has led to several research efforts, looking for the domestication and

commercialization of these species, in countries like Japan, Israel, The

Netherlands and New Zealand (Kim et al. 1998; Lancaster et al. 2000; Catley

2003; Walton et al. 2008; Olate and Schiappacasse 2013; Hoffmann et al. 2015).

In the same way, several studies have been conducted in Chile on Leucocoryne

sp., including botanical identification; chromosomal studies; self-incompatibility

processes; life cycle; plant propagation and breeding techniques. These Chilean

studies have been carried out primarily by the Pontificia Universidad Católica de

Valparaíso that has been able to register three new cultivars in addition to the ones

offered from The Netherlands (Bridgen et al. 2002; De la Cuadra and Mansur

2004; Verdugo and Teixeira 2006; Verdugo 2013; Olate and Schiappacasse 2013;

De la Cuadra et al. 2016).

Seeds are usually used for Leucocoryne propagation. However, as in other

geophytes species, such as Narcissus and Tulipa, these species have a very long

life cycle and juvenile period, requiring between 3 and 4 years to form a floral size

bulb from seed (Riedemannn and Aldunate 2001; Mansur 2002; Escobar et al.

2008; Bach and Sochacki 2012). Because it is self-incompatible (Mansur 2002;

Mansur et al. 2004), this genus presents a wide range of genetic variability and

hence, natural populations with individuals showing segregation can be easily

31

found. This fact offers a great potential for breeding purposes but at the same time,

it makes difficult to propagate heterozygous lines having traits of interest by sexual

reproduction (Mansur 2002). Seed propagation, as the most important technique

used for breeding and conservational purposes, has been largely studied in

aspects like germination requirements, and other aspects related to the growth and

development of the plants (De la Cuadra et al. 2002; De la Cuadra and Mansur

2004; De la Cuadra et al. 2016).

Until now vegetative propagation is the best way to maintain over time the genetic

characteristics of Leucocoryne plants, either naturally inherited or produced by

controlled crosses. This techniques allow to produce new individuals with identical

genotype to the original plant from which the propagule was obtained (Mansur

2002; Schiappacasse et al. 2002). It has been reported that natural vegetative

propagation occurs in Leucocoryne species, from either a two or three-year old

plant, which will produce lateral bulblets or droppers (a type of bulblet that grows

deep in the ground) developed from the base of the mother bulb (Riedemannn and

Aldunate 2001; Mansur 2002). Currently, bulblet separation is the propagation

technique commercially used, in spite of its low multiplication rates and its

dependence with the plant genotype (Escobar et al. 2008). On the other hand,

there are artificial techniques that enhance and accelerate the vegetative

propagation capacity in geophytes. These are essentially bulb-cutting methods

consisting in different levels and types of controlled mechanical damages

depending on the genotype to propagate. In the case of the tunicate bulbs,

sectioning, scoring, scooping and coring have been used (Schiappacasse et al.

32

2002). Schiappacasse et al (2002) conducted studies in Leucocoryne

coquimbensis and Leucocoryne ixioides, where the use of bulb sectioning made it

possible to increase the bulb-multiplication rates.

In Leucocoryne, as in other geophytes, natural bulblet multiplication rates are very

low due to the reduced number of axillary meristems existing in modified stems as

bulbs, corms and rhizomes (Bach and Sochacki 2012). In addition to bulb-cutting

methods, there are more sophisticated techniques like in vitro propagation, also

known as micropropagation, by which potentially higher multiplication rates can be

achieved (mass clonal propagation). Micropropagation is composed by several

techniques by which the totipotency of plant cells is used. During micropropagation

small plant pieces or explants are cultured under aseptic conditions onto a media

supplemented with nutrients and plant growth regulators. The explants are then

cultured in an environment with controlled light, temperature and humidity. Using

micropropagation, plant growers are capable to supply stocks for

commercialization by multiplying elite cultivars in large scale without altering the

genotype (Rout et al. 2006; George et al. 2008; Sharma and Agrawal 2012; Bach

and Sochacki 2012). This technique has five stages: Stage 0, which corresponds

to the selection of the genotype and the explant selection and preparation. Stage I

involves the disinfection of the explants and their in vitro establishment. During

Stage II the explants are mass propagated by different means and techniques.

During Stage III the in vitro rooting of the explants is achieved. Finally, in Stage IV

fully developed plants are acclimatized to ex vitro conditions. Duration and

33

conditions for each of these stages must be adapted to each plant genotype

(George et al. 2008; Bach and Sochacki 2012; Kyte et al. 2013).

In vitro culture has been widely used in several ornamental plants as Narcissus,

Lilium, Gladiolus, Freesia, Zantedeshia, Crocus, Hippeastrum, Allium, Tulipa and

Hyacinthus, among others (Bach and Sochacki 2012). In the case of Leucocoryne

only a few studies have been conducted on the in vitro propagation of these

species and specific conditions for a successful in vitro culture are still poorly

understood (Olate and Bridgen 2005; Verdugo and Teixeira 2006; Escobar et al.

2008).

In this context, the objective of this research is to improve the in vitro propagation

techniques for Leucocoryne species and cultivars, to obtain a more efficient

method of mass clonal propagation.

Materials and methods.

Plant material.

Dormant bulbs of Leucocoryne purpurea, L. ixioides, L. vittata and the ecotype

‘Pichicuy’ (reported as L. aff. vittata) were harvested from the Leucocoryne

breeding program of the Pontificia Universidad Católica de Valparaiso (Fig.1a-d).

Bulbs of 1.0 ± 0.2 g in weight of each genotype were selected and stored at room

temperature under dry conditions until their in vitro initiation.

34

Disinfection and in vitro establishment.

In order to establish the plant material under in vitro conditions, the dry protective

tunics of the bulbs were eliminated and bulbs were washed under running tap

water for 5 min. Then, the bulbs were submerged in a fungicide solution of 1gL-1

Benomyl® and 1gL-1 Captan® for 30 min, rinsed with distilled water and washed

with ethanol 95% for 1 min. Subsequently, the explants were disinfected under a

laminar flow chamber using a solution of 50 g·L-1 NaOCl with addition of 2 drops of

Tween® 20, during 30 min and constant agitation. Bulbs were rinsed three times in

sterile distilled water.

After the sterilization all the bulb storage scales and the outer basal plate section

were completely remove from the explant until exposing the vegetative growing

point. Thus, each explant consisted by the apical growing point and a section of the

basal plate (Fig.1e)

The explants were established in culture tubes containing culture media

supplemented with 30 gL-1 sucrose and pH adjusted to 5.7, and previously

sterilized in autoclave at 121°C for 15 min. Macro and micronutrient composition of

the media and the use of a gelling agent varied depending on the experiment.

Explants were cultured under growing chamber conditions at 20±1°C, 16 h

photoperiod and luminous intensity of 125 µmol·m-2·s-1.

35

Experiment 1: Effect of the basal media and BAP on the in vitro growth and

multiplication of Leucocoryne.

To determine a proper culture media for the in vitro propagation of Leucocoryne

purpurea, L. ixioides and L. vittata explants of each of these species were

established in 20 mL of three different basal culture media: MS (Murashige and

Skoog 1962), LS (Linsmaier and Skoog 1965) y B5 (Gamborg et al. 1968). The

media cultures were solidified by adding 0.6% agar. Half of the treatments were

supplemented with BAP 1.0 mgL-1. Each treatment consisted of fifteen individual

explants as repetitions. We evaluated the percentage of bulb sprouting as the

bulbs that developed sprouts, percentage of single shoot as the bulbs that

developed only one shoot, percentage of multiple shoots as the bulbs that

developed two or more shoots, percentage of bulb multiplication as the bulbs that

developed lateral bulblets, bulb production as the number of bulbs obtained per an

initial bulb and bulb fresh weight as the average final weight obtained per explant.

Experiment 2: Effect of the BAP concentration on the in vitro growth and


In order to determine an adequate BAP concentration for the in vitro growth and

multiplication of Leucocoryne twenty explants of L. purpurea, L. ixioides and L.

vittata were established onto 20 mL of MS medium solidified with 0,6% agar and

supplemented with 0 (control), 0.5, 1.0, 1.5 and 2.0 mg·L-1 of BAP. Percentage of

bulb sprouting, percentage of single shoot, percentage of multiple shoots,

36

percentage of bulb multiplication, bulb production and bulb fresh weight were

evaluated as described above.

Experiment 3: Effect of bulb-cutting methods, culture system and the addition of

BAP on the in vitro growth and propagation of Leucocoryne vittata.

L. vittata in vitro cultured-bulbs were used as explants and cultured in MS medium.

To evaluate two different in vitro bulb-cutting methods: scoring and sectioning.

Scoring consisted in vertical incisions in the basal plate of the bulbs in order to

damage the main growing point and thus to break the apical dominance (Fig. 1g).

Sectioning consisted in to divide each bulb longitudinally into four isolated identical

parts (Fig. 1h). Cutting treatments were compared to intact bulbs as controls (Fig.

1f). In addition to the bulb-cutting methods two different culture systems were

compared: semi-solid (0.6% agar) and liquid media imbibed in a cotton pad. Bulbs

were cultured onto MS medium, and half of the treatments were supplemented with

1.0 mgL-1 BAP. Five bulbs of 0.8+0.1 g were used in each treatment as replicates.

Percentage of bulb sprouting, percentage of bulb multiplication and bulb production

were evaluated as described above.

Experiment 4: Effect of BAP on the in vitro growth and multiplication of

Leucocoryne sectioned bulbs

In vitro bulbs of L. purpurea, L. ixioides, L. vittata and L. sp. Pichicuy of 0.8+0.1 g

in weight were sectioned in four parts and used as explants (Fig. 1h). Five

37

replicates per treatment were used, each replicate consisted of the four sections

that were obtained from the cut of the same bulb. Each of these four sections were

cultured in the same container onto MS medium solidified with 0.6% agar, and half

of the treatments were supplemented with 1.0 mgL-1 BAP. Percentage of bulb

sprouting, percentage of bulb multiplication, bulb production and bulb fresh weight

were evaluated as described above.

Statistical analysis.

Results of shooting and multiplication were statistically analyzed using Scheffé´s

procedure for multiple comparison of proportions (P≤0.05) (Zar 2010). In the case

of bulb production and bulb fresh weight data were analyzed performing an

analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test

(P≤0.05) using GraphPad Prism 5 software (GraphPad Software Inc., San Diego,

California, USA).

38

Results.

Experiment 1: Effect of the basal media and BAP on the in vitro growth and


The methods of disinfection and establishment in vitro resulted in a successful

initiation of the explants, with a contamination level of only 7% after 4 weeks of

culture.

Bulbs with vegetative sprouting were in average 91.9% of the total, varying

between 80 and 100% depending on the treatment. L. purpurea and L. ixioides

explants cultivated onto any of the three culture media assayed presented 100%

bulb sprouting when they were supplemented with 1.0 mgL-1 BAP. In the case of L.

vittata that level of vegetative sprouting was only achieved when MS, B5 and B5 +

1.0 mgL-1 BAP media was used (Table 1). After 13 weeks of culture the addition of

BAP produced the highest bulb sprouting with multiple shoots per bulb. This

difference between the controls and the media supplemented with BAP was even

clearer in L. ixiodes bulbs but not in the other genotypes (Table 1). After 30 weeks

of culture different multiplication rates were observed among the genotypes. In

general, L. ixioides bulbs showed the highest multiplication rate, reaching up to

100% when cultivated onto MS + 1.0 mgL-1 BAP medium (Fig. 2a). In this

treatment it was produced an average of 5.3 bulbs/explant, which was higher than

in MS without BAP, which produced a multiplication rate of 30.8% and only 1.9

bulbs/explant (Fig.2b).

39

In most of the genotype/medium combinations bulb multiplication was produced

except in the case of L. purpurea, where no multiplication of the bulbs cultured onto

B5 + 1.0 mgL-1 BAP medium was observed (Fig. 2a). The latter treatment also

produced the lowest bulb fresh weight. In the case of L. ixioides the highest values

of bulb fresh weight occurred in MS + 1.0 mgL-1 BAP, while in L. vittata no

difference among treatments was observed (Fig. 2c).

Experiment 2: Effect of the BAP concentration on the in vitro growth and


In this experiment the in vitro establishment protocol also resulted in a very low

contamination rate (7.7%) after 4 weeks of culture.

A high bulb sprouting rate (98.2% average) was produced, varying between 90 and

100% depending on the treatment (Table 2). L. purpurea and L. vittata sprouting

rates did not show any significant differences when different BAP concentrations

were added to the culture media. On the other hand, L. ixioides showed the lowest

sprouting rate (90%) when 1.0 mg·L-1 BAP was added to the medium (Table 2).

This genotype also showed a significant higher rate of multiple shoots by the

addition of BAP to the media compared to the control without BAP. However, no

difference was observed between the different BAP concentrations used (Table 2).

In the other two genotypes no clear tendency was observed between treatments in

terms of single or multiple shoot production after 13 weeks of culture (Table 2).

40

Multiplication varied according to the genotype and BAP concentration. The

highest multiplication rates were obtained in L. ixioides with values between 47.9%

(control plants) and 94.4% when bulbs were cultured onto MS + 2.0 mg·L-1 BAP

(Fig. 3a; Fig. 4). In this genotype it was also observed the highest bulb production

per explant, with 6.2 and 5.7 bulbs/explant when cultured onto MS plus 1.0 or 2.0

mg·L-1 BAP respectively. These results were significantly higher than the ones

measured in the control treatment, with only 2.9 bulbs/explant produced. In L.

purpurea and L. vittata no differences were observed between the different

treatments (Fig. 3b).

The highest bulb fresh weight was produced in L. purpurea and L. ixioides when

BAP was added to the culture media. In the case of L. vittata no significant

difference was observed among treatments (Fig. 3c).

Experiment 3: Effect of bulb-cutting methods, culture system and the addition of

BAP on the in vitro growth and propagation of Leucocoryne vittata.

After 8 weeks of culture 100% of sprouting was observed in either intact (control)

or treated bulbs with one of the cutting methods, when they were cultured onto

liquid media imbibed in cotton pads and supplemented with 1.0 mg·L-1 BAP (Table

3). The latter was also observed in sectioned bulbs and cultured onto MS media

solidified with agar and supplemented with 1.0 mg·L-1 BAP. The lowest sprouting

rates (40%) were produced in intact bulbs, when they were cultured onto media

supplemented with BAP, independently of the system used (Table 3).

41

A tendency to increase the multiplication rate was observed when bulb-cutting

treatments were used, especially in those including sectioning (Figure 5). Intact

bulb treatments showed multiplication rates below 20% (Table 3). The highest bulb

production occurred in sectioned bulbs cultured onto media solidified with agar

either supplemented or not with BAP, producing 13 and 9.2 bulbs/explant

respectively. Independently of the culture system used and the addition or not of

BAP, the lowest bulb production was clearly observed in intact bulbs, showing

values between 0 y 0.6 bulbs/explant (Table 3). In terms of culture system,

differences between treatments were observed only when sectioned bulbs were

cultured onto media without BAP. In this case, bulbs cultured on media with agar

produced significantly higher number of bulbs than the ones cultured on cotton

pads imbibed with liquid media, with values of 13 and 4.4 bulbs/explant

respectively (Table 3).

Experiment 4: Effect of BAP on the in vitro growth and multiplication of

Leucocoryne sectioned bulbs.

Considering the results from experiment 3, sectioning was used on four

Leucocoryne genotypes cultured onto growing media either supplemented or not

with BAP. In L. purpurea sprouting rates of 66.6% were observed in control plants

and 50% when BAP was added to the media, but showing no statistical difference

between those groups of plants. On the other hand, L. ixioides and L. vittata plants

showed higher sprouting rates reaching 80% and 100% but there was no influence

of BAP addition to the growing media. In the case of the plants belonging to the L.

42

sp. Pichicuy only 40% of them sprouted in the absence of BA, but 80% of them did

sprout when BAP was added to the media.

In terms of multiplication, after 6 months of culture it was possible to observe

different rates between genotypes, although no statistical difference was founded

when data were analyzed (Fig. 6a). Thus bulb production varied considerably

among genotypes from 2.6 to 16 bulbs/explant (Fig. 7). In L. sp. Pichicuy bulb

production was significantly larger in BAP treatments compared to the controls,

showing values previously mentioned. In L. purpurea and L vittata differences were

also observed but they all remained below the threshold for statistical difference. In

the last genotype, L. ixioides, no differences in bulb production were also observed

among treatments, with values between 12.4 and 14 bulbs/explant for the controls

and BAP treatments, respectively (Fig. 6b).

The addition of BAP to the growing media did not affect the fresh weight of the

bulbs. Values varied between 0.07 and 0.19 g/bulb with no significant differences

between treatments (Fig 6c).

Discussion.

We developed an effective disinfection and in vitro establishment protocol for

Leucocoryne bulbs (Stage 0 and 1). Explant disinfection is one of the most

challenging steps in the process of the in vitro clonal propagation. The protocol to

use will depend on the type and origin of the explant and its sensitivity to

43

disinfectant agents (Smith et al. 1999; Kyte et al. 2013). In addition, the

combination of explant, culture media and controlled environment gives the perfect

conditions for the proliferation of any pathogenic and non-pathogenic

microorganism. Therefore, a proper disinfection system must be implemented to

avoid both explant contamination and extensive tissue damage (Mroginski and

Roca 1991). The disinfection protocol developed in this work resulted in a very

effective and safe protocol for the in vitro establishment of Leucocoryne bulbs,

showing in average only 7% of contamination. This is quite remarkable knowing

the major difficulty that is faced when establishing vegetative tissues that grow on

the ground or even underground in direct contact with soil, such as bulbs, corms,

rhizomes and tubers. In such cases, disinfection protocols should be labor-

intensive and more aggressive for the explant, using combinations of agents and/or

longer disinfections periods (Pedersen and Brandt 1992; Slabbert et al. 1993;

Kritzinger et al. 1998; Smith et al. 1999; Chang et al. 2003; Sochacki and

Orlikowska 2005; Paredes et al. 2014). Studies in other geophytes species have

also reported the difficulty and importance of the disinfection process. In vitro

establishment of Zantedeschia sp. has evidenced low success when using

conventional disinfection techniques (Kritzinger et al. 1998; Chang et al. 2003). In

the case of Narcissus sp., which possess a tunicate bulb similar to Leucocoryne,

problems during the in vitro initiation have been also reported, with high bacterial

and fungal contamination rates, surpassed in some cases only by using hot water

treatments plus use of fungicides (Sochacki and Orlikowska 2005). High

contamination rates have been also reported in other bulb species: 20-40% in

Crinum macowanni (Slabbert et al. 1993), 90% in Eucomis autumnalis (Ault 1995),

44

20-90% in Hippeastrum sp. (Smith et al. 1999) and 50-80% in Traubia modesta,

another endemic species to Chile with ornamental potential (Paredes et al. 2014).

The disinfection protocol developed in this work resulted in low contamination

rates, eventhough it is a more aggressive and laborious procedure than the

conventional ones, survival of the explants was not negatively affected given the

high sprouting rates obtained (80%). In previous studies including Leucocoryne

bulbs, in vitro contamination has been the main limitation for further advance in a

suitable protocol, with values between 40-100% (Fuentevilla, unpublished). Due to

this problem, the few in vitro studies on Leucocoryne micropropagation have

started their plant material from seeds (Escobar et al. 2008).

The different experiments of this work resulted in the development of a suitable

protocol for mass clonal propagation for the Leucocoryne genus. Among the

different media tested, basal MS medium was the most suitable for in vitro growth

and multiplication. It was observed high levels of sprouting and a normal growth

and development of the explants. Previous works on the in vitro propagation of

Leucocoryne have only used MS medium (Escobar et al., 2008; Olate & Bridgen,

2005). The other two media used in this work, LS and B5, also showed to be

suitable for Leucocoryne in vitro culture but in the case of B5 media a lower fresh

weight of the bulbs was obtained. These three media have previously been used

for the micropropagation of other geophyte species. For instance, MS has resulted

in better results than B5 in terms of embryo germination in Alstroemeria (Lu and

Bridgen 1996) and also on callus formation and shoot differentiation in Lilium

longiflorum (Ramsay et al. 2003). Besides, MS medium has been widely used in

45

different ornamental geophytes species, for example, Zhephyra elegans (Vidal et

al. 2012), Narcissus sp. (Sochacki and Orlikowska 2005), Hippeastrum sp. (Smith

et al. 1999), Muscari muscarimi (Ozel et al. 2015) and Zephyranthes sp. (Smith et

al. 1999). B5 medium has been successfully used in other geophytes from the

Allium genus (Dunstan and Short 1977; Shahidul Haque et al. 1997; Barandiaran

et al. 1999; Martınez et al. 2000) in the same way as LS medium have been used

in Allium sp. (Nagakubo et al. 1993; Ayabe and Sumi 1998), Crocus sp. (Mir et al.

2011) and Narcissus sp. (Stone 1973).

The addition of BAP to the culture media contributed to a higher multiplication rate

like the one observed on L. ixioides. However, no significant differences were

observed over the sprouting and multiplication rates when using different BAP

concentrations. Bulb production values of 6.2 and 5.7 bulbs/explants were possible

to obtain when plants were established on MS medium supplemented with 1 or 2

mg·L-1 BAP respectively, which contrast with the much lower values observed in

natural populations. In nature Leucocoryne bulbs are able to produce 1-2 bulblets

per plant every other year but only when they have reached their mature stage

(Riedemannn and Aldunate 2001). A bulb would reach their maturity from seed

only after 2 or 4 years of growth (Riedemannn and Aldunate 2001; Mansur 2002).

We have also concluded that the addition of BAP to the growing media produced a

larger explant fresh weight in L. purpurea and L. ixioides than the controls. In

previous studies Escobar et al. (2008) reported obtaining 3-4 bulbs/explant after

seven months of culture of Leucocoryne plants onto MS medium without any plant

growth regulators. That particular study produced the in vitro bulbs via indirect

46

organogenesis and further induction of the bulbs on MS supplemented with 1.0

mg·L-1 BAP. In Allium cepa micropropagation, it has been reported that 1.0 mg·L-1

BAP added to the growing media resulted in the best treatment, however higher

BAP concentrations resulted in a lower production of new plantlets (Kamstaityte

and Stanys 2004). In Muscari azureum 1.0 mg·L-1 BAP plus 0.25 mg·L-1 IAA

resulted in a larger shoot and bulblet production (Uranbey 2011). Bridgen et al.

(2009) also reported the use of MS medium supplemented with 2.0 mg·L-1 BAP for

an adequate Alstroemeria sp. micropropagation.

Bulb-cutting methods allow accelerating or increasing the efficiency of vegetative

propagation of some geophyte species. These methods include bulb division or

"sectioning", "scoring", "scooping" and "coring". All these methods are focused in a

controlled damage of the main growing point and basal plate and thus favor new

bulblet production (Hartmann et al. 1997; Kumar et al. 2010; Schiappacasse et al.

2002). Hyacinthus is an important plant genus in which these techniques have

been commercially adopted, producing up to 60 bulblets from a single bulb using

scooping, although these bulblets will require four to five years of growth before

flowering (Hartmann et al. 1997). In many other bulb species it has been also

reported successful use of this kind of technique. van Leeuwen and van der

Weijden (1997) using sectioning was able to increase the natural propagation rates

not only in Chionodoxa (from 1.9 to 6.5), but also in Gelanthus (2.7 to 6.5), Muscari

(2.7 to 10.0) and Scilla (1.1 to 8.0). Species with larger bulbs like those belonging

to Hippeastrum, allow a more intensive sectioning obtaining up to 28.5 bulbs when

the initial bulb has been divided in 24 sections (Sandler-Ziv et al. 1997). Scoring

47

has been reported to be a successful technique in Nerine (Mori et al. 1997) and in

Crinum x powellii (Knippels 2012). In the present work we observed a higher

multiplication rate when bulb-cutting methods were applied compared to intact

bulbs, however by using scoring we obtained a lower bulb multiplication and

production rates than sectioning. Similar results were observed by Solgi et al.

(2015) in Fritillaria imperialis where the number of bulbs obtained from an initial

bulb was greater by using sectioning compared to scoring. Schiappacasse et al.

(2002) working with traditional propagation reported the production of 2.2 bulblets

per initial bulb in L. coquimbensis and 5 bulblets in L. ixioides by using sectioning

in two vertical sections. By using in vitro bulb sectioning we obtained up to 11.3

bulbs/explant in L. purpurea and 14 bulbs/ explant in L. vittata and L.ixioides. In the

case of L. sp. Pichicuy, the addition of BAP significantly increased bulb production

from 2.6 to 16 bulbs/explant. Previously Olate and Bridgen (2005) reported on

Leucocoryne coquimbensis 7 bulbs/explant using scoring, 8 bulbs using a 4-

sectioned bulb and almost no bulblets from intact bulbs, but in that case no BAP

was added to the media.

When comparing bulb-cutting methods and culture system in L. vittata, a higher

bulb production was achieved using MS medium solidified with agar than using

liquid MS media imbibed in cotton pads. This result contradicts the potential

advantages of the liquid media in terms of producing a higher multiplication rate

reported by Kim et al. (2003) in Allium sativum.

In conclusion, in this work we have developed a highly efficient mass clonal

micropropagation of Leucocoryne. Our protocol also includes an effective

48

disinfection and explant preparation method for a successful in vitro establishment.

By using different bulb-cutting methods, culture media and growth regulator

addition, we also have been able to culture four different Leucocoryne genotypes,

achieving the highest multiplication rates reported to date. These results represent

an important set of tools to be used by conservational or commercial purposes,

such as breeding programs or nurseries. These techniques may also contribute to

the advance in the knowledge of the propagation of other native and ornamental

geophyte species.

49

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58

Table 1 Effect of basal media and BAP addition on the in vitro sprouting of three Leucocoryne genotypes.

Genotype Basal medium

BAP Sprouting Single shoot Multiple shoots (mg*L-1) (%) (%) (%)

L. purpurea

MS 0.0 86.7 b 86.7 b 0.0 b 1.0 100.0 a 71.4 b 28.6 a

LS 0.0 83.3 b 75.0 b 8.3 ab 1.0 100.0 a 92.9 ab 7.1 b

B5 0.0 91.7 ab 91.7 ab 0.0 b 1.0 100.0 a 100.0 a 0.0 b

L. ixioides

MS 0.0 92.3 ab 92.3 a 0.0 b 1.0 100.0 a 53.3 b 46.7 a

LS 0.0 85.7 b 85.7 ab 0.0 b 1.0 100.0 a 64.3 b 35.7 a

B5 0.0 85.7 b 85.7 ab 0.0 b 1.0 100.0 a 53.3 b 46.7 a

L. vittata

MS 0.0 100.0 a 93.3 ab 6.7 bc 1.0 86.7 b 73.3 b 13.3 b

LS 0.0 93.3 ab 93.3 ab 0.0 c 1.0 86.7 b 40.0 c 46.7 a

B5 0.0 100.0 a 100.0 a 0.0 c 1.0 100.0 a 80.0 b 20.0 b

Sprouting indicates the percentage of bulbs that developed sprouts. Single shoot indicates the percentage of bulbs that developed only one shoot. Multiple shoots indicates the number of bulbs that developed two or more shoots. Different letters in the same column indicate statistical differences for each genotype based on Scheffé´s procedure for multiple comparison of proportions (P≤0.05).

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Table 2 Effect of different BAP concentrations added to the growing media on the in vitro sprouting of three Leucocoryne genotypes.

Genotype BAP Sprouting Single shoot Multiple shoots

(mg*L-1) (%) (%) (%)

L. purpurea

0.0 100.0 a 94.7 ab 5.3 bc 0.5 100.0 a 65.0 c 35.0 a 1.0 100.0 a 100.0 a 0.0 c 1.5 100.0 a 87.5 b 12.5 b 2.0 100.0 a 55.0 c 45.0 a

L. ixioides

0.0 100.0 a 89.5 a 10.5 b 0.5 100.0 a 47.4 bc 52.6 a 1.0 90.0 b 30.0 c 60.0 a 1.5 94.4 ab 50.0 bc 44.4 a 2.0 100.0 a 61.1 b 38.9 a

L. vittata

0.0 93.3 a 80.0 b 13.3 a 0.5 100.0 a 100.0 a 0.0 b 1.0 100.0 a 85.0 b 15.0 a 1.5 100.0 a 82.4 b 17.6 a 2.0 93.8 a 68.8 b 25.0 a

Sprouting indicates the percentage of bulbs that developed sprouts. Single shoot indicates the percentage of bulbs that developed only one shoot. Multiple shoots indicates the number of bulbs that developed two or more shoots. Different letters in the same column indicate statistical differences for each genotype based on Scheffé´s procedure for multiple comparison of proportions (P≤0.05).

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Table 3 Effect of the in vitro cutting method, culture system and the addition of BAP to the growing media on sprouting, bulb multiplication and bulb production in Leucocoryne vittata.

Cutting method

Culture system

BAP Sprouting Bulb multiplication

Bulb production

(mg*L-1) (%) (%) (bulbs/explant)

Intact bulb (control)

Agar 0.0 40.0 b 20.0 cd 0.6 c 1.0 80.0 ab 20.0 cd 0.2 c

Liquid (Cotton pads)

0.0 40.0 b 0.0 d 0.0 c 1.0 100.0 a 20.0 cd 0.6 c

Scoring Agar 0.0 80.0 ab 60.0 bc 2.8 bc

1.0 80.0 ab 60.0 bc 3.2 bc Liquid

(Cotton pads) 0.0 60.0 b 40.0 bc 0.4 c 1.0 100.0 a 80.0 ab 2.6 bc

Sectioning Agar 0.0 100.0 a 100.0 a 13.0 a

1.0 80.0 ab 80.0 ab 9.2 ab Liquid

(Cotton pads) 0.0 80.0 ab 80.0 ab 4.4 bc 1.0 100.0 a 100.0 a 7.8 ab

Sprouting indicates the percentage of bulbs that developed sprouts. Bulb multiplication indicates the percentage of bulbs that developed lateral bulblets. Bulb production indicates the number of bulbs obtained per an initial bulb. Different letters in the same column indicate statistical differences based on Scheffé´s procedure for multiple comparison of proportions (P≤0.05) and analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P≤0.05).

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Fig. 1 Leucocoryne genotypes and explant types. (a) Leucocoryne purpurea; (b) Leucocoryne ixioides; (c) Leucocoryne vittata; (d) Leucocoryne sp. Pichicuy; (e) Initiation explant composed by the inner basal plate section plus the vegetative growing point; (f) Intact bulb; (g) Scoring and (h) Sectioning done to in vitro bulbs.

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Fig. 2 Effect of basal media and the addition of BAP on three Leucocoryne genotypes. (a) Percentage of bulbs that developed lateral bulblets; (b) Number of bulbs obtained per an initial bulb; and (c) Average final fresh weight obtained per explant. Different letters indicate statistical differences for each genotype based, in the case of bulb multiplication, on Scheffé´s procedure for multiple comparison of proportions (P≤0.05) and for bulb production and bulb fresh weight, on analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P≤0.05).

63

Fig. 3 Effect of BAP concentration on three Leucocoryne genotypes. (a) Percentage of bulbs that developed lateral bulblets; (b) Number of bulbs obtained per an initial bulb; and (c) Average final fresh weight obtained per explant. Different letters indicate statistical differences for each genotype based, in the case of bulb multiplication, on Scheffé´s procedure for multiple comparison of proportions (P≤0.05) and for bulb production and bulb fresh weight, on analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P≤0.05).

64

Fig. 4 Effect of BAP concentration on in vitro Leucocoryne ixioides: (a) MS medium (control); (b) MS+0.5mg·L-1 BAP; (c) MS+1.0mg·L-1 BAP d) MS+1.5mg·L-1 BAP and (e) MS+2.0mg·L-1 BAP.

65

Fig. 5 Leucocoryne bulb production in response to different bulb-cutting methods. (a) Intact bulb (control); (b) Scoring; and (c) Sectioning.

66

Fig. 6 Effect of sectioning technique and BAP addition on four Leucocoryne genotypes. (a) Percentage of bulbs that developed lateral bulblets; (b) Number of bulbs obtained per an initial bulb; and (c) Average final fresh weight obtained per explant. Different letters indicate statistical differences for each genotype based, in the case of bulb multiplication, on Scheffé´s procedure for multiple comparison of proportions (P≤0.05) and for bulb production and bulb fresh weight, on analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P≤0.05).

67

Fig. 7 Visual appearance of a Leucocoryne bulb and adventitious bulblet produced after in vitro sectioning. (a) Sectioned original explant; (b) Adventitious new bulblets.

68

CHAPTER 3

AN EFFICIENT IN VITRO PRODUCTION SYSTEM FOR

Leucocoryne spp. PLANTS INITIATED FROM SEEDS

Alejandro Altamira1, Eduardo Olate1, Marlene Gebauer1, Levi Mansur2, Carlos De

la Cuadra2 and Gloria Montenegro1*.

1Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería


Santiago, Chile.

2Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Avda. San


This chapter will be sent to Propagation of Ornamental Plants (POP) –International Journal (March 2017)

69

An efficient in vitro production system for Leucocoryne spp. plants initiated

from seeds

Alejandro Altamira1*, Eduardo Olate1, Marlene Gebauer1, Levi Mansur2, Carlos De la

Cuadra2 and Gloria Montenegro1.

1 Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal,

Pontificia Universidad Católica de Chile. Vicuña Mackenna 4860, Macul, Santiago, Chile.

2 Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Avda. San


*Corresponding author:

Name: Alejandro Altamira

Address: Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería


Santiago, Chile

Telephone: +56 9 90915256

E-Mail: [email protected]

Keywords: Amaryllidaceae, in vitro germination, geophyte, sprouting, bulb, optimal

temperature

70

Abstract

Leucocoryne spp. are geophyte plants that belongs to Amaryllidaceae, which have

a great potential in the ornamental plant industry due to its colorful flowers,

distinctive shape and long vase life. The plants are suitable to be used as cut

flowers, potted plants and also as landscape plants. This research focused on

optimizing an in vitro seed germination protocol and the subsequent bulb growth

and development of three Leucocoryne genotypes. Culture temperatures of 15 and

20 °C were used from seed initiation until bulblet formation. Seeds were in vitro

initiated on 12.5% MS medium and subsequently transferred to 50% MS media

optionally supplemented with BAP. Germination rates varied between 85-98% at

15 ºC and 48-83% at 20°C, depending on the genotype. In all the genotypes, the

highest bulb growth and development occurred at 15°C. Use of 50% MS

supplemented with BAP broke down bulb dormancy and stimulated lateral bulblets.

Final fresh weight of the bulbs was higher at 15°C in two of the genotypes and

varied between 0.04 and 0.10 g per bulb.

Abbreviations: BAP - N6-benzylaminopurine; MS – Murashige and Skoog medium

(1962)

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Introduction.

The ornamental crop industry constantly demands for new cultivars or species with

new shapes and colors (Olate and Bridgen 2005). Leucocoryne is a geophyte

genus endemic to Chile which belongs to Amaryllidaceae and possess exceptional

qualities to be used as cut flower and potted plant, as well as in landscaping, due

to its long vase life and wide flower shape, design and color variety (Bridgen 2000;

Mansur 2002; Sassone et al. 2014; De la Cuadra et al. 2016). There are 15 to 20

species distributed throughout northern and central Chile. Its major diversity center

is located between the Coquimbo and Valparaiso regions (Zoellner 1972; Muñoz

and Moreira 2000; Mansur and Cisternas 2005; Olate and Schiappacasse 2013;

Jara-Arancio et al. 2014). Plants belonging to this genus have a small tunicate bulb

and a single floral scape 30-40 cm in height, which ends in an umbel carrying 3-12

flowers depending on the species. One of the main characteristics are its flower

pattern and color, varying between white, blue and purple (Mansur 2002;

Schiappacasse et al. 2002; Zoellner 2002; Olate and Bridgen 2005; Hoffmann et

al. 2015).

The ornamental value of Leucocoryne has brought special interest in the

knowledge and domestication of the species, with several studies and commercial

efforts done in Japan, The Netherlands, Israel, New Zealand and Chile (Kim et al.

1998b; Bridgen 2000; Lancaster et al. 2000; Schiappacasse et al. 2002; Catley

2003; Walton et al. 2008; Olate and Schiappacasse 2013). This particular interest

and the anthropic and climate threats that natural populations face, have led to the

72

development of a research and breeding program in Chile for the conservation and

commercialization of this genus. This particular program has already registered

three new cultivars in addition to the ones already developed in The Netherlands

(De la Cuadra et al. 2016).

Few studies on the propagation of Leucocoryne have been published to date.

It is therefore necessary to make progress in these techniques in order to help

commercial and conservation advances. has a slow life cycle that takes up to

three or four years from seed to floral bulb (Mansur 2002). It has been reported a

high genetic variation among species and ecotypes, which implies a great potential

to do breeding but, at the same time, it is a sign of the difficulties to maintain

interesting characteristics using sexual propagation (Mansur 2002; Mansur et al.

2004). Leucocoryne, as other geophyte genus, has the natural capacity of

vegetative propagation through the development of one or two lateral bulblets at

the bulb base, or by the production of “droppers”, a type of bulblet that come out as

an additional stem from the center of the bulb but instead grows deeper into the

ground, also from the second or third year of growth (Riedemannn and Aldunate

2001; Mansur 2002; Schiappacasse et al. 2002). Bulb-cutting methods have been

studied in order to increase propagation efficiency (Schiappacasse et al. 2002).

Some of these studies include specific in vitro culture research on Leucocoryne

species (Olate and Bridgen 2005; Escobar et al. 2008).

Sexual propagation of Leucocoryne spp. can be an interesting tool of propagation

for breeding purposes or when genetic stability of the plants is needed.

Nevertheless, seed propagation is still the easiest and inexpensive way to

73

propagate Leucocoryne plants in big quantities, either for conservation purposes or

during the first stages of plant breeding. The in vitro propagation from seeds adds

the extra advantage of a more reliable system under a controlled environment, and

independent from seasonal environmental conditions. It is important to fully

understand the seed propagation process and all the factors that control the

different phases of a particular methodology. In that sense, germination and

growing temperature is one of the main factors to determine. It has been reported

that different Leucocoryne species show different optimal temperatures for seed

germination measured either in natural growing habitats (Jara et al. 2006) or under

artificial conditions (De la Cuadra et al. 2016).

In this context, the objectives of this study are to develop and to optimize the

protocol for the in vitro seed germination of three Leucocoryne genotypes and to

determine the effect of the temperature on the in vitro plant and bulb production.

74

Materials and methods.

Plant material.

Seed belonging to three Leucocoryne species were used as explants: Leucocoryne

purpurea (Fig. 1a), Leucocoryne vittata (Fig. 1b) and ecotype Leucocoryne sp.

Pichicuy (Fig. 1c). Seeds were collected from plants under greenhouse conditions

in the germplasm bank of the Leucocoryne Breeding Program at the Pontificia

Universidad Católica de Valparaíso Experimental Station, located in Quillota (lat.

32°53´ S; long. 71°12’ W). Seeds were stored up to six months in paper bags at

20±2°C in dark conditions, until they were used.

The in vitro studies were conducted in the laboratories of the Plant Science

Department of the Pontificia Universidad Católica de Chile, in Santiago, Chile

(33°29’ S; long. 70° 36’W).

Disinfection and in vitro establishment.

To disinfect the seeds were submerged in a 10 g·L-1 hydric solution of sodium

hypochlorite for 15 min under a laminar flow chamber and rinsed 3 times with

sterile distilled water. Seeds were then imbibed in sterile distilled water for 96 h at

8°C in dark conditions (Ham 2002; De la Cuadra and Mansur 2004; Jara et al.

2006). After imbibition, seeds were sown in 200 mL glass culture vessels

containing 30 mL of 12.5% MS medium supplemented with 30 g·L-1 sucrose, 6 g·L-

1 agar and pH were adjusted to 5.7 (Murashige and Skoog 1962; Ham 2002).

75

In vitro seed germination.

A total of 300 seeds per Leucocoryne genotype were sown in vitro conditions.

Seeds were cultured under two temperature treatments: 15±1°C and 20±1°C, in a

growth chamber with a 16h photoperiod. Treatments consisted in six replicates of

five vessels each, containing five seeds/vessel. After 13 weeks of in vitro culture,

germination rate, bulbing rate, dormancy rate and bulb fresh weight were

evaluated.

Bulb development and multiplication.

After 13 weeks of in vitro culture, bulbs from seeds were transferred onto 25% MS

medium, and after 10 additional weeks they were transferred onto 50% MS

supplemented with 1 mg·L-1 BAP and 30 g·L-1 sucrose, 6 g·L-1 agar and pH

adjusted to 5.7. After 24 weeks, bulbing rate, bulb sprouting, bulblet production,

rooting rate and bulb fresh weight were evaluated.

Statistical analysis.

Bulb fresh weight data were analyzed by analysis of variance (ANOVA) and

Bonferroni's multiple comparison test (P≤0.05) using GraphPad Prism 5 software

(GraphPad Software Inc., San Diego, California, USA). In the case of germination,

bulbing, dormancy, sprouting, bulblet production and rooting data were analyzed

using Sheffé's multiple comparison for proportions test (P≤0.05) (Zar 2010).

76

Results and discussion.

Disinfection and in vitro establishment protocols for Leucocoryne seeds were

highly effective, showing less than 1% contamination and high germination rates in

all the genotypes used. This indicates that disinfection method used did not

damage seeds.

In terms of germination rate after 13 weeks of in vitro culture, the best results were

obtained at 15°C in the three genotypes, with values of 85.3% in L. purpurea,

96.7% in L. vittata and 98.7% in L. sp. Pichicuy, significantly higher than

germination rates at 20°C (Table 1). However, we observed that seed germination

occurred earlier at 20°C than at 15°C (data not showed). Ham (2002) reported 89%

germination in L. coquimbensis using the same culture medium but using 12°C and

darkness conditions, also reporting that 12.5% MS worked as the best MS

concentration for seed germination compared to higher concentrations (25%,

37.5%, 50% and 100%). Our results are also in accordance with reports of De la

Cuadra et al. (2016) that used the same three seed genotypes, but moist

chromatographic paper and darkness as germination conditions. They found that

germination rates were significantly higher at 10ºC and 15°C than at 20°C,

reporting also no seed germination at 25°C. As mentioned by Jara et al. (2006),

these optimal low temperatures for Leucocoryne seed germination and the

inhibitory effect caused by high temperatures under laboratory conditions coincide

with the natural desert and Mediterranean environments in which these species are

found.

77

We also evaluated the occurrence of bulbs (bulbing) from the seedlings produced.

The highest bulbing percentage occurred in seedlings cultured at 15°C (Table 1;

Fig. 2), thus repeating the positive effect of the lower temperature treatment for

seed germination. Bulbs obtained in both treatments were clearly different in size;

therefore bulb fresh weight was also evaluated. In the same way, better results

were obtained at the lowest culture temperature with higher bulb fresh weight at

15°C than at 20°C (Fig. 2; Fig. 3).

After seed germination, during bulb and plant development, we observed the

occurrence of premature senescence of the seedlings. Apparent dormancy started

to show up after 13 weeks of in vitro culture, thus the effect of temperature culture

on dormancy of the bulbs was also evaluated. Bulbs were considered as dormant

when more than 2/3 of the shoot showed senescence before transferring them to

fresh media. In average, L. purpurea showed the lowest dormancy rates between

28% and 53%, while L. vittata and L. sp. Pichicuy showed over 73% of dormant

bulbs (Table 1). Culture temperature affected dormancy rates, occurring lower

dormancy rates at 15°C in all genotypes. De la Cuadra and Mansur (2004)

reported seedling senescence 11 weeks after germination, and completely

senescent plants after 13 weeks of growth.

After transferring the bulbs onto 25% MS medium, we were able confirm that most

of the bulbs were dormant. After 10 weeks, the bulbs achieved a sprouting rate of

only 9.1% (Fig. 4a). During this period, bulb fresh weight did not significantly

change, showing 0.03 g/bulb in L. purpurea in both culture temperatures

78

treatments, and 0.04 and 0.02 g/bulb in L. vittata and L. sp. Pichicuy at 15ºC and

20°C respectively.

Transferring the bulbs onto 50% MS medium supplemented with 1.0 mg*L-1 BAP

triggered dormancy break. The interruption of the bulb dormancy could be verified

with the appearance of vegetative shoots from the explants, regardless of the

culture temperature (Table 2). This would confirm that BAP produces an effect on

bulb dormancy break in Leucocoryne species under in vitro conditions. BAP

addition to in vitro culture media has been used before for stimulating cell division,

shoot formation and growth from axillary buds in other geophyte species (Jha

2005; Maślanka and Bach 2014). We could also observe the appearance of

adventitious bulblets between 4.9% and 12.6% of the explants, independently of

the culture temperature (Table 2; Fig. 4b). Therefore, BAP could also promote and

accelerate adventitious bulblet formation in Leucocoryne species under in vitro

conditions. Although the adventitious bulblet formation observed in this study was

low, it is remarkable that naturally bulb formation in Leucocoryne species does not

occur until the second or third year of growth (Riedemannn and Aldunate 2001;

Mansur 2002). Therefore, the use of in vitro propagation and BAP could delay or

avoid bulbs entering in the natural early dormancy and shorten the time from seed

to adventitious bulblet appearance, thus allowing a faster and more efficient

propagation system. In terms of bulb rooting, statistical differences were observed

between temperature treatments in both L. purpurea and L. sp. Pichicuy, with

higher bulb rooting at 15°C (Table 2).

79

We would also like to highlight the fact that gradual increase of MS concentration

to the growing media (12.5% to 25% and then to 50%) allowed seedlings to

continue growing by gradually adapting to increasing osmotic levels produced by

higher salt contents of the culture media. Previously, Vidal et al. (2012) reported

that Zephyra elegans plants, another geophyte species endemic to Chile with

ornamental potential, were unable to grow in vitro when seedlings germinated on

water agar were directly subcultured onto 100% MS medium.

Final fresh weight of the bulbs varied between 0.04 and 0.1 g depending on both

genotype and culture temperature, but without statistical differences (Fig. 5). It has

been reported that Leucocoryne bulbs must have a fresh weight over 0.3 g in order

to produce quality cut flowers (Kim et al. 1998a) Nevertheless, it has been also

reported that bulbs 0.1-0.2 g in weight are able to increase their weight up to 1 g

after one growing season under greenhouse conditions, producing high quality cut

flowers in the subsequent season (Kim et al. 1998a).

Future works in Leucocoryne sexual propagation under in vitro conditions should

consider increasing the macro and micronutrient concentration of the media

gradually until reaching 100% MS. In that way bulbs will be able to increase and

accelerate their weight constantly. By maintaining this continuous in vitro bulb

growth, and avoiding dormancy, it should be possible to reduce the time needed to

obtain flowering size bulbs.

In conclusion, in this work we determined that 15ºC as culture temperature

produced a very efficient Leucocoryne plant production using in vitro seed

germination. In all the genotypes tested high germination and bulbing rates were

80

achieved at 15°C. Culture temperatures did not produce statistical differences on

bulb fresh weight, but temperatures lower than 15ºC could produce an increase on

this parameter. With the addition of BAP it was possible to break the dormancy of

the bulbs, thus favoring sprouting and the multiplication of the explants. The

protocol developed in this work leads to high in vitro plant production efficiency and

may reduce the time needed to produce flowering size bulbs.

81

References.

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Seeds: Trade, Production and Technology. pp 218–230

Catley JL (2003) Temperature and Irradiance Effects on Vegetative Growth of Two

Species of Leucocoryne. J Am Soc Hortic Sci 128:815–820.

De la Cuadra C, Mansur L (2004) Descripción de la primera etapa del ciclo de vida

de tres genotipos de Leucocoryne sp.: semilla a bulbo. Agric Técnica. doi:

10.4067/S0365-28072004000200009



51:412–415.


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Ham F (2002) Evaluación de la germinación in vitro de Leucocoryne coquimbensis

F. Phil., geófita enémica con potencial ornamental. Pontificia Universidad

Católica de Chile

Hoffmann A, Watson J, Flores AR (2015) Flora Silvestre de Chile, Cuando el

Desierto Florece Vol.I. Monocotiledóneas, 1st edn. Ediciones Fundación

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Jara-Arancio P, Arroyo MTK, Guerrero PC, et al (2014) Phylogenetic perspectives

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on biome shifts in Leucocoryne (Alliaceae) in relation to climatic niche

evolution in western South America. J Biogeogr 41:328–338. doi:

10.1111/jbi.12186

Jara P, Arancio G, Moreno R, Carmona M (2006) Factores abióticos que

influencian la germinación de seis especies herbáceas de la zona árida de

Chile. Rev Chil Hist Nat 79:309–319.

Jha TB (2005) Plant Tissue Culture: Basic and Applied. Universities Press

Kim HH, Ohkawa K, Nitta E (1998a) EFFECTS OF BULB WEIGHT ON THE

GROWTH AND FLOWERING OF LEUCOCORYNE COQUIMBENSIS F.

PHILL. Acta Hortic 341–346. doi: 10.17660/ActaHortic.1998.454.40

Kim HH, Ohkawa K, Nitta E (1998b) Fall flowering of Leucocoryne coquimbensis F.

Phil. after long-term bulb storage treatments. HortScience 33:18–20.



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Endemic Genus. J Amer Soc Hort Sci 129:836–838.

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Table 1 Effect of temperature on the in vitro germination, bulbing and dormancy of three Leucocoryne genotypes after 13 weeks of culture from initiation.

Genotype Temperature Seeds Germination Bulbing Dormancy (°C) (N°) (N°) (%) (N°) (%) (N°) (%)

L. purpurea 15 150 128 85.3 a 118 92.2 a 37 28.9 b 20 150 73 48.7 b 60 82.2 b 39 53.4 a

L. vittata 15 150 145 96.7 a 144 99.3 a 108 74.5 b 20 150 118 78.7 b 114 96.6 b 106 89.8 a

L. sp. Pichicuy 15 150 148 98.7 a 140 94.6 a 108 73.0 b 20 150 125 83.3 b 108 86.4 b 104 83.2 a

Different letters in the same column indicate statistical differences for each genotype based on Scheffé´s procedure for multiple comparison of proportions (P≤0.05).

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Table 2 Effect of temperature on bulb occurrence bulbing, bulb sprouting, bulblet production and rooting after 24 weeks in MS 50% + 1.0 mg*L-1 BAP of three Leucocoryne genotypes.

Genotype Temperature Bulbing Bulb sprouting

Bulblet production

Rooting

(°C) (N°) (N°) (%) (N°) (%) (N°) (%)

L. purpurea 15 107 71 66.4 a 7 6.5 a 33 30.8 a 20 53 36 67.9 a 4 7.5 a 11 20.8 b

L. vittata 15 143 94 65.7 a 7 4.9 a 44 30.8 a 20 107 76 71.0 a 7 6.5 a 28 26.2 a

L. sp. Pichicuy 15 135 97 71.9 a 17 12.6 a 66 48.9 a 20 85 63 74.1 a 7 8.2 a 28 32.9 b

Different letters in the same column indicate statistical differences for each genotype based on Scheffé´s procedure for multiple comparison of proportions (P≤0.05).

87

Fig. 1 Leucocoryne genotypes. (a) Leucocoryne purpurea; (b) Leucocoryne vittata; and (c) Leucocoryne sp. Pichicuy.

Fig. 2 genotypes

Fig. 2 Effect of temperature ongenotypes after 13 weeks

Effect of temperature on13 weeks of culture

Effect of temperature on the in vitroof culture from

in vitro bulb production of from seed initiation

bulb production of initiation.

bulb production of three three Leucocoryne

88

Leucocoryne

89

Fig. 3 Effect of temperature on the bulb fresh weight of three Leucocoryne genotypes after 13 weeks from seed in vitro initiation. Different letters indicate statistical differences for each genotype according to analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P≤0.05).

Fig. 4Dormant bulbsMS 50% + 1.0 mg*L

Fig. 4 Different vDormant bulbs onMS 50% + 1.0 mg*L

Different visual appearance of on MS 25%

MS 50% + 1.0 mg*L-1 BAP.

isual appearance of MS 25%; (b) Active bulb

BAP.

isual appearance of Leucocoryne(b) Active bulbs with shoot

Leucocoryne bulbs on with shoot and bulblet

bulbs on in vitro conditions: (a) and bulblet producti

conditions: (a) production on

90

conditions: (a) on on

91

Fig. 5 Effect of culture temperature on bulb fresh weight of three Leucocoryne genotypes after 24 weeks of in vitro culture on MS 50% + 1.0 mg*L-1 BAP. Different letters indicate statistical differences for each genotype according to analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P≤0.05).

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CHAPTER 4

CONCLUSIONS

93

In this study we have developed an efficient protocol for the in vitro propagation of

the Leucocoryne genus using different culture techniques.

Different culture media, supplemental addition of BAP, different culture systems

and type of explants were evaluated in order to develop mass clonal bulb

propagation. We are also reporting an effective disinfection method as part of the

in vitro establishment protocol, by which we have surpassed one of the main

barriers for the Leucocoryne in vitro culture.

By studying the effect of culture media and BAP addition, higher bulb production

rates than the naturally occurred were achieved. A higher bulb fresh weight was

also achieved, which allows the use of such larger bulbs in further propagation

techniques.

We have achieved the highest rate of propagation of Leucocoryne bulbs reported

to date, considering both in vitro and ex vitro conditions, when bulbs were

subjected to bulb-cutting methods.

The effects of the culture temperature BAP addition on the in vitro seed

germination and bulb development, was also established. Results regarding both

factors have great potential of use in obtaining active and large plant populations

from sexual reproduction.

Protocols developed in this study will help to overcome the need of a reliable mass

propagation method, which is one of the most important pending challenges for the

Leucocoryne genus. An efficient mass propagation method is an essential and

valuable advance and an important contribution to the domestication and

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sustainable use of this plant genetic resource. Therefore, the use of these

protocols will assure a high production of plants to be used for conservation,

breeding or commercial purposes. This will greatly increase the value of

Leucocoryne genus in the ornamental crop industry.

Documents

in vitro techniques as supportive tools for breeding and