CELL BIOLOGY AND MORPHOGENESIS

Micropropagation of ornamental Prunus spp. and GF305 peach,a Prunus viral indicator

Anna Kalinina Æ Daniel C. W. Brown

Received: 26 July 2006 / Revised: 23 December 2006 / Accepted: 24 January 2007 / Published online: 24 February 2007

� Springer-Verlag 2007

Abstract A micropropagation approach was developed

for nine ornamental Prunus species, P. americana, P. cis-

tena, P. glandulosa, P. serrulata ‘Kwanzan’, P. laurocer-

asus, P. sargentii, P. tomentosa, P. triloba, P. virginiana

‘Schubert’, commercially important in North America, and

GF305 peach, commonly used for Prunus virus indexing.

The micropropagation cycle based on proliferation of

vegetative tissues includes establishment of tissue culture

through introduction of shoot meristems in vitro, shoot

proliferation, root induction and plant acclimatization steps

and can be completed in 5 months. A meristem steriliza-

tion protocol minimized bacterial and fungal contamina-

tion. Multiple shoot formation in ornamental Prunus was

obtained through the use of 1 mg l–1 6-benzyladenine. For

GF305 peach, alteration in the sugar composition, fructose

instead of sucrose, and addition of 1 mg l–1 ferulic acid had

a significant impact on the shoot proliferation rate and

maintenance of long-term in vitro culture. Rooting and

plant acclimatization conditions were improved using a

two-step protocol with a 4-day root induction in indole-3-

butiric acid (IBA)-containing media with consequent 3-

week root elongation in IBA-free media. One-month

incubation of rooted shoots in a vermiculite-based medium

resulted in additional shoot and root growth and provided

better acclimatization and plant recovery. The microprop-

agation approach can be used for maintenance of the clonal

properties for Prunus spp. as well as a protocol to support

meristem therapy against viral infection.

Keywords P. americana � P. cistena � P. glandulosa �P. serrulata ‘Kwanzan’ � P. laurocerasus � P. sargentii �P. tomentosa � P. triloba � P. virginiana ‘Schubert’ �Virus indexing � Meristem therapy

Abbreviations

BA 6-Benzyladenine

IBA Indole-3-butiric acid

GA Gibberellic acid

TDZ 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea

(thidiazuron)

NAA a-Naphthaleneacetic acid

FA Ferulic acid (trans-4-hydroxy-3-methoxycinnamic

acid)

MES 2-(N-Morpholino)-ethanesulphonic acid

MS Murashige and Skoog (1962) basic salt medium

(PhytoTechnology Laboratory, KS, USA)

QL Quoirin and Lepoivre (1977) basic salt medium

(PhytoTechnology Laboratory, KS, USA)

SAM Shoot apical meristem

AxM Axillary shoot meristem

MP Meristem proliferation medium

SP Shoot proliferation medium

RM Rooting medium

Introduction

The Prunus genus includes more than 200 species of

deciduous or evergreen trees and shrubs widely distributed

in temperate zones and are valued for their ornamental

Communicated by R.J. Rose.

A. Kalinina � D. C. W. Brown (&)

Southern Crop Protection and Food Research Centre,

Agriculture and Agri-Food Canada, 1391 Sandford Street,

London, ON N5V 4T3, Canada

e-mail: browndc@agr.gc.ca

A. Kalinina

e-mail: kalininaa@agr.gc.ca

123

Plant Cell Rep (2007) 26:927–935

DOI 10.1007/s00299-007-0315-x

features. Ornamental Prunus species, such as P. americana,

P. glandulosa, P. tomentosa and P. triloba, are grown for

their white, pink or red flowers, saucer-, bowl- or cup-shaped

with five petals, often in semi-double or double forms.

Some, such as P. serrulata ‘Kwanzan’ and P. sargentii, have

shiny and colourful bark and beautiful autumn leaf colour;

others, such as P. x cistena and P. virginiana ‘Schubert’,

have attractive purple or green waxy foliage. Dense, bushy

species, such as P. laurocerasus, are useful for groundcover

and green fences. The commercial importance of most or-

namental Prunus in the North American climate is based on

their attractive appearance, extensive vegetation, unpreten-

tious growth conditions and high adaptation abilities. GF305

peach (P. persica ‘GF305’) is commonly used for grafting

different commercially important peach varieties as well as

an indicator plant for virus indexing (Bernhard and Mare-

naud 1969; Gentit et al. 1998). More than 30 years before

establishment of molecular biology approaches the only way

to identify and characterize several degenerating Prunus

virus and virus-like diseases, such as Plum pox potyvirus,

Apple chlorotic leaf spot trichovirus, Prune dwarf ilavirus,

Prunus necrotic ring spot ilavirus, Tomato ring spot nepo-

virus, Peach latent mosaic viroid, European stone fruit

yellow phytoplasma and Peach asteroid spot agent, was

based on the symptom analysis of GF305 peach seedlings

(Desvignes 1976; EPPO Panel on Certification of Fruit

Crops 1992).

Propagation of most Prunus spp. and selection of new

cultivars based on traditional crossing, seed and clonal (stem

cuttings) propagation are time and labour consuming and

often applicable for only a few species (Matt and Jehle

2005). Moreover, most ornamental Prunus do not produce

pollen or viable generative organs. Micropropagation tech-

niques for sour and sweet cherries (Borkowska 1983;

Hammat and Grant 1996; Matt and Jehle 2005), cherry and

peach rootstocks (Dalzotto and Docampo 1997; Marino

1997; Radice et al. 1999), plum (Mante et al. 1989, 1991;

Gonzalez Padilla et al. 2003) and peach (Hammerschlag

et al. 1987) have been described. There are only a few re-

ports on ornamental Prunus spp., P. tomentosa, P. fructi-

cosa, P. virginiana ‘Carrington’ and P. pensylvanica (e.g.

Pruski et al. 2000, 2005). Protocols for P. tomentosa and P.

virginiana ‘Carrington’ described a short-term (3-month)

tissue culture maintenance, terminated by rooting and had

low plant recovery efficiency. Most protocols are optimized

only for particular cultivar, and it is not clear if they are

applicable to other cultivars or species. Micropropagation of

other commercially used ornamental species, such as P.

americana, P.x cistena, P. glandulosa, P. serrulata

‘Kwanzan’, P. laurocerasus, P. sargentii and P. triloba,

have not yet been reported. There are a few reports on

propagation of GF305 peach confirming that it was a re-

calcitrant species under in vitro conditions, allowing only 2–

3 months of subculture (Lansac et al. 1998). The purpose of

this investigation was to develop a reliable system for mi-

cropropagation of nine ornamental Prunus spp., which have

a high commercial importance in North America and GF305

peach, which is a well-known species used as an indicator of

Prunus diseases for virus-free certification of plant material.

Materials and methods

Plant material

Nine ornamental Prunus cultivars, P. americana Marshall

(American plum), P.x cistena Koehne (P. cerasifera

‘Atropurpurea’ · P. pumila) (Purpleleaf sand cherry), P.

glandulosa Thunb. (Flowering almond), P. serrulata Lindl.

‘Kwanzan’, P. laurocerasus L. (Cherry laurel), P. sargentii

Rehder (Sargent cherry), P. tomentosa Thunb. (Nanking

cherry), P. triloba Lindl. (Flowering almond) and P. vir-

giniana L. ‘Schubert’ (Choke cherry) (Encyclopaedia of

Garden Plants 1997), obtained from nurseries in the

Niagara region (Ontario, Canada), and seeds of P. persica

L. (Batsch) ‘GF305’, obtained from the INRA research

station in Bordeaux, France, were used for establishing

in vitro cultures.

Establishment of Prunus in vitro culture

Shoot apical meristems (SAM) derived from greenhouse-

grown 1-year-old trees of P. americana, P.x cistena

(Fig. 1a), P. glandulosa, P. tomentosa, P. triloba, P. vir-

giniana ‘Schubert’ and GF305 peach (Fig. 1g), and axillary

shoot meristems (AxM) obtained from branches of field-

grown trees of P. serrulata ‘Kwanzan’, P. sargentii and P.

laurocerasus were used to establish in vitro cultures. Wood

sticks were forced to shoot using a bud-breaking medium

based on the half-strength MS basic salt medium (Mu-

rashige and Skoog 1962; PhytoTechnology Laboratory,

KS, USA), supplemented with 6-benzyladenine (BA),

indole-3-butiric acid (IBA) and gibberellic acid (GA)

(Table 1). Shoot tip explants with several leaf primordia

2–5 mm long were surface sterilised using 70% ethanol for

30 s followed by immersion in 15% Bleach and 0.02%

Tween-20 solution on a shaker for 15 min and then washed

with sterile distilled water. Explants were placed on mer-

istem proliferation media (MP1-5, Table 2). After 4 weeks,

explants were transferred into shoot proliferation media

(SP1 or SP2, Table 2) and subcultured each month.

Rooting in vitro and plant acclimatization

Shoot explants obtained from proliferating 4-week-old

Prunus tissue culture, 1–2 cm long, were placed in a

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vertical position into different rooting media (RM1-8, Ta-

ble 3) and incubated for 4 weeks under three conditions

with different duration periods (Table 4). Experiment 1

included shoot incubation in RM1 or RM5 for 28 days;

experiment 2 included a 4-day incubation in RM1 or RM5

and subsequent transfer of shoots into RM2 or RM6;

experiment 3 included a 4-day incubation in RM3 or RM7

and subsequent transfer of shoots into RM4 or RM8.

Successfully rooted explants were transferred into a ver-

miculite-based medium (medium size vermiculite, Therm-

O-Rock East Inc., PA, USA) containing 1/2 MS salts and

vitamins (see vitamins used in the Table 1) without sugar,

growth regulators and Bactoagar, dispensed in Magenta

vessels and incubated for an additional month to adapt

newly formed roots to soil conditions. After 1 month,

plants were transferred into pots with ProMix ‘BX’ soil

mixture (ProMix ‘BX’, Premier Horticulture Inc., Canada),

placed in a growth chamber (+25 ± 2�C, 16 h photoperiod,

800–850 lmol m–2 s–1, 75% humidity), continuously irri-

gated with 1/5 MS salts solution and maintained in the light

chamber for an additional month before transfer into the

greenhouse.

Media and growth conditions

All solid media consisted of 0.8% Bactoagar (Fisher, USA)

and 0.5 g l–1 MES (2-(N-Morpholino)-ethanesulphonic

acid); pH was adjusted to 5.7–5.8 by 1 M KOH before

autoclaving. For GF305 peach shoot proliferating culture,

instead of MS, QL basic salt medium (Quoirin and Le-

poivre 1977; PhytoTechnology Laboratory, KS, USA) was

used supplemented with 15 g l–1 fructose and 1 mg l–1

ferulic acid (trans-4hydroxy-3-methoxycinnamic acid, FA)

(Tables 2, 3). Vitamins and growth regulators were filter-

sterilized and added to the autoclaved media when re-

quired. Media were dispensed into 100 · 25-mm Petri

plates (40 ml per plate) or Magenta vessels (Magenta,

Chicago, Il, USA), 60 ml per vessel when specified. Tis-

sues were grown in a culture room at +25 ± 2�C, 16 h light

photoperiod with light provided by full-spectrum fluores-

cent lamps (General Electric, ‘‘Starcoat’’, F32T8/SP41,

Canada) at an intensity of 300–350 lmol m–2 s–1. All

chemical products were purchased from Sigma, USA, ex-

cept when specified.

Results

Establishment of in vitro meristem proliferation system

for ornamental Prunus and GF305 peach

The SAM and AxM sterilization protocol reduced the

frequency of bacterial or fungal contamination of green-

house and field-grown material to a very low level (we

estimated less than 1% visual culture contamination). SAM

of P.x cistena (Fig. 1b) and P. triloba had a high prolif-

eration rate in MP1 containing 0.5 mg l–1 BA, 0.5 mg l–1

IBA and 2 mg l–1 GA, respectively 94 and 66% (Table 5).

SAM and AxM of P. serrulata ‘Kwanzan’, P. americana

and P. virginiana ‘Schubert’ proliferated in MP2 containing

Fig. 1 P.x cistena (a-f) and

GF305 peach (g-l)micropropagation system. a, gA shoot tip with SAM. b, hSAM proliferation in tissue

culture. c, i Multiple shoot

formation. d, j Root induction.

e, k Plant acclimatization in

Magenta boxes. f, l Recovered

plant. Bars: 1 cm

Table 1 Medium used for excised dormant Prunus bud-wood

Components Concentration (mg l–1)

MS salts 1/2

Vitamins

Glycine 2.0

Myo-inositol 100

Nicotinic acid 0.5

Pyridoxine 0.5

Thiamin-HCl 1.0

Ascorbic acid 2.0

Growth regulators

BA 2.0

IBA 0.1

GA 5.0

MS Macro and microelements from Murashige and Skoog (1962)

(PhytoTechnology Laboratory, KS, USA)

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2 mg l–1 BA with the proliferation rate of 75, 100 and 50%,

respectively. SAM and AxM of P. glandulosa, P. sargentii

and P. tomentosa proliferated in MP3 containing

2.75 mg l–1 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (thidi-

azuron) (TDZ) and 0.5 mg l–1 IBA with the proliferation

rate of 100, 100 and 85%, respectively. For GF305 peach

SAM, the medium based on QL basic salts and 15 g l–1

fructose supplemented with 0.5 mg l–1 BA, 0.5 mg l–1 IBA

and 2 mg l–1 GA (MP4) was more successful, and over

75% of SAM proliferated (Table 5, Fig. 1h). While using

sucrose, shoot explants had a depressed growth of apical

and axillary shoots and often produced callus at the base of

the shoot and lower leaves (Fig. 2a). Addition of 1 mg l–1

FA provided earlier shoot tissue maturation, formation of

secondary tissues at the shoot stem and prevented oxidation

of the shoot base containing tissue scars (Fig. 2b). Axillary

Table 2 Meristem and shoot proliferation media used for ornamental Prunus and GF305 peach in tissue culture

Plant applied Meristem proliferation media Shoot proliferation media

Ornamental Prunus Peach Ornamental Prunus Peach

Components MP1 MP2 MP3 MP4 MP5 SP1 SP2

Macro and micro salts MS MS MS QL MS MS QL

Sugar (g l–1) Suc 30 Suc 20 Suc 25 Fru 15 Fru 15 Suc 30 Fru 15

Vitamins (see Table 1)

FA (mg l–1) 1.0 1.0 1.0

Growth regulators (mg l–1)

BA 0.5 2.0 0.5 1.0 1.0

IBA 0.5 0.5 0.5 0.5 0.05

GA 2.0 2.0 0.1

TDZ 2.75 2.75

MS Macro and microelements from Murashige and Skoog (1962) (PhytoTechnology Laboratory, KS, USA); QL macro and microelements from

Quoirin and Lepoivre (1977) (PhytoTechnology Laboratory); Suc sucrose added as a sugar component. Fru fructose added as a sugar component

Table 3 Root induction media used for ornamental Prunus and peach in tissue culture

Plant applied Rooting media

Ornamental Prunus Peach

Components RM1 RM2 RM3 RM4 RM5 RM6 RM7 RM8

MS salts 1/2 1/2 1/2 1/2 1/2 1/2

Sucrose (g l–1) 30 30 15 20 30 30 15 20

Vitamins (see Table 1)

FA (mg l–1) 1.0 1.0 1.0 1.0

Growth regulators

IBA (mg l–1) 3.0 3.0 3.0 3.0

MS Macro and microelements from Murashige and Skoog (1962) (PhytoTechnology Laboratory, KS, USA)

Table 4 Duration and transfer protocol for root induction in ornamental Prunus and GF305 peach

Plant applied Incubation time (days)

Ornamental Prunus Peach

Rooting media RM1 RM2 RM3 RM4 RM5 RM6 RM7 RM8

Experiment 1 28 28

Experiment 2 4 24 4 24

Experiment 3 4 24 4 24

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shoots were formed on proliferated shoot explants only

during the second passage on proliferation media SP1 and

SP2, respectively, for ornamental Prunus and peach (Ta-

ble 2). Particularly, P. glandulosa shoot explants had a

high axillary shoot formation rate of 4.1 ± 0.6 shoots per

explant within first 2 months (Fig. 3), whereas, the rest of

the species required additional subculturing on SP1 or SP2

(Fig. 1c, i). The number of axillary shoots formed per ex-

plant per month was evaluated within the first and the

second year after establishment of shoot meristem culture

and was stable for all investigated Prunus spp. (Fig. 4).

The highest rate of axillary shoot formation per month,

5.37 ± 0.3 shoots per explant, was observed in P. glan-

dulosa and less so, 2.01 ± 0.2 to 2.65 ± 0.6, in P. ameri-

cana, P. x cistena, P. tomentosa, P. triloba, P. virginiana

‘Schubert’ and GF305 peach. Whereas, in P. serrulata

‘Kwanzan’, P. laurocerasus and P. sargentii the rate was

within 1.5 ± 0.2 to 1.8 ± 0.2 (Fig. 4).

Root induction and plant recovery in ornamental

Prunus and GF305 peach

Several different media (RM1-8, Table 3) and approaches

(Experiments 1–3, Table 4) were applied to optimize a

rooting in vitro protocol for ornamental Prunus spp. and

GF305 peach. Successful root induction and elongation

was obtained in experiment 1 for P. americana, P.x cistena

(Fig. 1d), P. serrulata ‘Kwanzan’, P. triloba and P. vir-

giniana ‘Schubert’ using RM1 medium containing 3 mg l–1

IBA (experiment 1, Table 6). In P. laurocerasus, P. sar-

gentii and P. tomentosa, roots formed after a 4-day

induction in RM1 and a 3-week elongation in RM2 without

IBA (experiment 2). For P. glandulosa, roots successfully

formed using a 4-day induction in RM3, MS salts-free

medium contained 3 mg l–1 IBA, followed by root elon-

gation in RM4 without IBA (as outlined for experiment 3).

GF305 peach formed roots with a high efficiency (Fig. 1j)

with conditions used in experiment 2, i.e. a 4-day root

induction period in RM5 with 3 mg l–1 IBA, 1 mg l–1 FA

and 30 g l–1 sucrose instead of fructose and a subsequent

root elongation in RM6 without IBA (Table 6). The num-

ber of roots formed per shoot explant and the timing of root

formation was strongly dependant on the species and media

(Fig. 5). In general, roots appeared between 8 and 16 days

post induction. Early rooting species were P. americana,

P.x cistena, P. tomentosa, P. triloba, P. virginiana

‘Schubert’ and GF305 peach. With some delay, roots were

formed by P. serrulata ‘Kwanzan’, P. sargentii and

P. laurocerasus. Mostly, these latter species formed callus

at the explant base before root initiation.

Acclimatization to greenhouse conditions was based

on a two-step protocol. Firstly, rooted microshoots were

transferred into a vermiculite-based medium in Magenta

vessels to adapt roots to soil conditions for 1 month

(Fig. 1e, k). Then plantlets were exposed briefly to

ambient air conditions in the tissue culture room and

subsequently moved into pots. The survival rate was

generally high and species dependant (Table 7). P.x cis-

tena, P. laurocerasus, P. sargentii, P. serrulata ‘Kwan-

zan’, P. tomentosa and GF305 peach had more than 80%

survival rate in soil. In contrast, P. americana, P. glan-

dulosa, P. triloba and P. virginiana ‘Schubert’ were af-

fected by low humidity and had a reduced efficiency.

After 1–3 months cultivation in soil, plants produced

axillary shoots and had the same morphological tree-like

structure including an apical dominance growth pattern

and axillary branches (Fig. 1f, l).

Table 5 Meristem proliferation rate after 1 month incubation

Prunus spp. MP1 MP2 MP3 Recommended medium

for meristem proliferation

P. americana 25a 100 30 MP2

P. x cistena 94 13 29 MP1

P. glandulosa 37 76 100 MP3

P. laurocerasus NA NA NA MP1

P. sargentii 0 0 100 MP3

P. ‘Kwanzan’ 48 75 21 MP2

P. tomentosa 80 28 85 MP3

P. triloba 66 20 3 MP1

P. ‘Schubert’ 45 50 23 MP2

MP4 MP5

GF305 peach 76 62 MP4

NA Not attempteda The shoot proliferation rate is normalized by percent of the total and

calculated, for each case, as a ratio of explants with shoots prolifer-

ated divided by the total number of explants subjected to the exper-

iment. The sampling number analysed for percentage data included

25–88 explants per each species

Fig. 2 Influence of sugar composition on growth and development of

GF305 shoots in tissue culture. a Incubation of explants in shoot

proliferation medium containing 30 g l–1 sucrose. b Multiple shoot

formation in SP2 medium containing 15 g l–1 fructose and 1 mg l–1

FA. Bars: 1 cm

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123

Discussion

A micropropagation approach for ornamental Prunus and

GF305 peach was developed to preserve the clone/species

identity and, therefore, was based on propagation of veg-

etative tissues rather than embryonic tissue. All steps of the

micropropagation protocol were found to be dependant on

the species genetic background and media conditions,

including growth regulators, optimized for each species. A

shoot meristem proliferation approach with over 80%

proliferation efficiency was found for the Prunus species

tested here, and allowed the use of greenhouse and field-

grown material over the entire year. Protocols for P. tom-

entosa and P. virginiana ‘Carrington’ have previously been

reported to be limited by the season (Pruski et al. 2000,

2005). The shoot meristem sterilization protocol, one of the

critical steps for obtaining higher proliferation rate, was

effective for both ornamental Prunus and GF305 peach and

minimized visible bacterial and fungal contamination

in vitro. The shoot proliferation media had minor but sig-

nificant variations for ornamental Prunus and GF305

peach, allowing the maintenance of tissue culture with a

Fig. 3 Number of axillary shoots formed in meristem proliferating

culture after 2 months. During the first 4 weeks, explants were

incubated in meristem proliferation media (MP1-5, Table 2) and,

thereafter, transferred into shoot proliferation media (SP1 or SP2,

Table 2) for additional 4 weeks. White, grey and dark bars

correspond, respectively, to MP1, MP2 and MP3. For GF305 peach,

the bar set corresponds to MP4 (white bar) and MP5 (dark bar).

Values represent the mean with the standard error of three replicates

with 5–21 shoot explants in each

Fig. 4 Number of axillary

shoots per month formed in

shoot proliferation media (SP1

or SP2, Table 2) after 1 year in

culture. Values represent the

mean number of shoots formed

per each shoot explant and the

standard error of three replicates

with 11–45 shoot explants in

each

Table 6 Root formation in Prunus tissue culture

Prunus spp. Exp1a Exp2a Exp3a Recommended

protocol for rooting

P. americana 100b 100b 69b Exp 1

P. x cistena 93 93 86 Exp 1

P. glandulosa 67 89 96 Exp 3

P. laurocerasus 100 80 50 Exp 2

P. sargentii 50 61 27 Exp 2

P. Kwanzan 100 85 46 Exp 1

P. tomentosa 100 79 77 Exp 2

P. triloba 87 50 56 Exp 1

P. ‘Schubert’ 70 36 30 Exp 1

GF305 peach 93 100 53 Exp 2

80c 100c

a See Table 4 for duration and transfer protocol of rooting experi-

mentsb The root proliferation rate is normalized by percent of the total and

calculated, for each case, as a ratio of rooted explants divided by the

total number of shoots subjected to the experiment. The sampling

number analysed for percentage data included 16–122 explants per

each speciesc No FA added

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stable proliferation rate over a 2-year period. Particularly,

alteration in the sugar composition, fructose instead of

sucrose, and addition of 1 mg l–1 FA prevented shoot

necrosis and withering and had a significant impact on the

shoot rate and survival of GF305 peach tissue culture. A

treatment with antioxidant compounds such as ascorbic

acid, citric acid, activated charcoal, FA, polyvinilpyrroli-

done and silver nitrate has been reported for several mi-

cropropagation systems (Komalavalli and Rao 2000;

Huang et al. 2002; Wen and Deng 2005). Formation of

morphologically well-developed axillary shoots was ob-

tained at the optimal BA concentration of 1 mg l–1. The

same BA level was also suggested for P. tomentosa and P.

virginiana ‘Carrington’ by Pruski et al. (2000, 2005).

Treatments with elevated BA concentrations and TDZ

promoted the shoot number per explant but decreased the

shoot length and negatively affected shoot development

(Pruski et al. 2000; Sarwar and Skrivin 1997; our obser-

vations).

Ex vitro root induction by application of 2 mg l–1 IBA

has been reported for some Prunus spp. such as P. tom-

entosa, P. fruticosa, P. virginiana ‘Carrington’ and P.

pensylvanica (Pruski et al. 2000, 2005). Our observations

confirmed this approach for P. glandulosa, P. tomentosa

and P. virginiana ‘Schubert’ (data not shown), whereas,

root induction in P. americana, P.x cistena, P. serrulata

‘Kwanzan’, P. sargentii, P. laurocerasus and P. triloba

was only achieved by use of an in vitro protocol. Most

Prunus spp. produced a high number of roots in IBA-

containing media, but roots were often shorter and thicker

and did not form lateral roots (data not shown). Rooting

was improved using the two-step protocol with a 4-day root

induction in IBA-containing media with a subsequent 3-

week root elongation in IBA-free media. As shoot height

and number of leaves had been reported to be critical

factors for acclimatization of plum plantlets (Gonzalez

Padilla et al. 2003), an additional 1-month incubation of

rooted shoots in the vermiculite-based medium provided

extensive shoot and root growth and resulted in better

acclimatization for most Prunus spp.

In summary, the micropropagation cycle based on pro-

liferation of vegetative tissues includes establishment of

tissue culture through introduction of shoot meristems

in vitro with reduced fungal and bacterial contamination,

meristem and shoot proliferation, root induction and plant

acclimatization steps (Fig. 6). The complete cycle from

introduction of meristems into tissue culture to recovery of

greenhouse grown plants requires about 5 months. The

overall efficiency for plant recovery during the minimum

time period required per each step depends on the genotype

and ranges from 2.38 to 0.51 (Table 8). The maximum

efficiency occurred for P. glandulosa and P.x cistena, 2.38

and 2.0, respectively; the second group including P. tom-

entosa, GF305 peach, P. laurocerasus and P. serrulata

Fig. 5 Numbers of adventitious roots formed in Prunus spp. after a

4-week incubation in root induction media (RM1-8, Table 3). White,

grey and dark bars correspond to experiment 1, experiment 2 and

experiment 3 (Table 4). For GF305 peach, the second bar set

corresponds to experiments 1 and 2 without FA in the media. Values

represent the mean number of roots formed per each shoot explant

with the standard error of three replicates with 5–22 shoot explants in

each

Table 7 The survival rate of Prunus tissue culture-derived plantlets

after rooting and acclimatization for greenhouse conditions

Prunus spp. Number of plants Survival rate

P. americana 38 29a

P. x cistena 50 80

P. glandulosa 67 46

P. laurocerasus 12 92

P. sargentii 14 79

P. ‘Kwanzan’ 33 85

P. tomentosa 60 84

P. triloba 22 57

P. ‘Schubert’ 38 57

GF305 peach 52 83

a The survival rate is normalized by percent of the total and calcu-

lated, for each case, as a ratio of rooted plants growing in ProMix soil

(ProMix’BX’, Premier Horticulture Inc., Canada) after 1 month di-

vided by the total number of plantlets subjected to the experiment

Plant Cell Rep (2007) 26:927–935 933

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Fig. 6 Five-month Prunus micropropagation cycle. All stages of the

protocol are genotype dependent; the efficiency of meristem

proliferation ranges 0.5–1.0 explants surviving; shoot proliferation

ranges 1.5–5.4 shoots formed per explant; root induction ranges 0.6–

1.0 explants rooted and acclimatization ranges 0.3–0.9 plantlets

recovered. Bars: 1 cm

Table 8 The calculated efficiency of the micropropagation cycle based on the efficiency of each step of the protocol

Prunus spp. Micropropagation steps Total efficiency

Meristem proliferation Shoot proliferation Rooting Acclimatization

P. americana 1.0a 2.1b 1.0c 0.29d 0.61e

P. x cistena 0.94 2.86 0.93 0.8 2.0

P. glandulosa 1.0 5.37 0.96 0.46 2.38

P. laurocerasus NA 1.49 1.0 0.92 1.37

P. sargentii 1.0 1.76 0.61 0.79 0.85

P. ‘Kwanzan’ 0.75 1.72 1.0 0.85 1.10

P. tomentosa 0.85 2.64 1.0 0.84 1.88

P. triloba 0.66 2.45 0.87 0.57 0.8

P. ‘Schubert’ 0.5 2.58 0.7 0.57 0.51

GF305 peach 0.76 2.28 1.0 0.83 1.44

The efficiency of each step was calculated at the minimum time period used for each step, i.e meristem proliferation, shoot proliferation, rooting

and acclimatizationa Value calculated from Table 5 divided by 100%b Value taken from Fig. 4c Value calculated from Table 6 divided by 100%d Value calculated from Table 7 divided by 100%e Total efficiency is calculated by multiplication of the efficiency of each step of the protocol

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‘Kwanzan’ had an efficiency of 1.1–1.9 and the third group

ranges from 0.5–0.8 for P. sargentii, P. triloba, P. ameri-

cana and P. virginiana ‘Schubert’. In addition, Prunus

tissue culture at the shoot proliferation stage can be ex-

tended for at least 2 years without visible changes in the

shoot formation rate and shoot development properties. An

extended shoot proliferation phase can result in an im-

proved plant multiplication rate. For example, in P. glan-

dulosa, if a 1-month proliferation phase was used then 2.38

plants are recovered, whereas, in a 3-month proliferation

phase up to 70 plants can be recovered. The micropropa-

gation approach could also be used for other applications

such as genetic transformation. The high rate of multiple

shoot proliferation in most Prunus spp. and fast root

induction protocols might be useful for micropropagation

and recovery of putative transformants. Another applica-

tion of the micropropagation cycle is that recovered orna-

mental Prunus plants could be used as part of a protocol for

self-rooted or grafted viral-free material in nurseries and

orchards. Manganaris et al. (2003) has showed that an

in vitro based meristem-tip culture/heat therapy protocol

was effective for virus elimination. The established pro-

tocols for tissue culture and plant recovery of P. glandul-

osa, P. tomentosa, P. serrulata ‘Kwanzan’ and GF305

peach plantlets might be useful for propagation of the plant

material for virus indexing in vitro culture, greenhouse or

field conditions.

Acknowledgments This work was supported by an NSERC re-

search fellowship for Canadian Government Laboratories for A.K.

The authors thank Paul Moote for technical assistance and Alex

Molnar for picture graphic design.

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