In vitro germination and seedling development of

cryopreserved Dendrobium hybrid mature seeds

Wagner A. Vendrame a,*, V.S. Carvalho b, J.M.M. Dias b

a Tropical Research and Education Center, University of Florida, 18905 SW 280th Street, Homestead, FL 33031-3314, USAb Departamento de Fitotecnia, Universidade Federal de Vicosa, Vicosa, MG 36570-000, Brazil

Received 18 April 2006; received in revised form 2 April 2007; accepted 5 June 2007

Abstract

In vitro germination and seedling development from Dendrobium Swartz. hybrid ‘Sena Red’, ‘Mini WRL’, ‘Jaquelyn Thomas’, and ‘BFC Pink’

seeds cryopreserved through vitrification (PVS2) were evaluated. Germination percentages after cryopreservation (LN) were variable among

different controls and treatments, despite the initial high seed viability for all hybrids. Seeds exposed to PVS2 at ice temperature from 1 to 3 h prior

to LN exhibited significantly higher germination than seeds exposed to PVS2 at room temperature for the same time periods. No significant

differences in germination percentages were observed for time exposure to PVS2 at 1, 2 or 3 h for ‘Sena Red’, ‘Mini WRL’, and ‘BFC Pink’. Seeds

of ‘Jaquelyn Thomas’ exposed to 1 h prior to LN showed higher germination percentage than for exposure to 2 or 3 h. The combination of a pre-

cooling treatment (ice) with a dehydration treatment (PVS2) for a period of time of 1–3 h was essential to allow proper germination of

cryopreserved seeds. Although variability in seed germination among different hybrids and treatments was observed, germination was above 50%

of the controls and all germinated seeds developed into normal seedlings with healthy shoot and root formation. No abnormalities, nutritional

deficiencies, or diseases were observed in developed seedlings and no significant differences were observed for seedling growth and development

from germinated seeds among the different hybrids. Seedlings transplanted to pots acclimatized well and developed into normal plants within 6–8

months in greenhouse. Transplanted seedlings exhibited 100% survival for all hybrids.

# 2007 Elsevier B.V. All rights reserved.

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Scientia Horticulturae 114 (2007) 188–193

Keywords: Orchids; Cryopreservation; Vitrification; Orchidaceae

1. Introduction

Hybridization in orchids is a common means for producing

new and improved material, including new flower colors, color

patterns, flower size, number, and a number of additional

characteristics of commercial value. Over 100,000 commercial

hybrids are registered worldwide to date, being grown as cut

flowers and potted plants. The demand for orchid cut flowers

increased and recent data indicate that orchids represent 8% of

the global floriculture trade, with Dendrobium hybrids being

commercially desirable due to the number of flowers per

inflorescence and recurrent flowering (Martin and Madassery,

2006). Furthermore, the variety of flower colors and color

Abbreviations: LN, liquid nitrogen; FDA, fluorescein diacetate; PVS2,

plant vitrification solution number 2

* Corresponding author. Tel.: +1 305 2467001; fax: +1 305 2467003.

E-mail addresses: vendrame@ufl.edu (W.A. Vendrame),

virginia@vicosa.ufv.br (V.S. Carvalho).

0304-4238/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2007.06.006

patterns, and the relatively short production cycle from seedling

to a full bloom plant for Dendrobium hybrids increase their

commercial value. In vitro germination of hybrid seeds is a

common practice among orchid growers and most orchid seeds

can either be readily germinated after harvest from the mother

plant or stored for later germination. Seed storage plays an

important role for long-term conservation of orchid species

seeds, allowing both preservation and easy distribution of

germplasm at reduced costs (Pritchard and Seaton, 1993).

Likewise, for orchid breeders long-term storage of hybrid seeds

represents an important tool for breeding programs. Vitrifica-

tion is used as an efficient and suitable means for cryopre-

servation of plant tissues and organs (Fahy et al., 1984; Sakai

et al., 1990; Thammasiri, 2000). Cryopreservation is reported in

orchids (Pritchard, 1984; Pritchard et al., 1999; Popova et al.,

2003) and vitrification is used (Ishikawa et al., 1997; Wang

et al., 1998; Tsukasaki et al., 2000) to cryopreserve orchid

protocorms, zygotic embryos, and cell suspensions. The use of

small storage containers and the low maintenance costs make

W.A. Vendrame et al. / Scientia Horticulturae 114 (2007) 188–193 189

cryopreservation potentially valuable for the conservation of

orchid seeds (Thammasiri, 2000). Cryopreservation of orchid

seeds is reported for the preservation of Dendrobium candidum

Wall. ex Lindl. (Wang et al., 1998), Doritis pulcherrima Lindl.

(Thammasiri, 2000), and Bletilla striata Rchb. f. (Hirano et al.,

2005), among others. Vitrification is an appropriate and

practical approach for conservation of many accessions of

orchid seeds, being quick, simple, reliable, and low-cost. The

aim of the present research was to evaluate in vitro germination

and plant growth from several Dendrobium hybrid seeds

cryopreserved through vitrification.

2. Materials and methods

2.1. Plant material

Mature seed capsules from four self-pollinated Dendrobium

Swartz. hybrids, ‘Sena Red’, ‘Mini WRL’, ‘Jaquelyn Thomas’,

and ‘BFC Pink’ were obtained from Ellenton Growers

(Palmetto) and Kerry’s Bromeliads (Homestead), FL, USA.

The capsules were collected in the fall 2004 and stored at room

temperature (27 � 2 8C) in a desiccator for 24 h. Capsule size

(length � width) was measured using an Electronic Digital

Caliper (Control Company, Friendswood, TX, USA). Seeds

were removed from capsules and seed size was evaluated with a

micrometer by measuring length � width of individual seeds

under a Leica MZ12.5 stereoscope (Leica Microsystems,

Buffalo, NY, USA) at 50� magnification. Capsule weight with

and without the seeds was calculated. Seeds were weighted and

oven-dried at 103 8C for 17 h to constant weight, and initial

seed moisture content was determined. Final seed moisture

content was also determined after seed were disinfected. Seed

viability was determined using the fluorescein diacetate (FDA)

staining technique (Pritchard, 1985). Briefly, orchid embryos

immersed in distilled water are mixed 1:1 (v/v) on a microscope

slide with a solution of FDA at 0.5% (w/v) in absolute acetone.

Embryos are observed under UV light for fluorescence, shown

as a bright yellow color, thus indicating viability. A seed

germination test was also performed to verify the consistency of

the FDA viability test. Seed germination percentage was

determined for each hybrid. The number of seeds per capsule

was estimated by weight difference (capsule weight with

seeds � capsule weight without seeds) and by calculating the

weight of 100 seeds.

2.2. Seed disinfection and preparation

About 250 mg of seeds from each hybrid were placed inside

a 100-ml sterile syringe containing 50 ml of 70% ethanol (v/v)

and agitated for 1 min. The ethanol was removed with a plastic

disposable transfer pipette and 50 ml of 0.6% sodium

hypochlorite were added, followed by agitation for 20 min.

After removal of the sodium hypochlorite solution, seeds were

rinsed in sterile deionized water twice and transferred to a

sterile 150-ml flask. They were left in the flasks overnight at

room temperature (27 � 2 8C). The following day, seeds were

distributed in 2-ml cryovials, containing about 1 ml of seed

suspension per vial. Through serial dilutions, 1 ml of solution

was calibrated to contain approximately 1000 seeds. Prior to

serial dilutions, seed samples were removed, weighed and

oven-dried as described above to verify the variation in seed

moisture content as affected by the seed disinfection technique.

2.3. Vitrification procedure and treatments

For each treatment, sterile deionized water was removed

from each cryovial and 1 ml cryoprotective solution (2 M

glycerol and 0.4 M sucrose, pH 5.7) was added. Cryovials were

left for 30 min at room temperature (27 � 2 8C) prior to the

addition of a plant vitrification solution. The plant vitrification

solution, designated PVS2 (Sakai et al., 1990), consisted of

30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v)

dimethyl sulfoxide (DMSO) in half-strength MS medium

(Murashige and Skoog, 1962) with 0.4 M sucrose (pH 5.7).

Treatments consisted of seeds left in PVS2 either at room

temperature (27 � 2 8C) or pre-cooled in ice (0 8C) for 1, 2, 3,

4, or 5 h prior to immersion in liquid nitrogen (LN).

Controls consisted of seeds placed for germination into Petri

dishes containing half-strength MS medium immediately after

exposure to—control 1: no PVS2, room temperature, no LN;

control 2: 3 h in PVS2, ice temperature, no LN; control 3:

addition of PVS2 followed by immediate immersion in LN for

14 days (30-s exposure period to PVS2 prior to LN); control 4:

no PVS2, room temperature, LN for 14 days. Control 1

contained 1 ml liquid half-strength MS medium with 58.5 mM

sucrose (pH 5.7) replacing PVS2.

2.4. Seed germination

For controls with no exposure to LN, seeds were placed on

germination medium after each control procedure. For controls

and treatments submitted to LN, after 14 days cryovials were

rapidly re-warmed in a 40 8C water bath for 3 min and the

PVS2 solution was removed using a plastic disposable transfer

pipette. About 1 ml of half-strength liquid MS medium with

1.0 M sucrose was added to each vial and held for 1 h at room

temperature, followed by two rinses in 1 ml half-strength liquid

MS medium with 58.5 mM sucrose (pH 5.7). Seeds were placed

in Petri dishes containing half-strength MS medium with

58.5 mM sucrose and solidified with 0.6% agar (Fisher1,

Chicago, IL, USA) under controlled environmental conditions

(27 � 2 8C; 60 mmol m�2 s�1; 18/6 light/dark; 2� 9A Philips1

fluorescent bulbs). Plates were visually monitored on a weekly

basis for germination occurrence. Germination percentage was

determined from 6 to16 weeks for all controls and treatments by

counting the number of germinated seeds under a Leica

MZ12.5 stereoscope at 50� magnification. Seed survival was

measured by germination.

2.5. Seedling growth and transplantation

Germinated seedlings were monitored weekly for shoot and

root development and after 16 weeks they were transplanted

into either Magenta GA7 boxes (Sigma–Aldrich Co., St. Louis,

W.A. Vendrame et al. / Scientia Horticulturae 114 (2007) 188–193190

MO, USA) or Phytotech P700 culture boxes (Phytotechnology

Laboratories, Shawnee Mission, KS, USA) containing the same

medium as described above for germination, where they were

maintained for further growth and development. Light and

temperature conditions were the same as for germination;

27 � 2 8C; 60 mmol m�2 s�1; 18/6 light/dark; 2� 9A Philips1

fluorescent bulbs. Fully developed seedlings were transplanted to

pots (10.16 cm diameter) with 3–4 plants per pot containing coir

dust (Coco Gro-Brick, OFE International, Miami, FL, USA) as

the substrate. Pots were maintained in an environmentally

controlled Percival E30B incubator (Percival Scientific, Inc.,

Perry, IA, USA) at 27� 2 8C; 320 mmol m�2 s�1; 18/6 light/

dark; 6� 9A Philips1 fluorescent bulbs for acclimatization and

further growth and development. Pots were irrigated every other

day with a solution of Peters Orchid Food (Spectrum Group, St.

Louis, MO, USA) consisting of 30% total N, 10% P2O5, 10%

K2O, 0.5% Mg, 0.02% B, 0.05% chelated Cu, 0.1% Fe, 0.05%

Mn, 0.0005% Mo, and 0.05% Zn. Plant survival was determined

by growth and development of normal seedlings into plants,

which were transplanted to greenhouse.

2.6. Experimental design and data analysis

The experimental design consisted of a 2 � 5 factorial for

vitrification treatments plus 4 controls, with 10 replicates of

approximately 1000 seeds per treatment/control. The experiment

was replicated twice. Seed germination data was normalized

using square root and arcsine transformation and subsequently

subjected to analysis of variance (ANOVA). Plant survival was

calculated by the number of germinated seeds developing into

seedlings and subsequently into plants. Means were compared

using Duncan’s Multiple Range Test at a = 0.05.

3. Results

3.1. In vitro germination of cryopreserved seeds

Seed characteristics are summarized in Table 1. The FDA

staining assay showed between 68 and 96% viability. Viability

was assessed by embryos exhibiting bright yellow color under

Table 1

Characteristics of mature seeds of four Dendrobium hybrids

‘Sena Red’ ‘M

Capsule sizea (L (cm) �W (cm)) 3.19 � 1.54 2

Capsule weighta (g) 3.04 0

Seed sizeb (L (mm) �W (mm)) 0.52 � 0.10 0

Weight of 100 seedsc (mg) 0.15 0

Initial seed moisturec (% FW) 18 1

Final seed moisturec (% FW) 19 1

Seed viability (FDA)c (%) 95 7

Seed germination testc (%) 96 7

Number of seedsd 690,066 3

L: length; W: width; FW: fresh weight.a Values are means of two capsules.b Values are means of 50 seeds.c Values are means of three replications.d Values are means of two capsules based on estimates per capsule.

UV light. The seed germination test revealed a range of 65–

96% germination, showing consistency with the FDA viability

test for this experiment. Initial seed moisture content ranged

from 9 to 18%. Final seed moisture determined after seed

disinfection showed a small increase in seed moisture content

up to 1% for all samples, except for Dendrobium ‘Jaquelyn

Thomas’, which exhibited a 2% increase in seed moisture

(Table 1). However, the increase in moisture content apparently

did not affect seed germination after cryopreservation. Seeds

observed under microscope (50�) contained well-formed

embryos and no abnormalities were apparent. Capsule size

and weight, seed size, and estimated number of seeds were

variable among the different hybrids (Table 1) and apparently

did not affect germination of cryopreserved seeds.

Despite the high seed viability for all hybrids, germination

percentages were variable among different controls and treat-

ments. Fig. 1 illustrates germination for Dendrobium ‘Jaquelyn

Thomas’ seeds after cryopreservation. Germinating seeds after

cryopreservation showed enlargement, change togreen color, and

development into protocorms (Fig. 1A). Protocorms developed

leaf primordia and rhizoids (Fig. 1B). All hybrids exhibited the

same germination characteristics after cryopreservation and were

morphologically and developmentally similar to the germination

of seeds that were not cryopreserved.

Table 2 summarizes the germination percentages for controls

and for seeds submitted to pre-cooling treatments prior to LN.

The highest germination percentages for all hybrids were

obtained for seeds maintained at room temperature and germ-

inated directly onto MS medium with no exposure to PVS2, nor

LN (control 1), and for seeds exposed to PVS2 for 3 h at ice

temperature without subsequent exposure to LN (control 2).

Germination percentages in these controls varied from 63.7 to

96.1% (Table 2). In contrast, seeds maintained at room temp-

erature either submitted to LN after brief exposure (30 s) to PVS2

(control 3), or submitted to LN without exposure to PVS2

(control 4), exhibited highly reduced germination percentages

(0.4–26.9%) or completely failed to germinate (Table 2).

Seeds maintained at room temperature, but exposed to PVS2

prior to LN showed no or very low germination percentages (0–

3.8%) as exposure time to PVS2 was extended from 1 to 5 h

ini WRL’ ‘Jaquelyn Thomas’ ‘BFC Pink’

.55 � 1.02 3.41 � 1.54 1.80 � 1.24

.91 7.75 1.14

.38 � 0.08 0.60 � 0.08 0.40 � 0.10

.10 0.20 0.14

6 9 17

7 11 18

4 96 68

2 94 65

53,600 259,100 1,062,000

Fig. 1. Seed germination, seedling development, and plant growth after cryopreservation. (A) Germinating seeds after cryopreservation showing enlarged protocorms

(P) at different stages of development. Bar: 1.0 mm. (B) Detail of enlarged protocorm (P) showing leaf primordial (L) and rhizoids (R). Bar: 0.4 mm. (C) Seedling

development in Phytotech culture boxes. (D) Plant growth in greenhouse.

W.A. Vendrame et al. / Scientia Horticulturae 114 (2007) 188–193 191

(data not shown). Similarly, seeds maintained in ice for 4 or 5 h

showed very low germination percentages as exposure time to

PVS2 increased from 4 to 5 h (4.0 and 1.6%, respectively) prior

to LN (data not shown). For treatments where seeds were

exposed to PVS2 at ice temperature from 1 to 3 h prior to LN

germination was significantly higher (47.2–63.8%) than for

seeds exposed to PVS2 at room temperature for the same time

periods. Although germination percentages for these treatments

were still lower than controls 1 and 2, they represented over

50% germination of these controls.

For Dendrobium ‘Sena Red’, ‘Mini WRL’, and ‘BFC

Pink’, no significant differences in germination percentages

were observed for time exposure to PVS2 at 1, 2 or 3 h prior

to LN.

However, Dendrobium ‘Jaquelyn Thomas’ seeds exposed

to PVS2 and ice for 1 h prior to LN exhibited higher

Table 2

Germination (%) of cryopreserved Dendrobium hybrid seeds submitted to dehydra

Dendrobium hybrids Controlsa

1 2 3

‘Sena Red’ 96.1 a 95.4 a 26.9 c

‘Mini WRL’ 70.6 a 73.1 a 6.1 c

‘Jaquelyn Thomas’ 95.7 a 82.9 b 0 e

‘BFC Pink’ 63.7 a 65.1 a 0.4 c

Values are means of 10 replicates (1000 seeds per replicate). Means followed by the s

Data for PVS2 + ice for periods over 3 h are excluded due to the low-germinationa Control 1: room temperature, no PVS2, no LN; control 2: ice, PVS2 3 h, no L

temperature, no PVS2, LN (room temperature = 27 � 2 8C; ice temperature = 0 8C

germination (60.4%) than for seeds exposed to 2 h (52.4%) or

3 h (51.2%).

3.2. Seedling growth and transplantation

Despite some variability in seed germination among

different hybrids and treatments, all seeds germinated from

LN developed into normal seedlings with healthy shoot and

root formation. Fig. 1C illustrates well developed seedlings

of Dendrobium ‘Jaquelyn Thomas’ from cryopreserved

seeds. No abnormalities, nutritional deficiencies, or diseases

were observed. No significant differences were observed

for seedling growth and development from germinated

seeds among the different hybrids (data not shown). After

8 weeks, some seedlings were transplanted to pots and

acclimatized well, developing into normal plants (Fig. 1D).

tion (PVS2) and pre-cooling (ice) treatments prior to cryopreservation (LN)

PVS2 + ice (LN)

4 1 h 2 h 3 h

0 d 63.6 b 60.2 b 63.8 b

0 d 55.2 b 52.6 b 50.3 b

0 e 60.4 c 52.4 d 51.2 d

0 c 49.8 b 51.5 b 47.2 b

ame letter along rows are not significantly different by Duncan’s test (P = 0.05).

percentages (<0.4%) for all hybrids.

N; control 3: room temperature, brief exposure to PVS2, LN; control 4: room

).

W.A. Vendrame et al. / Scientia Horticulturae 114 (2007) 188–193192

Transplanted seedlings exhibited 100% survival percentages

for all hybrids.

4. Discussion

Plant development and survival was directly related to the

successful germination of cryopreserved seeds. Therefore, the

development of a successful cryopreservation protocol for the

different Dendrobium hybrids was an essential aspect of this

study. Initial seed viability is indicated as an important factor

for long-term seed storage (Seaton and Hailes, 1989). Seed

viability varied among hybrids and it was assessed through the

FDA viability test. In this study, seed viability assessed by FDA

was comparable and similar to germination percentages

exhibited through the seed germination test performed, and

also through direct germination of seeds without LN (control

1). The FDA staining technique was efficient for this study and

it is reported to be accurate for viability assessment of orchid

seeds (Pritchard, 1985). Also essential for successful cryopre-

servation is the determination of moisture content in tissues

(Pence, 1992). Initial seed moisture content for Dendrobium

hybrids in this study varied from 9 to 18%, with a small increase

of 1–2% (11–19%) after rehydration post-disinfection

(Table 1). Wang et al. (1998) reported 95% survival percentage

for cryopreserved D. candidum seeds placed directly in LN,

with initial seed moisture content between 8 and 19%, although

seed growth was slower in samples with less than 12%

moisture. An optimal level of 12–19% moisture was reported

for cryopreservation of D. candidum via vitrification (PVS2). In

this study, no seeds germinated when placed directly in LN,

even though the range of moisture content in the seeds were

similar to those reported by Wang et al. (1998). Our results

indicated that regardless of moisture content, seeds were

damaged by direct immersion in LN (control 4) and could not

survive LN temperatures without cryoprotection (Table 2).

However, by exposing seeds to PVS2 for a period of time,

sufficient dehydration and cryoprotection for vitrification upon

rapid cooling in LN was provided to seeds to allow good

germination percentages after cryopreservation. This approach

was used by Sakai et al. (1990) who successfully cryopreserved

nucellar cells of navel orange through vitrification (Sakai et al.,

1991a,b). Likewise, dehydration remains as one of the most

important steps in cryopreservation and is critical for survival,

allowing the reduction of water content in the cells to avoid

physical damage caused by ice crystals upon freezing (Sakai

et al., 1991a). The exposure to PVS2 prior to cryopreservation

contributed to minimize any possible damage by chemical

toxicity, osmotic stress, or ice crystallization. In fact, seeds

exposed briefly to PVS2 prior to LN (control 3), or submitted to

LN without exposure to PVS2 (control 4) did not germinate

compared to seeds exposed to PVS2 under ice for 1, 2, or 3 h

prior to LN, which provided the best germination percentages.

Furthermore, the exposure of seeds to PVS2 between 1 and 3 h

(pre-cooling) was essential for successful cryopreservation and

subsequent germination of cryopreserved seeds. Pre-cooling

treatments are essential for proper cryopreservation and post-

rewarming survival of cells and/or tissues (Panis and Thinh,

2001). In treatments when pre-cooling was provided by

maintaining seeds in ice for a period between 1 and 3 h while

exposed to PVS2, germination percentages were significantly

higher than for seeds maintained at room temperature.

Genotypic differences may also have contributed for the

differences in germination percentages observed among

different hybrids. The effect of exposure time to PVS2 is

species-specific for the cryopreservation of shoot tips of pear

and apple (Niino et al., 1992) and interspecific variation is

reported for orchid seed longevity (Pritchard et al., 1999),

although in this study genotypic effects were not directly

evaluated. Plant growth and development in pots was

successfully achieved for all hybrids using coir as the substrate.

5. Conclusion

The procedure described for cryopreservation of Dendro-

bium hybrid mature seeds is relatively simple and reliable

whereby cryopreserved seeds germinated and seedlings

developed into normal and healthy plants. Although germina-

tion percentages after cryopreservation were not as high as for

the germination test and some of the controls performed, they

were above 50% of the controls, therefore high enough to

validate this protocol as viable for cryopreservation of different

Dendrobium hybrids here described. The combination of low

temperature with dehydration treatments prior to cryopreserva-

tion showed to be necessary for the success of the vitrification

method in this study. Our results indicate that a pre-cooling

treatment (ice) combined with a dehydration treatment (PVS2)

for a period of time between 1 and 3 h was essential to allow

proper germination of cryopreserved seeds. In the present study,

regardless of treatment, all germinated seeds developed into

normal seedlings and plants. Plant growth and development

was not adversely affected by cryopreservation, but germina-

tion of cryopreserved seeds dictated the success for seedling

survival and growth.

Acknowledgments

The authors thank the Coordenacao de Aperfeicoamento de

Pessoal de Nıvel Superior (CAPES-Brasil) and the University

of Florida for providing funding to support this project. The

authors also thank Mr. Ian Maguire, Ms. Pamela Moon, and

Ms. Helen Fitting, Biological Scientists at the Tropical

Research and Education Center, University of Florida, for

their assistance during the development of this research

project, Mrs. Lisa Smith of Ellenton Growers, Palmetto,

Florida, and Mr. Kerry Herndon of Kerry’s Bromeliads

Nursery, Inc., Homestead, FL, for providing the plant material

for this experiment.

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