Seedling Development and Evaluation of Genetic Stabilityof Cryopreserved Dendrobium Hybrid Mature Seeds

Renato Fernandes Galdiano Jr. & Eliana Gertrudes de Macedo Lemos &Ricardo Tadeu de Faria & Wagner Aparecido Vendrame

Received: 18 September 2013 /Accepted: 25 December 2013# Springer Science+Business Media New York 2014

Abstract Vitrification, a simple, fast, and recommended cryopreservation method for orchidgermplasm conservation, was evaluated for Dendrobium hybrid “Dong Yai”mature seeds. Thegenetic stability of regenerated seedlings was also evaluated using flow cytometry. Matureseeds from this hybrid were submitted to plant vitrification solution (PVS2) for 0, 0.5, 1, 2, 3,4, 5, or 6 h at 0 °C. Subsequently, they were plunged into liquid nitrogen (LN) at −196 °C for1 h and recovered in half-strength Murashige and Skoog culture medium (1/2 MS), and seedgermination was evaluated after 30 days. Seeds directly submitted to LN did not germinateafter cryopreservation. Seeds treated with PVS2 between 1 and 3 h presented the bestgermination (between 51 and 58 %), although longer exposure to PVS2 returned moderatedgermination (39 %). Germinated seeds were further subcultured in P-723 culture medium anddeveloped whole seedlings in vitro after 180 days, with no abnormal characteristics, diseases,or nutritional deficiencies. Seedlings were successfully acclimatized under greenhouse condi-tions with over 80 % survival. Flow cytometry analysis revealed no chromosomal changes onvitrified seedlings, as well as seedlings germinated from the control treatment (direct exposureto LN). These findings indicate that vitrification is a feasible and safe germplasm cryopreser-vation method for commercial Dendrobium orchid hybrid conservation.

Appl Biochem BiotechnolDOI 10.1007/s12010-013-0699-8

E. G. de Macedo LemosDepartment of Technology, Univ Estadual Paulista, Via de Acesso Prof. Dr. Paulo Donato Castellane,s/n, Jaboticabal, SP 14884-900, Brazile-mail: renatofgaldianojr@yahoo.com.br

R. T. de FariaDepto. de Ciências Agrárias, Universidade Estadual de Londrina,Caixa Postal 6001, Londrina, PR 86051-990, Brazil

W. A. VendrameTropical Research and Education Center, University of Florida,18905 SW 280th St, Homestead, FL 33031-3314, USA

Present Address:R. F. Galdiano (*)Department of Technology, Univ Estadual Paulista, Via de Acesso Prof. Dr. Paulo Donato Castellane,s/n, Jaboticabal, SP 14884-900, Brazile-mail: renatofgaldianojr@yahoo.com.br

Keywords Vitrification . Commercial orchid . Germplasm conservation . Flow cytometry

Abbreviations

LN Liquid nitrogenPVS2 Plant vitrification solution1/2 MS Half-strength Murashige and Skoog culture mediumTTC 2,3,5-Triphenyl tetrazolium chlorideFCM Flow cytometryDMSO Dimethyl sulfoxide

Introduction

Orchids are among the most diversified of the flowering plant families, with over 800described genera, 25,000 species, and over 100,000 registered hybrids. They are valued bytheir beautiful long lasting flowers exhibiting an intense diversity in color, shape, size, andfragrance. The large-scale multiplication of species and hybrids using in vitro plantmicropropagation techniques has led orchids to be positioned among the top 10 cut and pottedflowering plants worldwide [1]. The increasing demand for orchid cut flowers acts as a catalystto the various breeding programs of orchids [2].

Currently, orchids are the second most valuable potted flowering plant in the USA, thelargest importer of potted orchids, with a total wholesale of over $200 million in 2011 [3]. Themost commercially important orchid genera are Phalaenopsis, Dendrobium, Cymbidium,Oncidium, Vanda, and Cattleya [4]. The genus Dendrobium is one of the largest genera inthe family Orchidaceae represented by more than 1,000 species distributed from the foothillsof the Himalayas through Southeast Asia to Japan, Australia, Tasmania, and the PacificIslands. Dendrobium hybrids have desirable horticultural characteristics and, consequently,are the most popular cut flower orchid [5].

Advances in biotechnology, especially in the area of in vitro culture techniques andmolecular biology, provide some tools for improved conservation and management of plantgenetic resources [6]. Due to the rate at which the consumer market is demanding change andinnovation, much pressure is placed on breeders to develop methods to rapidly insert genes ofinterest into important orchid cultivars [7]. Therefore, plant genetic resources conservation isvery useful to ensure important genes for plant breeding programs.

Cryopreservation, the long-term storage of genetic resources at liquid nitrogen temperature(−196 °C) or in its vapor phase (−150 °C) is desirable, providing a simple, safe, and cost-effective method with minimal space and maintenance required [7, 8]. Vitrification-basedcryopreservation protocols are easy, reliable, and applicable to a wide range of genotypes. ForDendrobium orchids, cryopreservation by vitrification methods is widely applicable and hasbeen reported for pollen [9], seeds [10, 11], protocorms [12], and protocorm-like bodies [13].

Seed viability plays a major role in the determination of the efficiency of a cryopreservationprotocol for orchid seeds. The use of 2,3,5-triphenyl tetrazolium chloride (TTC), the vital stain,allows the enzymatic dehydration of the colorless TTC to form the red color insoluble triphenylformazan, which allows the differentiation of viable and nonviable tissues [14]. This test iscommonly used to evaluate seed viability and cryopreserved orchid explants [8, 11, 15, 16].

Exposure to excessive harsh physical environment such as very low temperature, highosmotic pressure, dehydration, and exposure to certain chemicals such as cryoprotectantsduring cryopreservation procedures may cause physical stress and consequently result ingenetic instability of explants [17]. Such stressful events might cause the formation of free

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radicals that cause damage, for example, by lipid peroxidation, denaturation of protein, andDNA mutations [18], leading to genetic alterations. Potential somaclonal variation could occurin the cryopreserved explants and, therefore, phenotypic and genotypic analyses of theregenerated plants should be performed [19].

Flow cytometry (FCM) represents a feasible technique for fast and large-scale analysis ofDNA content and genetic stability of plants. FCM is particularly well suited for the determi-nation of ploidy variation in samples with low mitotic indices, such as in vitro plant cellcultures, where chromosome counting cannot provide a representative picture of a heteroge-neous population of cells [20].

The objectives of this study were to evaluate the effectiveness of the vitrification method forthe cryopreservation ofDendrobium Swartz. hybrid “Dong Yai”mature seeds and the evaluationof the in vitro development and genetic stability of regenerated seedlings using flow cytometry.

Materials and Methods

Seed Capsules, Sterilization, and Viability Test

Four-month-old mature seed capsules of artificially self-pollinated Dendrobium Swartz. hybridDong Yai obtained from greenhouse grown plants were used. Capsule and seed surfacesterilization, preparation, and moisture content evaluation were performed according toVendrame et al. [9]. About 200 mg of seeds were submitted to serial dilutions using sterile-distilled water, whereby 1 ml of seed aqueous solution was calibrated to contain approximately1,000 seeds. Seed solutions were maintained in 2 ml cryovials. Seed viability was assessedusing a modified TTC test with sucrose [21]. Seeds were counted using a Leica MZ12.5stereoscope, and only rose-red seeds were counted as viable.

A seed germination test was performed in semi-solid 1/2 MS medium containing0.088 M sucrose (pH 5.7) and maintained under controlled environmental conditions(27±2 °C; 60 μmol m−2 s−1; 18/6 light/dark; 4× 9A Philips® fluorescent bulbs).

Vitrification Treatments

For each treatment, sterile-deionized water was removed from each cryovial containing seedsand 1 ml cryoprotective loading solution was added consisting of 2.0 M glycerol and 0.4 Msucrose [22]. Cryovials were left for 30 min at room temperature (27±2 °C) prior to theaddition of a plant vitrification solution (PVS2). The PVS2 [23] contains 30 % (w/v) glycerol,15 % (w/v) ethylene glycol, and 15 % (w/v) dimethyl sulfoxide (DMSO) in 1/2 MS mediumcontaining 0.4 M sucrose (pH 5.7).

The effect of different exposure times (0, 0.5, 1, 2, 3, 4, 5, and 6 h) in PVS2 at 0 °C wasassessed for seed germination. Treatment control consisted of direct exposure of seeds to liquidnitrogen (LN) (time 0 min) with no cryoprotective solution.

After 60 min in LN, cryovials were removed and rapidly rewarmed in a 40 °C water bathfor 90 s. Cryopreservation solutions were removed from cryovials with a sterile disposabletransfer pipette under a laminar flow hood. Seeds were rinsed with liquid 1/2 MS culturemedium + 1.2 M sucrose (pH 5.7) for 15 min, transferred to Petri dishes containing semi-solid1/2 MS medium + 0.088 M sucrose (pH 5.7), and cultured under the same controlledenvironmental conditions as described above.

Treatments were comprised of six replicates with 1,000 seeds each. Petri dishes werevisually monitored weekly for germination occurrence. Germination percentage was assessed

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after 30 days for the control and the different treatments by counting the number of germinatedseeds under a Leica MZ12.5 stereoscope. Seed survival was assessed by counting the numberof germinated seeds that formed protocorms.

Seedling In Vitro Growth and Subsequent Acclimatization in Greenhouse

Seedlings developed from cryopreserved seeds were transferred to 180 ml flasks containing40 ml of semi-solid P-723® culture medium (Phytotechnology Labs, Shawnee Mission, KS,USA) for further growth and development. Flasks were maintained under the same conditionsas described above, and seedlings were subcultured to fresh medium every 60 days. At180 days of culture, fully developed seedlings were transplanted to individual 7 cm plasticpots (140 ml per pot) containing coconut coir (Coco Gro-Brick, OFE International, Miami, FL,USA) as the growing mix.

Seedlings were watered daily and sprayed weekly with a solution of Peter’s orchid food(Spectrum Group, St. Louis, MO, USA) consisting of 30 % total N, 10%P2O5, 10 % K20, 0.5 %Mg, 0.02%B, 0.05 chelated Cu, 0.1%Fe, 0.05%Mn, 0.05 Zn, and 0.0005%Mo. Plant survivalwas determined by growth and development of seedlings into normal plants, which weresuccessfully transplanted and evaluated 90 days after transferred to greenhouse conditions.

Flow Cytometry Analysis

The relative DNA content was evaluated for plants regenerated from both untreated andcryopreserved seeds using flow cytometry analysis. Nuclear suspensions from young leaveswere prepared, and nuclei were released from the cells by chopping samples of this materialwith a razor blade in Marie’s isolation buffer [24]. The suspension of nuclei was first filteredthrough a 50-μm nylon filter to remove cell fragments and large tissue debris, and then50 μg ml−1 of propidium iodide (PI) (Sigma, St. Louis, Mo, USA) and 20 μg ml−1 ofRNAse (Sigma) were added to the samples to stain the DNA. Samples were analyzed withina 15-min period in a flow cytometer.

The fluorescence of samples was measured using a BD Accuri C6 flow cytometer(BD Biosciences, California, USA). Histograms were generated after analyses of at least4,000 nuclei using the software FSC 3.0 Flow Cytometry Express.

Experimental Design and Data Analysis

All experiments were arranged in a completely randomized design. The data were subjected toanalysis of variance using the SAS software (Release 9.2; SAS Institute, NC, USA), andmeans were separated by Tukey’s test (P<0.05). Seed germination data were normalized usingthe square root and arcsine transformation. The experiment was entirely repeated three times.For FCM analysis, 20 seedling samples each from control (not cryopreserved), PVS2 for 1 hand PVS2 for 6 h cryopreserved mature seeds were evaluated.

Results and Discussion

Seed characteristics and germination are summarized in Table 1. Before cryopreservation, theTTC staining assay (Fig. 1a) revealed 83±2.37 % (mean ± standard deviation) seed viabilityand correlated positively with the germination test, which revealed 85.7±3.86 %. This positivecorrelation confirmed the accuracy and safety of the staining test used in this study.

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At 15 days after removal from cryopreservation and culture in 1/2 MS semi-solid culturemedium, embryos showed enlargement and were similar to those from not cryopreservedmature seeds (control) (Fig. 1b, c). Afterwards, they developed into a greenish protocormstructure (Fig. 1d) with the presence of leaflets in the upper portion and rhizoids in the lowerportion. At this time, protocorms were counted and germination percentage estimated.

The best germination percentage was observed in seeds treated with PVS2 for 1 to 3 h(between 51 and 58 %), although longer vitrification solution exposure time (6 h) also returnedmoderate germination (39 %, Fig. 2). Seeds submerged in to LN but without treatment inPVS2 did not germinate. In contrast, Wang et al. [25] successfully cryopreserved Dendrobiumcandidum seeds submitted directly to the LN without prior treatment, with seed water contentbetween 12 and 19 %, values comparable to those observed in this study with Dendrobiumhybrid Dong Yai. Similarly, other authors [15, 16] reported seed germination of orchid speciesafter direct submersion to LN without vitrification treatment. The longevity of orchid seedsmay be influenced by interspecific variations [26]. Thus, genotypic differences may havecontributed to the discrepancy between the results of this study compared with similar studies.

Table 1 Characteristics of 4-month-old capsules and matureseeds of Dendrobium Swartz. hy-brid Dong Yai artificially self-pollinated

aValues are means of three capsulesbValues are means of 50 seedscValues are means of three replica-tions of 200 mgdValues are means of sixreplications

Characteristics Mean ± SD

Capsule length (cm)a 5.53±0.25

Capsule width (cm)a 1.47±0.12

Seed length (mm)b 1.19±0.28

Seed width (mm)b 0.09±0.025

Seed moisture (%)c 12±0.65

Seed viability (TTC) (%)c

Seed germination test (%)d83±2.3785.7±3.86

A B C

D

R

L

P

PP

Fig. 1 Mature seeds and protocorms of Dendrobium Swartz. hybrid Dong Yai. Stained seeds after 2,3,5-trifenyltetrazolium chloride test (a); germinating embryos from non-cryopreserved (b) and cryopreserved mature seeds(c); developing seedlings (d) after seed cryopreservation showing 30-day-old protocorms (P), rhizoids (R), andleaflets (L). Bars, 1 mm

Appl Biochem Biotechnol

The results presented here confirm that the dehydration caused by exposure of tissues toPVS2 is an essential step in cryopreservation. The dehydration allows the reduction of watercontent in the cells to avoid physical damages caused by ice crystals upon freezing [27]. In thisstudy, the PVS2 treatment acted as a critical step for Dendrobium hybrid seeds survival andrecovery.

The key to successful cryopreservation by vitrification is the careful control of theprocedures for dehydration and cryoprotectant permeability to prevent injury by chemicaltoxicity or excess osmotic stress during dehydration [28]. Therefore, optimizing the exposuretime to PVS2 is the most important parameter for ensuring successful plant regeneration fromcryopreserved seeds following vitrification. The exposure duration of plant cells to thevitrification solution is critical for their survival because over dehydration may result in cellinjury by chemical toxicity and excess osmotic stress from cryoprotectants. The suitabledehydration duration can be related to several factors, including the nature and the size ofthe sample, and the composition and loading procedure of cryoprotectants [29].

The incubation time and temperature of the PVS2 solution affect the germination rate ofcryopreserved seeds because overexposure to PVS2 can cause chemical toxicity. By deter-mining the best dehydration time of explants at 0 °C, the toxicity of the vitrification solution

0

20

40

60

80

100

0 1 2 3 4 5 6

Ger

min

atio

n (

%)

PVS2 exposure time (h)

a aa

d

b b bc

Fig. 2 Germination percentage of Dendrobium Swartz hybrid Dong Yai mature seeds after cryopreservation byvitrification under different exposure times to PVS2. Seeds were cryopreserved in LN for 1 h. Values withdifferent letters are significantly different according to Tukey’s test after square root and arcsine transformation(P<0.05). Approximately, 1,000 seeds were used in each of the six replications. Bars indicate standard deviation

Control PVS2 0.5h

PVS2 1h

PVS2 2h

PVS2 3h

PVS2 4h

PVS2 5h

PVS2 6h

Fig. 3 Regenerated seedlings from control (not cryopreserved) and cryopreserved mature seeds of DendrobiumSwartz. hybrid Dong Yai after 180 days of in vitro culture

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can be reduced and, consequently, the exposure time to PVS2 solution can be increased for asuccessful cryopreservation protocol [2, 13]. Vendrame et al. [9] showed that pre-coolingtreatment (ice) combined with PVS2 treatment for a period of time between 1 and 3 h wasessential to allow proper germination of cryopreserved seeds of four commercial Dendrobiumhybrids genotypes. Likewise, in this study, the dehydration period was similar and 1 h in PVS2can be recommended. A moderate germination rate for the 6-h treatment in PVS2 wasobserved, leading to the hypothesis that this Dendrobium hybrid could have an intrinsic traitof high dehydration tolerance. However, this was not evaluated in this study.

In vitro seedlings developed shoots and roots in P-723 culture medium without the use ofplant growth regulators. The morphological characteristics of regenerated seedlings betweencontrol (from seeds not cryopreserved) and treatments (from cryopreserved seeds) presentedno variation (Fig. 3). Seedlings were successfully acclimatized in greenhouse and a survivalrate of over 80 % after 90 days was recorded. Plants from control and cryopreserved seedspresented the same growth pattern with 3–4 shoots each (Fig. 4), and no abnormalities, diseasesusceptibility, or nutritional deficiencies were observed.

Control PVS2 1h PVS2 6h

Fig. 4 Regenerated seedlings from control (not cryopreserved) and cryopreserved mature seeds of DendrobiumSwartz. hybrid Dong Yai after 90 days of acclimatization in greenhouse

a b

Relative DNA content Relative DNA content

Fig. 5 Histograms of relative fluorescent intensity of propidium iodide (PI) using nuclei from leaves ofDendrobium Swartz. hybrid Dong Yai seedlings from control seeds (a) and cryopreserved seeds with PVS2for 6 h (b)

Appl Biochem Biotechnol

Nuclei isolated from leaves of seedlings regenerated from control and treated seeds showedsimilar pattern of ploidy levels by FCM analysis (Fig. 5). This confirms that in this study theuse of PVS2 under different exposure times was feasible to maintain the genetic integrity andstability of the regenerated seedlings with no differences observed.

The FCM analysis is a simple and practical method for rapid evaluation of ploidy levels byfast scanning the whole genome using very small amounts of plant material. The resultsobtained in this study are similar to those obtained in previous studies using orchid seedlingsregenerated from cryopreservation [16, 30]. In this study, FCM analysis revealed that evenseedlings regenerated from seeds submitted to a longer PVS2 exposure time (6 h) revealed atrue-to-type characteristic, and therefore, confirmed the genetic stability of plants.

Conclusion

In the present study, mature seeds of a commercial Dendrobium orchid hybrid were success-fully cryopreserved by vitrification, whereby post-recovery germination and seedling accli-matization percentages in greenhouse were high and genetic integrity was maintained.Dehydration in PVS2 at 0 °C was an essential step for seed survival and recovery. The highestseed germination was observed after vitrification for 1 to 3 h. However, even after vitrificationfor 6 h, germinability was observed, showing a potential intrinsic trait of high dehydrationtolerance for this genotype. The FCM evidence of genetic stability provided the confirmationthat this is a feasible and safe protocol for the cryopreservation of mature seeds ofDendrobiumhybrid Dong Yai and can be recommended for other commercial orchids aiming at germplasmstorage.

Acknowledgments The authors thank the National Council of Research and Technology Development(Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq), Ministry of Science and Technology,Brazil, and the University of Florida for providing support for this study. Thanks also to Mrs. Alba Myers for thetechnical assistance and Dr. Allan Meerow, ARS-USDA and Mr. Deal Neal, UF/ICBR for the assistance withflow cytometry analysis.

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