Cryopreservation the seeds of a Taiwanese terrestrial orchid, Bletilla formosana (Hayata) Schltr. by vitrification
© Hu et al.; licensee Springer. 2013
Received: 11 October 2012
Accepted: 6 September 2013
Published: 12 September 2013
The cryopreservation of orchid seeds is an important conservation method, studies of the effects of cryopreservation on the seeds of wild orchids are scant. This investigation was to establish a method for the vitrification and cryopreservation of seeds of B. formosana that may be suitable for the long term storage of Taiwan native orchid germplasm for conservation purposes.
The germination rate and morphological stability of seeds from spontaneous-dehiscent capsules of Bletilla formosana (Hayata) Schltr. were evaluated after cryopreservation by vitrification. The germination rates of cryopreserved seeds varied according to immersion time and the vitrification method used. Seeds that were dehydrated by immersion in loading solution (LS; 2.0 M glycerol, 0.4 M sucrose) for 10 min to 30 min then transferred to plant vitrification solution 2 (PVS2) for 30 min prior to freezing in liquid nitrogen (LN) showed significantly higher germination rates than seeds immersed in PVS2 only. The optimal immersion times were 10 min for LS and 30 min for PVS2, resulting in an in vitro germination rate of 91%. Germination was not observed for cryopreserved seeds that were dehydrated by immersion in LS only. Seed viabilities and germination rates did not vary significantly for cryostorage times from 10 minutes to 1 year.
This study improve, an efficient protocol was established that maintained seed viability and enhanced the germination rates of seeds, compared with previously described cryopreservation methods, and the germinated seeds showed normal morphology of both vegetative and reproductive organs.
KeywordsGermination Loading PVS2 Viability Vitrification
There are many techniques available for the conservation of plant genetic resources of endangered species. These include in-situ and ex-situ conservation practices, micropropagation, seed germination, regeneration from explants, and cryopreservation (Nitzsche, 1983; Rick, 1984; Stanilova et al., 1994; Chang et al., 2000). The cryopreservation of wild orchid seeds is an important conservation method. Seeds are heterogeneous in most wild orchids, and preservation of seeds can help conserve the genetic diversity of plant populations. Compared with traditional methods of storage, cryopreservation is cost-effective, requires little space, and is efficacious for the long-term storage of plant genetic resources (Engelmann, 2011). Therefore, seed cryopreservation is an effective and powerful tool for ex-situ conservation (Thammasiri and Soamkul, 2007; Hirano et al., 2009).
Though many reports of the cryopreservation of orchids exist in the literature, studies of the effects of cryopreservation on the seeds of wild orchids are scant (Pritchard, 1984; Popova et al., 2003; Hirano et al., 2005a, 2005b, 2009). Vitrification is a simple, fast, and effective method for cryopreservation. It eliminates the need for controlled slow freezing, and permits cells and meristems to be cryopreserved by direct transfer into liquid nitrogen (LN) (Thammasiri and Soamkul, 2007; Tsai et al., 2009).
Bletilla formosana (Hayata) Schltr. is a species of terrestrial orchid that is native to Taiwan and southern China. It is found on seashore cliffs, lowland river banks, and mountain slopes (Su, 2000). Previous studies have shown that germination rates of B. formosana decreased to 47-57% after storage at 3°C of 6 months (Chang et al., 2006). The dry pseudobulbs of the closely-related B. striata are widely used in Chinese traditional medicine (Lin et al., 1994; Chen et al., 2009). The long term storage methods developed for the seeds of Bletilla may also be useful for the conservation of other orchid species of the same genus (Ishikawa et al., 1997).
The aim of our investigation was to establish a method for the vitrification and cryopreservation of seeds of B. formosana that would result in increased viability, compared with conventional cryostorage methods. An analysis of potential detrimental effects on the future development of the seedlings from cryopreserved B. formosana seeds was also performed. Such a method of seed cryopreservation may be suitable for the long term storage of Taiwan native orchid germplasm for conservation purposes.
Mature seeds from dehiscent capsules of B. formosana were collected from Puli Branch Station of the Taichung District Agricultural Research and Extension Station in Nantou, Taiwan. The seeds were stored in screw cap tubes (SCT-50MC-R-S) with silica gel at 4°C.
Osmoprotection and cryopreservation of seeds
For loading treatments, approximately 2000–3000 seeds were transferred to 2.0 ml plastic cryotubes with 1.0 ml loading solution (LS, Matsumoto et al., 1994) containing 2 M glycerol and 0.4 M sucrose in 1/2 Murashiage and Skoog basal medium (MS, Murashige and Skoog, 1962) at room temperature (27 ± 2°C) for different periods of 0, 10, 20, or 30 min. In order to separate the floating seeds from the LS in the cryotube, make sure that the tip of the pipet touches the bottom of the cryotube before draining the LS out. After the LS was removed, the seeds were dehydrated on ice for 30 min with 1.0 ml plant vitrification solution 2 (PVS2, Sakai et al., 1990) containing 30% glycerol, 15% ethylene glycol, 15% dimethyl sulfoxide in 1/2 MS basal medium supplemented with 0.4 M sucrose. The pH of all media was adjusted to 5.8 with 0.1 M NaOH before autoclaving at 121°C for 15 min, 117.7 kPa. The PVS2 was replaced with 0.5 ml fresh solution after upon treatment, and the seeds in the cryotubes were directly plunged into LN for 30 min. The cryotubes were rapidly re-warmed in a 40°C water bath for 1 min. About 0.5 ml of 1/2 MS medium supplemented with 1.2 M sucrose was added to each cryotube, and the tubes were incubated for 10 min at room temperature. The liquid was decanted from the cryotubes, and 1 ml of fresh 1/2 MS medium supplemented with 1.2 M sucrose was added. The tubes were then incubated for 30 min at room temperature.
For vitrification treatments, seeds in cryotubes were immersed in LS for 30 min, and then immersed in PVS2 for 0, 10, 20, 30, or 120 min. Other conditions were identical to the loading treatments.
For cryopreservation duration, all procedures and conditions were same as in above- mentioned loading treatment except that seeds in cryotubes were immersed to LS for 30 min combined with PVS2 30 min, and then plunged into LN for 0, 10, 20, 30 min, 1 week, or 1 year.
After completing all the cryopreservation treatments, seeds from each treatment were surface-sterilized in a lamina flow cabinet with 1% sodium hypochlorite and one drop of Tween-20 for 10 min, prior to rinsing three times with sterile distilled water. The aseptic seeds were then sown in 20 × 150 mm test tubes (Pyrex, No. 9820) with 8 ml of 1/2 MS salts modified medium (SD medium) supplemented with 0.5 mg/l niacin, 0.5 mg/l pyridoxine HCl, 0.1 mg/l thiamine HCl, 100 mg/l myo-inositol, 15% coconut milk, 0.6% potato powder (PhyotoTechnology Laboratories, Shawnee Mission, KS), 0.1% peptone, 2% sucrose and 0.8% agar (Medray Biotech, Taiwan) (pH 5.8). Seeds were maintained at 25 ± 1°C for 12 h photoperiods with the light intensity of 44.5 μmol m-2s-1 (daylight fluorescent tubes FL-30D/29, 40 w, China Electric Co, Taipei, Taiwan). After 4 weeks, the number of germinations was counted by observation under a stereomicroscope. Seed germination was defined as embryo swelling the coat and turning green. Germination rate was calculated as the ratio of germinations to the total number of seeds sown. 200–300 seeds were recorded per replicates. Values are means of 5 replicates.
TTC staining for viability assessment
The viability of the seeds following each treatment was evaluated using the 2,3,5-triphenyltetrazolium chloride (TTC) test. Seeds were incubated in 1% TTC solution for 1 day at 27 ± 2°C in the dark. The number of embryos stained by TTC was counted, and the percentage of TTC-stained seeds represented the survival rate.
The survival rate and germination rate of the seeds were analyzed by using ANOVA. Means were compared using Duncan’s Multiple Range Test at α = 0.05.
In vitro seedling culture and transplanting
Eight-week-old protocorms were transferred to flasks containing 100 ml of SD medium. After three rounds of sub-culture in SD medium at 8-week intervals, plantlets 4–6 cm tall were transplanted in 5 cm diameter plastic pots with 1:1 (v/v) peat moss and fern chips, and grown in a pan and fan greenhouse.
Effects of PVS2 treatments on the TTC staining and the germination rate of B. formosana cryopreserved seeds
PVS2 treatment period1
TTC staining (%)2
Germination rate (%)3
The germination rates of seeds of B. formosana using different periods of cryostorage
TTC staining (%)2
Germination rate (%)3
The beneficial effects of loading solution pretreatment in cryopreservation methods have been reported for numerous plant species (Takagi, 2000). Loading minimizes injurious membrane changes resulting from severe dehydration with PVS2, and is applied in many of plant vitrification procedures (Ishikawa et al., 1997; Kim et al., 2009). LS with high concentrations of sucrose were reported to increase the survival of cryopreserved orchid seeds (Ishikawa et al., 1997; Hirano et al., 2005a 2005b 2009). Our results show that, when PVS2 combined with LS treatments, germination rates were enhanced (Table 1). It is also important to decrease the deleterious effects of PVS2 treatment and enhance the acute dehydration tolerance of seeds. Different periods of loading treatment did not have a significant influence on germination rates (Table 1). However, the seeds lost viability after treated with LS 30 min and without PVS2 dehydration treatment. We conjectured that loading solution raise seeds moisture content and leading to seeds didn’t survive after cryostorage. An appropriate LS is crucial for plant species and explants that are sensitive to PVS2. Our data show a loading time of 10 minutes was optimal. The small diameter of the orchid seeds may contribute to this relatively short treatment period.
Vitrification is a simple, low cost, and practical method for long term preservation of orchid seeds (Thammasiri, 2000). Hirano et al. (2009) reported that Phaius tankervilleae survival rates decreased as a result of PVS2 treatment, suggesting that the seeds were damaged by the cryoprotectant. However, seed moisture content can affect viability and germination following cryopreservation because it must be low enough to minimize ice-crystal formation and high enough to avoid desiccation damage (Ozden-Tokatli et al., 2007). In addition, concentrated PVS2 is toxic to seed embryos. Therefore, it is essential to optimize the PVS2-dehydration procedure to prevent damage to the seed embryo caused by chemical toxicity and sudden osmotic stress (Panis et al., 2001). For the Thai orchid Doritis pulcherrima, it was shown that seeds exposed to PVS2 solution for 50 min had the highest germination rate, with longer or shorter exposure times resulting in a decrease in survival (Thammasiri, 2000). In a similar study of B. striata, embryos precultured in 0.3 M sucrose for 3 days, produced the highest survival when dehydrated in PVS2 for 3 h at 0°C prior to storage in LN (Hirano et al., 2005a 2005b). In our study, seeds that were treated with PVS2 for 10 min prior to storage in LN, showed a significantly higher germination rate (78%) than that observed for PVS2 treatments of 20 min to 120 min (Table 1). Our results also showed that seeds did not survive storage in LN without cryoprotection. According to previous studies, an optimal PVS2 exposure times of 30 minutes to several hours have been reported for the cryopreservation of orchid seeds using vitrification procedures (Ishikawa, 1997; Thammasiri, 2000; Hirano et al. 2005a 2005b; Thammasiri and Soamkul, 2007; Vendrame et al., 2007). Our data show that a shorter PVS2-exposure time (10 min) resulted higher viability in B. formosana that were cryopreserved using vitrification. The shorter PVS2-exposure time likely contributed to viability by reducing potential damage due to chemical toxicity or/and osmotic stress.
The TTC test has been previously used for fast and early evaluation of orchid seed viability (Van Waes and Debergh 1986a 1986b; Lauzer et al., 1994). The test result is determined by the appearance of a red color after soaking the seeds in the TTC solution. In our study, seed viabilities assessed by TTC staining were higher than the observed germination rates (Tables 1 and 2). Thus, viability testing based on TTC staining was not an accurate predictor of germination for B. formosana after cryopreservation. Rasmussen (1995) reported that viability testing using TTC staining does not necessarily correlate well with the germination rates of orchid seeds. In our study, we propose that the split in the seed coat may have lead to an overestimation of seed viability because the split may have allowed TTC to penetrate the seed coat more easily.
Regardless of the cryopreservation technique used, storage at ultralow temperature under optimal conditions only slighted affected seed germination rate of many species (Pritchard, 1984; Wang et al., 1998; Wood et al., 2000; Popova et al., 2003; Ozden-Tokatli et al., 2007; Hirano et al., 2009; Voronkova and Kholina, 2010). In the literature, cryostorage has been shown to increase germination rates of orchid seeds, compared with other storage methods (Nikishina et al. 2001a, 2001b, 2007; Popov et al., 2004). The germination rate of the seeds from LN treatment in our study was significantly higher (86.5-90.1%) than non-LN treatments (79%, Table 2). As both Nikishina et al. (2001a) and Popova et al. (2003) suggested, the effect of ultralow temperature followed by thawing can cause damage to the seed coat. The culture medium may reach the embryo more easily through the damage seed coat, thereby enhancing germination rates.
Seed banks have traditionally been used for germplasm conservation. However, seed banks often used storage conditions that may cause some seeds to lose viability, as was reported in a previous study of B. formosana in which the germination rate decreased after 6 months storage at 3°C (Chang et al., 2006). Thus, cryopreservation provides an effective long-term storage method for the conservation of plant genetic resources because it pauses essentially all biological processes (Gonzalez-Benito et al., 1998b; Benson, 1999). Previous studies of other species have also shown that cryopreservation periods did not affect germination rates (Gonzalez-Benito et al., 1998a, 1998b; Pence, 2003). Our results also indicated cryostorage times from 10 min to 1 year did not result in significant variation in germination rates (Table 2), and our method of vitrification and cryopreservation was also effective for maintaining both seed viability and germination rates for all storage times longer than 10 min. However, Walter et al. (2004) refuted the commonly held idea that all biological activity ceases at ultralow temperatures, and proposed that such activity is not only factor that contributes to seed deterioration. Therefore, measurements of seed viability after long term (several decades) cryostorage remains necessary to ensure germplasm survival.
Cryopreservation imposes a series of stresses to plant materials. It is thus necessary to verify that the genetic stability of the cryopreserved material is not altered before routinely using such techniques for long term conservation of plant genetic resources (Engelmann, 2011). Recent studies have been performed that compared the seedling morphology of different orchid species cultured from non-cryopreserved and cryopreserved seeds. Among these species, D. pulcherrima (Thammasiri, 2000), B. striata (Ishikawa, 1997), Vanda coerulea (Thammasiri et al., 2007), Dendrobium hybrid (Vendrame et al., 2007), and P. tankervilleae (Hirano et al., 2009) did not display significantly different vegetative characteristics. Hirano et al. (2005a, 2005b) showed that B. striata plantlets developed from cryopreserved seeds produced normal flowers. In our study, the morphology of both vegetative and reproductive organs of B. formosana obtained from cryopreserved seeds was normal with no significant variation observed (Figure 2). Plants derived from cryopreserved seeds were fertile, and the capsules were harvested after manual pollination. New generation plantlets exhibited similar morphology and levels of viability to seeds of plants grown from non-cryopreserved seeds (data not shown). Our experiments have demonstrated that no morphological variation was observed as a result of our method of cryopreservation by vitrification.
In conclusion, we have shown that B. formosana seeds can be preserved for long periods by using cryopreservation and vitrification, and that the vitrification method used in this study is an efficient means by which to preserve orchid seeds that are difficult to preserve under dry and low-temperature conditions. When seeds are treated with LS and PVS2 the germination percentage of B. formosana seeds during cryopreservation period was enhanced.
Plant vitrification solution 2
The authors would like to thank the Puli Branch Station of the Taichung District Agricultural Research and Extension Station, Taiwan for providing the plant material for this experiment.
- Benson EE: Cryopreservation. In Plant Conservation Biotechnology. Edited by: Benson EE. Taylor & Francis Ltd., London; 1999:83–95.
- Chang C, Chen CT, Tsai TC, Chang WC: A tissue culture protocol for propagation a rare plant species Lilium speciosum Thunb var. gloriosoides Baker. Bot Bull Acad Sin 2000, 41:139–142.
- Chang C, Sung PG, Chang CH, Chen YC, Lin YH: Seed development and storage of Bletilla formosana (Hayata) Schltr. Seed & Nursery Taiwan 2006, 8:29–38.
- Chen YC, Lee TH, Hung HC, Chang C, Chang LZ, Wei FM: The development, cultivation and chemical constituents in pseudobulbs of Bletilla formosana (Hayata) Schltr. Bull Taichung District Agr Res Ext Sta 2009, 103:31–39.
- Engelmann F: Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev Biol Plant 2011, 47:5–16.View Article
- Gonzalez-Benito ME, Fernandez-Llorente F, Perez-Garcia F: Interaction between cryopreservation, rewarming rate and seed humidifcation on the germination of two Spanish endemic species. Ann Bot 1998, 82:683–686.View Article
- Gonzalez-Benito ME, Iriondo JM, Perez-Garica F: Seed cryopreservation: an alternative method for the conservation of Spanish endemics. Seed Sci Technol 1998, 26:257–262.
- Hirano T, Godo T, Miyoshi K, Ishikawa K, Ishikawa M, Mii M: Cryopreservation and low-temperature storage of seeds of Phaius tankervilleae . Plant Biotechnol Rep 2009, 3:103–109.View Article
- Hirano T, Godo T, Mii M, Ishikawa K: Cryopreservation of immature seeds of Bletilla striata by vitrification. Plant Cell Rep 2005, 23:534–539.PubMedView Article
- Hirano T, Ishikawa K, Mii M: Cryopreservation of immature seeds of Ponerorchis graminifolia var. suzukiana by vitrification. Cryo-Letters 2005, 26:139–146.PubMed
- Ishikawa K, Harata K, Mii M, Sakai A, Yoshimatsu K, Shimonura K: Cryopreservation of zygotic embryos of a Japanese terrestrial orchid ( Bletilla striata ) by vitrification. Plant Cell Rep 1997, 16:754–757.View Article
- Kim HH, Lee YG, Park SU, Lee SC, Baek HJ, Cho EG, Engelmann F: Development of alternative loading solutions in droplet-vitrification procedures. Cryo-Letters 2009, 30:291–299.PubMed
- Lauzer D, St-arnaud M, Barabe D: Tetrazolium staining and in vitro germination of mature seeds of Cypripedium acaule (Orchidaceae). Lindleyana 1994, 9:197–204.
- Lin YJ, Chen CC, Yeh FT, Chiu NY, Tsay HS: Tissue culture of Bletilla formosana I. The influence of seed maturity and pretreatment on seed germination and seedling development. J. Agr. Res. China 1994, 43:40–50.
- Matsumoto T, Sakai A, Yamada K: Cryopreservation of in vitro -grown apical meristems of wasabi ( Wasabia japonica ) by vitrification and subsequent high plant regeneration. Plant Cell Rep 1994, 13:442–446.View Article
- Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1962, 15:473–497.View Article
- Nikishina TV, Popov AS, Kolomeitseva GL, Golovkin BN: Effect of cryopreservation on seed germination of rate tropical orchids. Russ J Plant Physiol 2001, 48:810–815.View Article
- Nikishina TV, Popov AS, Kolomeitseva GL, Golovkin BN: Cryopreservation of seed of some tropical orchids. Dokl Biochem Biophys 2001, 378:555–557.View Article
- Nikishina TV, Popova EV, Vakhrameeva MG, Varlygina TI, Kolomeitseva GL, Burov AV, Popovich EA, Shirokov AI, Shumilov VY, Popov AS: Cryopreservation of seeds and protocorms of rare temperate orchids. Russ J Plant Physiol 2007, 54:121–127.View Article
- Nitzsche W: Germplasm preservation. In Handbook of Plant Cell Culture, vol 1. Edited by: Evans DA, Sharp WR, Ammirato PV, Yamada Y. Collier Macmillan Publishers, London; 1983:782–805.
- Ozden-Tokatli Y, Ozudogru EA, Gumusel F, Lambardi M: Cryopreservation of Pistacia spp. seeds by dehydration and one-step freezing. Cryo-Letters 2007, 28:83–94.PubMed
- Panis B, Swennen R, Engelmann F: Cryopreservation of plant germplasm. Acta Hort. 2001, 560:79–86.
- Pence V: In vitro growth of embryo axes after long-term storage in liquid nitrogen. In Seed conservation: turning science into practice. Edited by: Smith RD, Dickie JB, Linington SH, Pritchard HW, Probert RJ. Royal Botanical Garden Kew, London; 2003:483–492.
- Popov AS, Popova EV, Nikishina TV, Kolomeitseva GL: The development of juvenile plants of the hybrid orchid Bratonia after seed cryopreservation. Cryo-Letters 2004, 25:205–221.PubMed
- Popova EV, Nikishina TV, Kolometseva GL, Popov AS: The effect of seed cryopreservation on the development of protocorms by the hybrid orchid Bratonia . Russ J Plant Physiol 2003, 50:672–677.View Article
- Pritchard HW: LN preservation of terrestrial & epiphytic orchid seed. Cryo-Letters 1984, 5:295–300.
- Rasmussen HN: Cultivation of immature seed. In Terrestrial orchids from seed to mycotrophyic plant. Edited by: Rasmussen HN. Cambridge University Press, Cambridge, UK; 1995:24–27.View Article
- Rick CM: Plant germplasm resources. In Handbook of Plant Cell Culture, vol 2. Edited by: Ammirato PA, Evans DA, Sharp WR, Yamada Y. Collier Macmillan Publishers, London; 1984:9–37.
- Sakai A, Kobayashi S, Oiyama I: Cryopreservation of nucellar cells of navel orange ( Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep 1990, 9:30–33.View Article
- Stanilova MI, Ilcheva VP, Zagorska NA: Morphogenetic potential and in vitro micropropagation of endangered plant species Leucojum aestivum L. and Lilium rhodopaeum Delip. Plant Cell Rep 1994, 13:451–453.View Article
- Su HJ: Bletilla formosana (Hayata) Schltr. In Flora of Taiwan, vol 5. 2nd edition. Edited by: Huang TC. Department of Botany, National Taiwan University, Taipei, Editorial committee of the flora of Taiwan; 2000:758–760.
- Takagi H: Recent developments in cryopreservation of hoot apices of tropical species. In Cryopreservation of Tropical Germplasm. Edited by: Engelman F, Takagi H. Current Research Progress and Application. JIRCAS, Rome; 2000:178–193.
- Tsai SF, Yeh SD, Chan CF, Liaw SI: High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines. Plant Cell Tissue Org Cult 2009, 98:157–164.View Article
- Thammasiri K: Cryopreservation of seeds of a Thai orchid ( Doritis pulcherrima Lindl.) by vitrification. Cryo-Letters 2000, 21:237–244.PubMed
- Thammasiri K, Soamkul L: Cryopreservation of Vanda coerulea Griff. ex Lindl. seeds by vitrification. ScienceAsia 2007, 33:223–227.View Article
- van Waes JM, Debergh PC: Adaptation of the tetrazolium method for testing the seed viability, and scanning electron microscopy study of some Western European orchids. Physiol Plant 1986, 66:435–422.View Article
- van Waes JM, Debergh PC: In vitro germination of some Western European orchids. Physiol Plant 1986, 67:253–261.View Article
- Vendrame WA, Carvalho VS, Maguire I, Dias JMM: In vitro germination and seedling development of cryopreservation Dendrobium hybrid mature seeds. Sci Hort 2007, 114:188–193.View Article
- Voronkova NM, Kholina AB: Conservation of endemic species from the Russian far east using seed cryopreservation. Biol Bull 2010, 37:469–501.
- Walter C, Wheeler L, Standwood PC: Longevity of cryogenically stored seeds. Cryobiology 2004, 48:229–244.View Article
- Wang JH, Ge JG, Liu F, Bian HW, Huang CN: Cryopreservation of seeds and protocorms of Dendrobium candidum . Cryo-Letters 1998, 19:123–128.
- Wood CB, Pritchard HW, Miller AP: Simultaneous preservation of orchid seed and its fungal symbiont using encapsulation-dehydration is dependent on moisture content and storage temperature. Cryo-Letters 2000, 21:125–136.PubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.