CryoLetters 26 (3), 139-146 (2005) © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK CRYOPRESERVATION OF IMMATURE SEEDS OF Ponerorchis graminifolia var. suzukiana BY VITRIFICATION T. Hirano1, K. Ishikawa2 and M. Mii1* 1 Laboratory of Plant Cell Technology, Faculty of Horticulture, Chiba University, 648 Matsudo, Matsudo-City, Chiba, 271-8510, Japan. 2 Japan Horticultural Production and Research Institute, 2-5-1 Kamishiki, Matsudo-City, Chiba, 270-2221, Japan. *For correspondence: [email protected] Abstract Ponerorchis graminifolia var. suzukiana is a terrestrial orchid that is an endangered species native to Japan, and it germinates more readily in immature seeds than in mature seeds. To preserve this orchid, an efficient protocol was established for the cryopreservation of immature seeds of P. graminifolia var. suzukiana. When immature seeds of 6 weeks after pollination, which showed higher germination and protocorm formation than mature seeds, were precultured on New Dogashima (ND) medium with 0.3M sucrose for 3 days and cryopreserved by vitrification method (treated with PVS2 for 60 min), the viability after preservation as assessed with 2,3,5-triphenyltetrazolium chloride staining test was about 86%. Immature seeds thus treated showed equal rates of germination and protocorm formation to the untreated control immature seeds, and they developed into normal plantlets on ND medium. Keywords: cryopreservation, endangered species, immature seeds, Ponerorchis graminifolia var. suzukiana, vitrification INTRODUCTION Ponerorchis graminifolia var. suzukiana is a terrestrial orchid native to Japan and cultivated ornamentally because of its beautiful flowers and long blooming period. The plant is also used as a breeding material for P. graminifolia due to the merits to have large number of flowers and high temperature tolerance. Because of an extensive hunting for horticultural demands and disturbance of natural habitat, P. graminifolia var. suzukiana is one of the endangered orchid species which may soon extinct in Japan. Therefore, it is urgently needed to establish the reliable methods for preservation of this orchid. In vitro culture for seed germination and embryo rescue of commercial orchids is now a routine method. However, in some orchid species, mature seeds are difficult to germinate, and P. graminifolia var. suzukiana is one of the recalcitrant species for seed germination. Recent review of Rasmussen (15) described that some orchid species, which have a difficulty in 139 germinating mature seeds, sometimes readily germinate in immature seeds. For example, the seeds of P. graminifolia become mature about 120 days after pollination, but the highest germination rate is obtained at 35 to 40 days after pollination (12). In Orchis papilionacea, immature seeds also germinate more readily in vitro than mature seeds (14). Thus establishment of a method for preservation of immature seeds is invaluable not only for conservation of germplasm but also for propagation and breeding of various orchid species. Cryopreservation has been considered as an important tool for the long-term storage of germplasms. As a simple and reliable cryopreservation procedure, vitrification method, which does not involve the use of expensive facilities, has been developed (11, 17). Vitrification method dehydrates a major part of the freezable water from the tissues at non-freezing temperature and enables them to cryopreserve by plunging into liquid nitrogen. The keys for successful cryopreservation are the acquisition of osmotolerance and the mitigation of injurious effects during dehydration. Osmoprotection by treating with a mixture of 2 M glycerol and 0.4 M sucrose (cryoprotective solution) for 20 min at 25ºC before dehydration with vitrification solution significantly increased osmotolerance (13). Vitrification method has been applied to different plant materials of some orchid species such as zygotic embryos of Bletilla striata (9), protocorms of Dendrobium candidum (21), seeds of Doritis pulcherrima (10), and suspension culture cells of Doritaenopsis (19). As a model for cryopreservation of immature orchid seeds, we have succeeded in cryopreservation of immature seeds of Bletilla striata by vitrification (7). The aim of the present study is to develop a method for cryopreservation of immature orchid seeds of an endangered and recalcitrant species of P. graminifolia var. suzukiana. MATERIALS AND METHODS Plant materials Greenhouse-grown plants of Ponerorchis graminifolia Rchb.f. var. suzukiana (Ohwi) Soó were supplied by K. Ohtani and M. Hashimoto, Kankyo Engineering Co., Ltd, Japan. Immature pods were collected from these plants 6 and 9 weeks after self-pollination (WAP), whereas mature pods were collected 17 WAP. The pods of these three developmental stages were surface sterilized with a sodium hypochlorite solution (1% available chlorine) containing a few drops of Tween-20 for 10 min. Following surface sterilization, the pods were washed once with sterile distilled water. The seeds were then taken out from each pod and sown on 0.2% gellan gum-solidified New Dogashima (ND) medium (18) with 58 mM sucrose, and cultured at 25ºC under continuous illumination at 62.0 µM m-2 s-1 or continuous dark condition. Length and width of ten pods were measured at each developmental stage. The length and width of embryos contained in the pods at each stage were also measured under optical microscope. Cryopreservation procedures and culture condition Immature seeds taken out from pods were sown as a preculture treatment on 0.2% gellan gum-solified ND medium with 0.3 M sucrose, and cultured at 25ºC for 3 days under continuous dark condition (9). About 300-400 seeds, which were treated with or without preculture, were transferred to 2.0 ml cryotubes, and then immersed in 1.5 ml cryoprotective solution (2 M glycerol and 0.4 M sucrose in ND medium) for 15 min at 25ºC. After removing the cryoprotective solution, the seeds were dehydrated at 0ºC for various periods with 2.0 ml PVS2 (17), which contained 30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) 140 dimethyl sulfoxide in ND medium supplemented with 0.4 M sucrose (pH 5.4). Then the seeds in cryotubes were directly plunged into liquid nitrogen. After storing there for at least 30 min, cryotubes were thawed in a water bath at 38ºC for 2 min. After draining PVS2 from the cryotubes, 1 ml of liquid ND medium supplemented with 1.2 M sucrose was added to each tube and held for 10 min. Then, 0.5 ml of fresh liquid ND medium supplemented with 0.4 M sucrose was added to the cryotubes every 10 minutes twice. After removing the solution from cryotubes, 1 ml of liquid ND medium supplemented with 0.4 M sucrose was added to each tube and held for 10 min. The seeds were then sown on 0.2% gellan gum-solidified ND medium with 58 mM sucrose, and cultured at 25ºC under continuous dark condition. Survival rate Survival rate after each procedure was estimated by 2,3,5-triphenyltetrazolium chloride (TTC) stainability test (9). After treating with each procedure and culturing for 3 days on 0.2% gellan gum-solidified ND medium with 58 mM sucrose, about 100 immature seeds were incubated in 1% (w/v) TTC solution for 1 day at 25ºC in the dark. The number of embryos stained by TTC was counted, and the percentage of TTC-stained seeds was calculated as TTC stainability (%). The experiment was repeated 3 times. Germination and protocorm formation rate Seed germination was defined as embryo emerged from the seed coat, and protocorm was defined as germinated embryo with developed rhizoids. After 1 month of culture, germination and protocorm formation rates were calculated. The experiment was repeated 3 times. Statistical analysis The rates of seed germination and protocorm formation were subjected to analysis of variance (ANOVA) after arc sin transformation, and the means were compared by the least significant difference (LSD) test. RESULTS The changes in pod size, embryo size, and TTC stainability of embryos were investigated during the development of seeds after pollination of P. graminifolia var. suzukiana (Table 1). From 6 to 9 WAP, the pod length and width increased from 8.8 mm to 9.4 mm and 2.2 mm to 2.4 mm, respectively. During this period, the embryo length and width also increased drastically from 149 µm to 227 µm and 104 µm to 169 µm, respectively. On the other hand, from 9 to 17 WAP, the pod size did not change and the embryo length and width slightly decreased (Table 1). TTC stainability of untreated seeds of 6, 9 and 17 WAP were 88, 94 and 88%, respectively. Table 2 shows the effect of light condition in cultivation on frequencies of germination and protocorm formation in seeds of different developmental stages. In immature seeds of 6 WAP, germination and protocorm formation rates under continuous dark condition were 42% and 9%, respectively, which were significantly higher than those under continuous light condition (Table 2). Protocorm formation in immature seeds of 9 WAP was only observed under dark condition. Therefore, immature seeds after cryopreservation were cultured under dark condition for further experiments. When mature seeds of 17 WAP were asymbiotically cultured, they showed equal germination rate and protocorm formation rate regardless of light condition (Table 2). Germination and protocorm formation rates of 17 WAP under continuous dark condition were 12% and 2%, respectively. 141 Table 1. Relationship between pod size and embryo size and TTC stainability of embryos at each developmental stage. Developmental stage (weeks after pollination) 6917 length of pods (mm) 8.8 ± 0.2 9.4 ± 0.3 9.3 ± 0.2 width of pods (mm) 2.2 ± 0.1 2.4 ± 0.1 2.5 ± 0.1 length of embryos (µm) 149.1 ± 3.1 227.1 ± 4.2 215.6 ± 5.2 width of embryos (µm) 104.1 ± 2.8 169.4 ± 3.1 160.3 ± 4.8 TTC stainability (%) 88.3 ± 4.1 94.2 ± 0.8 88.1 ± 1.0 Data represent mean ± standard errors.