CryoLetters 26 (3), 139-146 (2005) © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF IMMATURE SEEDS OF graminifolia var. suzukiana BY VITRIFICATION

T. Hirano1, K. Ishikawa2 and M. Mii1*

1 Laboratory of 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 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, 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 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 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. Length and width of embryos and pods at each development stage are shown as the averages of 10 pods and 40 embryos, respectively. TTC stainability is shown as the average percentage of three replicates.

Based on these results, the optimum conditions for cryopreservation were investigated. Immature seeds of 6 and 9 WAP were immediately immersed in 1.5 ml cryoprotective solution for 15 min, and then soaked into PVS2 for 0 to 90 min. In the seeds of 6 WAP, the highest survival rate (71%) as determined by TTC staining was obtained at 15 min exposure to PVS2 (Fig. 1a). The survival rate decreased from 71 to 4% as exposure time to PVS2 increased from 15 to 90 min (Fig. 1a). In the seeds of 9 WAP, high level of survival rate (88- 94%) was retained in immature seeds treated with PVS2 for 30-90 min (Fig. 1a). Preculture with 0.3M sucrose for 3 days before vitrification treatment increased the survival rate of immature seeds of 6 WAP after cooling in liquid nitrogen, and the highest survival rate (86%) was obtained when they were treated with PVS2 for 60 min (Fig. 1b). In immature seeds of 9 WAP, however, preculture treatment did not have the positive effect on the survival rate after cryopreservation with PVS2 for 60-90 min and gave equal level of survival rate (81-91%) to those without preculture treatment (Fig. 1b). In survived immature seeds after cryopreservation, entire part of embryo was stained by TTC (Fig. 2a).

Table 2. Effect of light on germination and protocorm formation rates after 1 month of culture.

Light Developmental stage (weeks after pollination) condition 6 917 germination protocorm germination protocorm germination protocorm (%) formation (%) (%) formation (%) (%) formation (%) light 18.5a 0.4a 9.1a 0.0a 13.1a 0.5a

dark 41.9b 9.3b 19.5a 2.0b 12.0a 2.1a

Data represent mean of three replicates. Values in each followed by the same letter are not significantly different at P < 0.01.

142

100

80 a ) % (

y t i l 60 abi n i a 40 st C T

T 6 WAP 20 9 WAP

0 0 5 15 30 60 90 Exposure time to PVS2 (min)

100

80 b ) ) % % ( ( y y t t i i l l 60 bi bi a a n n ai ai 40 st st C C T T T 20 6 WAP 9WAP 0 015306090 Exposure time to PVS2 (min)

Figure 1. Effects of preculture on medium with high concentration of sucrose and exposure time to PVS2 on survival rate (%) of immature seeds after cooling at –196ºC. Immature seeds of 6 and 9 WAP were treated by vitrification either (a) without or (b) with preculture. Data represent mean ± standard errors of three replicates. When immature seeds of 6 and 9 WAP were cryopreserved by vitrification with preculture, they showed equal rates of germination and protocorm formation in comparison to the corresponding untreated controls (Table 3). After cryopreservation, germinated immature seeds developed into protocorms (Fig. 2b, c) and normal seedlings after 6 months of culture on gellan gum-solidified ND medium (Fig. 2d).

143 Table 3. Germination and protocorm formation rates of cryopreserved immature seeds after 1 month of culture.

Treatment Developmental stage (weeks after pollination) 69 germination protocorm germination protocorm (%) formation (%) (%) formation (%) untreated control 51.5a 12.9a 19.2a 1.8b x z cryopreservation 50.1a 12.2a 19.0a 0.0a

preculture and 48.4a 14.1a 20.9a 1.1b y cryopreservation

Data represent mean of three replicates. Values in each column followed by the same letter are not significantly different at 0.01 level. xImmature seeds of 6 and 9 WAP were immersed in PVS2 for 15 and 30 min, respectively. yImmature seeds were immersed in PVS2 for 60 min. zProtocorm formation rate after 2 month of culture was 1.3%.

a b

c d

Figure 2. Development of plant from immature seeds of 6 WAP after cryopreservation with preculture and vitrification treatment. Immature seeds were immersed in PVS2 for 60 min. (a) TTC stained immature seeds. (b) 1 month-old germinated embryo and (c) protocorm. (d) 6 month-old plantlets. Bar = (a) 1.0 mm, (b) 0.3 mm, (c) 0.5 mm, (d) 1.0 cm.

144 DISCUSSION

When seeds of three different developmental stages (6, 9 and 17 WAP) were asymbiotically cultured, the highest germination and protocorm formation rates were obtained in 6 WAP (Table 2). Some orchid species are known to have a difficulty in germinating mature seeds because of the dormancy mechanisms (15). In P. graminifolia var. suzukiana, the seeds of 9 and 17 WAP showed lower germination and protocorm formation rates than those of 6 WAP (Table 2) although seed viability of 9 and 17 WAP was almost the same as that of 6 WAP (Table 1). Since embryo size of P. graminifolia var. suzukiana reached maximum at 9 WAP (Table 1), embryogenesis is considered to complete during the period from 6 to 9 WAP. Hence it is suggested that seed dormancy of this species was induced immediately after completing embryogenesis. When immature seeds of 6 and 9 WAP were cryopreserved by vitrification without preculture, survival rate of 9 WAP seeds was higher than that of 6 WAP seeds and long period of osmotic dehydration by PVS2 (90 min) decreased survival rate of 6 WAP seeds (Fig. 1). In general, the acquisition of dehydration tolerance is an essential part of the maturation program in most types of seeds (1,2,6). Therefore, it is suggested that immature seeds of P. graminifolia var. suzukiana acquired some tolerance to dehydration already at 6 WAP and it increased with seed maturation thereafter to 9 WAP. Our previous results of immature seeds of Bletilla striata also showed that higher survival rate after cryopreservation by vitrification was obtained on immature seeds which were collected near the time of completion of embryogenesis (7). Accordingly, it is possible that dehydration tolerance rises at the high level near the time of completion of embryogenesis in . With the vitrification method, a highly concentrated solution, PVS2, is used to induce vitrification of the cells. Consequently, key factor for the successful cryopreservation is an increase in the tolerance against osmotic dehydration of the cells. Preculture with medium containing a high level of sorbitol and/or sugar was reported to be extremely useful in improving survival of cryopreserved cells and tissues (20,23). In immature seeds of Bletilla striata, the highest survival rate was also obtained when they were treated with vitrification procedure after preculture (7). In the present study, preculture with 0.3 M sucrose for 3 days before vitrification treatment increased the survival rate of immature seeds of 6 WAP when cooled in liquid nitrogen, suggesting that immature seeds of 6 WAP acquired osmotic dehydration tolerance during the preculture. It has been reported in Spathoglottis plicata (Orchidaceae) that dehydration tolerance was induced by large accumulation of sucrose in protocorms after culture on 10% (0.29 M) sucrose medium (22). Therefore, it is also possible that the increase in dehydration tolerance of immature seeds of 6 WAP during the preculture treatment of this species was also associated with accumulation of sucrose. However, the mechanism involved in the effectiveness of high concentration of sugar or sugar alcohols in preculture medium has not been well known. During preculture step, important cellular changes might have occurred such as induction of endogenous absisic acid synthesis (3), stabilization of the membranes (4, 8), and gene expressions necessary for adapting to the high osmotic condition (16). It is possible that these complex changes might favor for inducing the tolerance of immature seeds of 6 WAP against vitrification treatment. When immature seeds of 6 WAP were cryopreserved by vitrification with preculture, they showed equal germination rate and protocorm formation rate to the corresponding untreated control (Table 3). Although protocorm was not formed after 1 month of culture when immature seeds of 9 WAP were cryopreserved by vitrification without preculture, it was observed after 2 months of culture (Table 3). Therefore, it is suggested that cryopreservation by vitrification had no harmful effect on germination and protocorm formation of immature seeds. In cryopreservation of meristems of Pea (Pisum sativum cv. Century), the meristematic

145 dome was damaged and shoot regeneration occurred from the remaining tissues of the meristem (5). In the present study, embryos survived after cryopreservation were stained entire by TTC (Fig. 2a) and entire part of the survived embryos swelled and grew into protocorms and plantlets during culture directly (Fig. 2b), indicating that they had no partial damage during the cryopreservation treatment. Morphological abnormalities were also not observed in any of the plants obtained after cryopreservation (Fig. 2b, c, d). Therefore, it can be concluded that cryopreservation by vitrification is an efficient method for preservation of immature seeds. The present data indicate that immature seeds of P. graminifolia var. suzukiana, which germinate more readily in vitro than mature seeds, can be cryopreserved by vitrification. Further study is necessary to confirm the applicability of this cryogenic protocol to other endangered orchid species which are recalcitrant in germinating mature seeds but easy in immature seeds.

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Accepted for publication 8/4/05

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