Cryopreservation of Paphiopedilum bellatulum (Rchb.f.) Stein and P. callosum (Rchb.f.) Stein Seeds and Protocorms

Abstract

Paphiopedilum Pfitzer is an attractive terrestrial lady’s slipper orchid being threatened with extinction due to over-collection and loss of habitats. Consequently, exsitu conservation methodology and strategy for this orchidare urgently needed. Two different Paphiopedilum species native to Thailand, namely Paphiopedilum bellatulum (Rchb.f.) Stein and P. callosum (Rchb.f.) Stein, were representatives of relevant subgenus. These two Paphiopedilum are characterized by different growth habits and thrive in different habitat types. Possible applications of cryopreservation as long-term ex-situ conservation strategy were explored. Three main elements were explored in this study. First, capsules and seeds were investigated in light of morphological maturity and behavior of tolerance on dehydration. Second, various techniques for cryopreservation of seeds, i.e. desiccation, vitrification and modification with aluminum foil wrap, were tested. Third, protocorms induced from cultured seeds were subjected to cryo-plate and alginate encapsulation-based methods, which involved the process of dehydration by desiccation and vitrification. For the capsules harvested at one month intervals from 5 to 9 MAP, results indicated that dimension of P. bellatulum and P. callosum capsules at 5 to 8 MAP showed no significant difference on average length and width, but the weight significantly decreased. Likewise, dimensions of P. bellatulum and P. callosum seeds at 5 to 8 MAP showed no significant differenceon average length and width, while seed moisture content (MC) continuously decreased. Seeds harvested at prolonged timing inclined to have more total seed germination percentage. P. bellatulum 9 MAPs seeds showed the highest seeds germination at 37.08% while P. callosum 8 MAPs seeds showed the highest seed germination at 32.69%. For seed cryopreservation by desiccation technique, which was investigated against increased time of desiccation under air flow of a laminar air-flow cabinet at 0-7 hour intervals, seed MC decreased to ca. 4%. After thawing from liquid nitrogen (LN), P. bellatulum 9 MAPs seeds showed the highest germination rate at 30.58% after being desiccated for 4 hours, whereas P. callosum at 8 MAPs seeds showed the highest germination rate at 33.40% after being desiccated for 4 hours. An application of modification with aluminum foil wrap technique did not show different results. For vitrification technique, in seeds, which were pretreated with LS at 25 °C for 15 min and then exposed to PVS2, it showed that P. bellatulum 9 MAPs seeds exposed to PVS2 for 60 minutes yieled the highest germination rate at 30.96% and P. callosum 8 MAPs seeds gave the highest germination rate at 44.03%. Cryopreservation of protocorms of P.bellatulum and P. callosum by encapsulation-dehydration, encapsulation-vitrification, D-cryo-plate, and V-cryo-plate technique did not present any regrowth after having been stored in LN for 1 day and recultured on ¼ MS medium for 1 month. Cryopreservation of Paphiopedilum by the application of fully mature seeds with split capsule directly immerse into LN is suitable becauseit is the most simplified, pragmatic and efficient in cost and time, especially for ex-situ long-term storage. This method can be further applied to ensure their continuous existence by preserving genetic diversity and should be an integral part in conservation strategies.