5. Case study for cryopreservation of Bletilla formosana seeds (Orchidaceae) by desiccation

B. formosana belongs to genus Bletilla in the family Orchidaceae. The species is distributed widely in Taiwan and is known for its medicinal and ornamental value [5, 15, 16]. B. formosana is endangered during the destruction of habitat and over collection for ornamental use. Therefore, preservation of B. formosana is urgent to be proceeded.

Seeds of Bletilla genus had been studied in the pretreatment method prior to cryopreservation. However, the procedure of common vitrification method is more complex and high concentrations of cryoprotectants may be toxic to plant tissues [10, 11]. Therefore, the purpose of this study is to establish a practical method of long-term preservation for B. formosana seeds and to provide potential cryopreservation methods for other orchid species.

Mature seeds of B. formosana obtained from capsules 3 months after hand-pollination (3 MAP) were collected as the test materials, then dried at room temperature (27 ± 1°C) for 24 h in the laboratory with air conditioning and dried in a sealed container using silica gel for 0–24 h, respectively. Seeds were then stored in LN for 24 h, to investigate the viability rate and the germination rate of seeds after warming.

viability. This contrasts with results that have indicated that desiccating orchid seeds over silica gel for 24 h induces a significant increase in seed germination. The effect of longer seed desiccation durations remains to be determined. For example, the seeds of P. tancarvilleae are difficult to preserve with a low WC (2–5%) at 4 and 25°C [4]; however, the germination rates of B. formosana dried seeds with a low WC are 68.5% after cryopreservation [35]. Thus, the appropriate WC that orchid seeds require for successful cryopreservation varies among the

The values for the pollen germination and WC of the English walnut (Juglans regia L.) were the highest before storage. Subsequently, the values decreased along with an increase in storage time at room temperature. However, they did not decrease linearly with time. The ability to germinate was significantly reduced during drying, though some pollen retained some viability until a 3.2% MC [23]. Pollinia of L. macrantha subjected to 0–30 min dehydration within a laminar air flow cabinet showed maximum germination of 65–67% in desiccated controls and 54% in LN treated samples. The germination rate decreased with

Long-term preservation of plant genetic resources is not easy. Orchid seeds and pollens are ideal materials suitable for cryopreservation because of the characteristics of tiny volume and low water content. This article describes common pretreatment techniques used in orchid cryopreservation and the factors affecting material viability. Besides, this describes Taiwan's endangered native medicinal and ornamental plants, and the desiccation method applied in a case study of Bletilla formosana. The genetic resources of other economic orchids will continue to be tested by the desiccation method in the future. The ultimate object of our study is to provide a more practical potential cryopreservation method and apply in the other economic

B. formosana belongs to genus Bletilla in the family Orchidaceae. The species is distributed widely in Taiwan and is known for its medicinal and ornamental value [5, 15, 16]. B. formosana is endangered during the destruction of habitat and over collection for ornamental use. There-

Seeds of Bletilla genus had been studied in the pretreatment method prior to cryopreservation. However, the procedure of common vitrification method is more complex and high concentrations of cryoprotectants may be toxic to plant tissues [10, 11]. Therefore, the purpose of this study is to establish a practical method of long-term preservation for B. formosana seeds and to

Mature seeds of B. formosana obtained from capsules 3 months after hand-pollination (3 MAP) were collected as the test materials, then dried at room temperature (27 ± 1°C) for 24 h in the

species.

210 Cryopreservation in Eukaryotes

prolonged desiccation time [44].

(Orchidaceae) by desiccation

orchids for sustainable development of orchid industry.

fore, preservation of B. formosana is urgent to be proceeded.

provide potential cryopreservation methods for other orchid species.

5. Case study for cryopreservation of Bletilla formosana seeds

The viability and germination rates of fresh seeds of B. formosana are 89.9 and 76.8%, respectively. The initial water content of fresh seeds is 49.5% but decreasing significantly within 1–4 h by silica gel desiccation and then getting stable to 1.9%, 1 day after desiccation. When fresh seeds placed directly into LN for cryopreservation, the viability and germination rates are suddenly went down to 2.3 and 1.8%, respectively. However, for the fresh seeds pretreated by silica gel desiccation for 24 h or air-dried for 24 h at room temperature before putting in LN, the viability rates dramatically increased to 86.8 and 84.9%, respectively, and germination rates are up to 68.5 and 68.6%, respectively (Figure 1). These data show that the seed viability of B. formosana after cryopreservation is affected by the water content of storage seeds. When the water content of orchid seeds decreases to 24.8% by 24 h air drying or to 21.9–31.2% by 1–2 h silica gel drying, high viability and germination rates still remain after long-term

Figure 1. Effect of desiccation on viability of Bletilla formosana seeds after cryopreservation by TTC staining method. (A) Harvested fresh seeds; (B) fresh seeds plunged into liquid nitrogen (LN, -196°C); (C) fresh seeds dried for 24 h with silica gel prior to LN; (D) fresh seeds dried for 24 h with air-drying in laboratory conditions prior to LN. Bar =1 mm [49].

Figure 2. Seedling growth of Bletilla formosana for 6 wk after sowing on MS medium. (A) Fresh seeds; (B) fresh seeds were dried for 24 h with air-drying in laboratory conditions prior to LN; (C) fresh seeds were dried for 24 h with silica gel prior to LN; (D) fresh seeds were dried for 24 h with silica gel, vitrification (0.06 M sucrose solution+LS+PVS2 1 h) prior to LN. Bar =1 mm [49].

cryopreservation. Therefore, those desiccation methods are recommended for long term storage of B. formosana. Fresh seeds of orchid pretreated in sucrose solution or vitrification before LN treatment are unsuitable for cryopreservation in our previous study [35]. Seedlings derived from the seeds desiccated by these two methods and then preserved in LN grew well 6 weeks after seed sowing (Figure 2). In addition, B. formosana seeds with 1.9–24.8% water content were found to be suitable for cryopreservation [35].
