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
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,
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.