Jarret and Fernandez (1984) have reported storage of V. planifolia shoot tips as tissue cultures for 10 months and Philip (1989) has discussed the possibility of using root cultures for conservation of vanilla germplasm for assured genetic stability. In vitro conservation of V. planifolia (Jarret and Fernandez, 1984) and V. walkeriae using slow growth method (Agrawal et al., 1964) has been reported and the effects of polyamines on in vitro conservation of V. planifolia have been studied by Thyagi et al. (2001).
Conventional and in vitro genebanks are complementary as the active and base collections of genetic resources. Although in vitro conservation cannot be viewed as a method to replace in situ conservation, the advantages of in vitro conservation as a component that can be incorporated into an overall vanilla long-term conservation strategy for a safe and economical storage of the germplasm were demonstrated.
Cryopreservation
Protocols for conservation of gene pools have been developed for slow growth as well as cryopreservation of vanilla accessions as encapsulated shoot tips, pollen, and DNA (Minoo, 2002). Combining the available gene pool in the genus will help in broadening the genetic base and in converging the useful genes into cultivated vanilla from wild species. Interspecific hybridization requires synchronized flowering between the species and availability of viable pollen. Pollen from two asynchronously flowering species of Vanilla, namely, cultivated V. planifolia and its wild relative V. aphylla, were cryopreserved after desiccation, pretreated with cryoprotectant dimethyl sulfox-ide (5%) and cryopreserved at −196°C in liquid nitrogen (LN). This cryopreserved pollen was later thawed and tested for their viability both in vitro and in vivo. A germination percentage of 82.1% and 75.4% in V. planifolia and V. aphylla pollen, respectively, were observed indicating their viability. These cryopreserved pollens of V. planifolia were used successfully to pollinate V. aphylla flowers resulting in fruit set. The seeds thus obtained were successfully cultured to develop hybrid plantlets (Minoo, 2002). Viability and fertility assessment of cryopreserved pollen (Figure 5.10) from Vanilla species thus showed that it is possible to use cryogenic methods for conservation and management of the haploid gene pool in this species. This is of great importance for the facilitating crosses in breeding programs, for distribution and exchange of germplasm, and for preserving nuclear genes of the germplasm.
FIGURE 5.10 Germination of cryopreserved pollen.
A procedure for storage of vanilla germplasm by cryopreservation of shoot tips using encapsulation/dehydration method has been standardized (Figure 5.11). The in vitro-grown shoot tips were encapsulated in 4% sodium alginate. The encapsulated beads were subjected to pretreatment by progressive increase of sucrose concentration from 0.1 to 1.0 M, followed by dehydration for 8 h to a moisture content of 22%. This was followed by rapid freezing by plugging into LN. The cryopreserved shoot tips were thawed after 12 h in LN by keeping them in water bath at 40°C for 3 min. The thawed propagules were allowed to recover on MS with 3% sucrose, 1 mg L−1 BAP, and 0.5 mg L−1 IBA in dark for one week and then transferred to light for regrowth and multiplication. Seventy percent of the propagules have for recovered, grown, and multiplied into full-fledged plants (Ravindran et al., 2004).
FIGURE 5.11 Germination of cryopreserved shoots.
Cryopreservation, once fully implemented will provide an expeditious and cheaper means to duplicate the base collection for safety reasons, as well as for the distribution of germplasm sets to other countries/continents.
Production of Synthetic Seeds
Synthetic seed technology was standardized by encapsulating 3–5 mm in vitro regenerated shoot buds and protocorms in 4% sodium alginate, to produce good quality rigid beads ideal for withstanding low temperatures and cryopreservation. Higher concentrations of alginate were not suitable as they produced very hard matrix, which hindered the emergence of shoot buds and thereby affecting the rate of germination and recovery, while at lower concentrations of alginate, the beads were difficult to handle during cryopreservation and retrieval. The synthetic seeds were stored at 5°C, 15°C, and 22°C to study the effect of temperature on their storage and viability. Low temperatures (5°C and 15°C) were not suitable for synthetic seed. Shoot buds of 0.4–0.5 cm size were suitable for encapsulation as smaller buds failed to survive the storage and lost their viability within a month. However at 22 ± 2°C, synthetic seeds could be stored for 10 months (Figure 5.12). The plants derived from these encapsulated buds were apparently healthy and developed into normal plants.
FIGURE 5.12 Synthetic seeds.
Clonal propagation of V. planifolia using encapsulated shoot buds have been reported by George et al. (1995). Synthetic seeds are ideal for germplasm conservation and exchange, especially in vanilla, where there is no natural seed set.
Protoplast Isolation and Fusion
The techniques of protoplast isolation and fusion are important because of the far-reaching implications in studies of plant improvement by cell modification and somatic hybridization. The possibility of protoplast systems in spice crops such as cardamom, ginger, and vanilla was studied by Triggs et al. (1995) and Geetha et al. (2000).
Protoplasts were successfully isolated from V. planifolia and V. andamanica when incubated in an enzyme solution containing macerozyme R10 (0.5%) and cellulase Onozuka R10 (2%) for 8 h at 30°C in dark (Table 5.4). In vitro leaves were plasmolyzed in a solution containing cell protoplast washing salts with 9% mannitol before enzymatic digestion. Since it was difficult to peel off the lower epidermis in vanilla, the plasmolyzed leaf tissue was mechanically macerated by scraping the lower surface of the leaf with a sharp blade and incubating in different concentrations and combinations of enzyme solutions. Periodical microscopic observations showed the liberation of cell clusters and individual cells after 2 h of incubation in enzyme solution.
| Species | Enzyme Solution | Incubation Conditions | Protoplast Yield | Viability (%) |
|---|---|---|---|---|
| V. planifolia | 0.5% macerozyme R10 + 2% onozuka cellulase R10 | 8 h at 30°C in dark | 2.5 × 105/g of leaf | 72 |
| V. andamanica | 1% macerozyme R10 + 3% hemicellulase + 6% onozuka cellulase R10 | 8 h at 30°C in dark | 1 × 105/g of leaf | 55 |