FIGURE 5.7 RAPD profiles of callus-regenerated progenies of vanilla using OPERON primer OPA10. 1: 1 kb ladder, 2–23: callus-regenerated plants, 24: control (V. planifolia).
Callus cultures initiated from leaf explants of V. planifolia showed better callus initiation than those from nodal explants with callus biomass production maximal when cultured on MS basal medium containing 2,4-dichlorophenoxy acetic acid and BA. Callus transferred to MS basal medium supplemented with 3 mg L−1 BA and 2.5 mg L−1 μM NAA showed superior growth response. Davidonis et al. (1996) have patented the production of callus of V. planifolia, extraction of vanillin, and the use of ferulic acid to increase the content of vanillin.
Heritable somaclonal variations with respect to various resistance traits have been reported, namely, resistance to methionine sulfoxime (Carlson, 1973) and Pseudomonas syringae (Thanutong et al., 1983), in tobacco, resistance to Fusarium oxysporum in tomato (Evans et al., 1984), and resistance to Helminthosporium sati-vum (Chawla and Wenzel, 1987) in wheat. In future attempts to genetically transform vanilla, the ability of transformed tissue to regenerate is a crucial prerequisite. The regeneration protocol optimized (Minoo, 2002) could shorten the length of genetic transformation experiments while inducing a high frequency of regeneration.
Ex Vitro Establishment of Seedlings
Most plant species grown in vitro require a gradual acclimatization and hardening for survival and growth in the natural environment. The survival of in vitro plants depends upon their ability to withstand water loss and carry out photosynthesis. However in vanilla, the survival rate of transferred plants is currently over 80% during hardening process (Minoo, 2002). Plantlets should be removed from culture vessels (Figure 5.8), washed, treated with fungicide, transferred to polybags containing potting mixture (sand, soil, and vermiculite) and hardened for 30 days under controlled conditions (26–28°C, 80–90% RH). Initiation of new growth occur through development of the axillary branch. These plants are successfully transferred to soil after initial hardening period of three weeks (Figure 5.9) and can be. They were later field planted with proper shade and support.
FIGURE 5.8 Hardening of in vitro developed plantlets.
FIGURE 5.9 Tissue-cultured plants growing in pots.
Interspecific Hybridization
Interspecific hybridization is an age-old mechanism by which useful genes from wild progenitors and species can be brought into cultivated species. The cultivated types of many crop species were improved through interspecific hybridization and backcrossing. Interspecific hybridization is very common in orchids to produce new and novel varieties of flowering plants.
Natural occurrences of interspecific hybrids have been reported in vanilla by Nielsen and Siegsmund (1999) between V. claviculata and V. barbellata in localities in Puerto Rico where they coexist. Progenies were discovered having morphological characters intermediate between the two parents.
The cultivated species of V. planifolia has been crossed with other American species including V. pompona and V. phaeantha, which are resistant to Fusarium (Purseglove et al., 1981). Interspecific hybridization was also conducted in Java between cultivated and wild vanilla to develop lines resistant to stem rot caused by Fusarium oxysporum (Mariska et al., 1997).
In India, V. aphylla and V. pilifera flower synchronously but V. aphylla occurs naturally in South India and V. pilifera in Assam, North East India. When cultivated in Kerala, flowers of both species opened sequentially and lasted for one day in V. pilifera, whereas it lasted for two days in V. aphylla. In the former, signs of fruit set were observed even without manual pollination whereas V. aphylla flowers did not set fruit. Since rostellum is present in both species, natural pollination without an aid is ruled out. It can be suspected, that the fragrance of the V. pilifera flowers attracts insects (which were found to frequent the flowers often) to visit them and bring about effective pollination (D. Minoo, unpublished data). Self and interspe-cific hybridizations between the two species were done manually and fruits set was observed.
Successful attempts were made to increase the spectrum of variation of V. plani-folia by interspecific hybridization with V. aphylla which is tolerant to Fusarium (Minoo, 2002). Morphological characters and molecular profiles revealed the true hybridity of the interspecific hybrid progenies. Seedling progenies of V. planifolia, and interspecific hybrids obtained from crosses between V. planifolia (female) and V. aphylla (male) were evaluated using a number of different loci as markers by using AFLPs and RAPDs loci. The profiles indicated similarity between the parents, selfed progenies, and interspecific hybrids and that all the progenies tested were variable when compared to each other, which can be exploited for crop improvement in vanilla (Minoo et al., 2006a).
Thus, these successful introgressions of male and female characters into the hybrids (Minoo et al., 2006a) by interspecific hybridization, confirmed by molecular profiles are promising to help solve the major bottlenecks in vanilla breeding.
In Vitro Conservation
Effective procedures for in vitro conservation by slow growth in selected species of vanilla have been standardized (Minoo et al., 2006b). The addition of mannitol (10–15 g L−1) and reduction of sucrose to lower levels (15–10 g L−1) induced slow growth and subsequently 80–90% of the cultures could be maintained for a period of 360 days, when the culture vessels were closed with aluminum foil. Supplementing mannitol and sucrose in equal proportions at 10 or 15 g L−1, could help to maintain the cultures for one year and thus were maintained in vitro for more than seven years with yearly subculture. The plantlets maintained in this medium showed reduced growth rate and maximum survival. The conserved material was transferred to MS medium fortified with 30 g L−1 sucrose and supplemented with 1 mg L−1 BA and 0.5 mg L−1 IBA, for retrieval of normal shoots and their multiplication. The conserved material was transferred to the multiplication medium (MS + 30 g L−1 sucrose and 1 mg L−1 NAA) for normal growth. The small-sized plantlets kept in the conservation medium for over one year showed good growth and developed into normal-sized plants with good multiplication rate (1:5). These plantlets were transferred to soil (garden soiclass="underline" sand:perlite in equal proportions) and established easily with 80% success when kept in a humid chamber for 20–30 days after transfer. They developed into normal plants without any deformities and defi-ciency symptoms and exhibited apparent morphological similarities to the mother plants. After more than seven years of slow growth storage, involving over five subculture cycles, the genotypic stability of few species was assessed using molecular markers. No changes were observed in DNA fingerprinting vis-à-vis nonconserved controls in the authors’ laboratory.