FIGURE 5.2 Flowers of Indian species of Vanilla: (a) and (b) V. andamanica with varying label colors, (c) V. pilifera showing indication of insect visits, and (d) V. aphylla.
AFLP profiles were developed to analyze Vanilla species, interspecific hybrids, and selfed progenies (Bory et al., 2008c; Lubinsky et al., 2008a; Minoo et al., 2006b). All these analyses converged in showing that most of the V. planifolia accessions cultivated outside of Mesoamerica exhibit very low levels of genetic diversity, as they derived from a single accession, possibly the Mexican cultivar Mansa from Papantla.
The patterns of diversification of the cultivated species were also studied and compared with other cultivated (V. tahitensis) and wild (V. aphylla, V. bahiana, V. insignis, V. odorata, and V. pompona) species. Clear polymorphism was detected in these related species, interspecific hybrids, and selfed progenies.
The development of SSR markers (microsatellites) have been reported by Bory et al. (2008b). The isolation and characterization of 14 microsatellite loci from V. planifolia have been described. These were monomorphic within cultivated accessions, as expected based on the probable single clonal origin of this crop and previous genetic studies. These markers were transferable to V. tahitensis and 11 loci were polymorphic between these two closely related species. Furthermore, some of these markers were transferable and polymorphic across 15 other wild American, African, and Asian species and revealed consistent relationships between species, together with a strong pattern of Old World versus New World differentiation in the genus. Furthermore, the use of microsatellites allowed the first molecular-based estimation of heterozygosity levels in this species, which was not possible when dominant markers such as AFLP or RAPD was used.
Sequencing of neutral genes has been used for reconstructing the evolutionary history of Vanilloid orchids, including a few Vanilla species (Cameron, 2000, 2004, 2009; Cameron and Molina, 2006; Cameron et al., 1999). Nuclear (internal transcribed spacer, ITS) and plastid (rbcL gene) DNA sequences were also used for unraveling the origin of the Tahitian vanilla (Lubinsky et al., 2008b). Recently, the length polymorphism of the nonneutral caffeic acid O-methyl transferase gene was also used to analyze 20 Vanilla species, and confirmed the strong differentiation of Old World versus New World species in the genus (Besse et al., 2009). On the basis of sequencing data for nuclear and plasmidic DNA, Cameron (2005) suggested in setting up a bar code system (Lahaye et al., 2008) for vanilla using the ITS region and the psbA-trnH intergenic spacer. This system may allow routine identification of vanilla specimens to the species level, and perhaps even to the accession level. To build a robust phylogeny for the Vanilla genus, reference herbarium specimens will need to be included. For this purpose, the development of plastid mononucle-otide microsatellites should be considered for vanilla (particularly when using degraded DNA samples extracted from herbarium material), as have already been successfully used for biogeographical studies of orchids (Fay and Krauss, 2003; Micheneau, 2002).
Given the difficulty in using classical phenotypic markers for perennial crops such as vanilla, molecular markers are powerful tools for studying the variability in cultivated vanilla, unraveling species interrelationships, identifying interspecific hybrids, and fingerprinting important genotypes (Minoo et al., 2006a). They are, therefore very helpful for monitoring and evaluating the achievements resulting from biotechnologies.
Propagation and Breeding Methods
Commercial vanilla is always propagated by stem cuttings of healthy vigorous plants and may be cut from any part of the vine. The length of the cutting is usually determined by the amount of planting material available. Short cuttings, 20 cm in length, will take 3–4 years to flower and fruit. Cuttings of 90–100 cm in length are usually preferable as they tend to flower earlier. When available, with their free ends hanging over supports, these will flower and fruit in 1–2 years. Cuttings are usually planted in situ, but they may be started in nursery beds when necessary. Because of their succulent nature, cuttings can be stored or transported for a period of up to two weeks, if required.
Traditionally, vanilla germplasm is conserved in clonal repositories belonging to botanical gardens and scientific institutions. The high costs of traditional conservation systems limit the number of accessions that can be preserved. In order to reduce the losses of biodiversity, attempts to conserve Vanilla species, in vitro, were made (Jarret and Fernandez, 1984; Minoo et al., 2006b) and have been extended to conserve the endangered species.
For breeding purposes, vanilla can be grown from seeds. Hybridization and the production of plants from seeds have been carried out in Puerto Rico and Madagascar. The seeds should be disinfected, washed in sterile distilled water, and cultured in nutrient medium (Knudson, 1950). The germination of vanilla seeds is better if the cultures are maintained in a dark incubator at 32°C. Seeds of interspecific crosses between V. planifolia and V. pompona required a higher temperature of 34°C for germination.
In Vitro Seed Germination
Vanilla produces numerous minute seeds that do not germinate under natural conditions. Tissue culture technique can be used to successfully germinate the seeds. Protocols for seed and embryo culture of vanilla have been standardized (Gu et al., 1987; Knudson, 1950; Minoo et al., 1997; Withner, 1955).
Seed culture in different basal media indicated that vanilla seeds had no stringent nutritional requirements for the initiation of germination unlike some terrestrial orchids of temperate climate (Minoo et al., 1997). The germination of seeds began within four weeks of culture and the initial stages of germination were typical of most orchids, such as swelling of the embryo followed by rupturing of the seed testa, and the subsequent emergence of protocorms (Figure 5.3). Seeds germinated directly into plantlets in the medium supplemented with benzyladenine (BA) (0.5 mg L−1) alone, without any intervening callus phase, and could thus be utilized for the production of selfed progenies/seedlings. The addition of tryptone had a growth-promoting effect on the size and development of protocorm, irrespective of the basal medium to which it was added. In treatments with BA, most of the protocorms remained the same with the scale-like leaf primordial and developing into shoots, whereas treatment with auxin supplements showed the gradual disorganization of the protocorms into callus. Murashige and Skoog’s (MS) medium gave a better response than Knudson’s medium, for in vitro cultures of vanilla. The minimum germination (26%) was observed in MS medium at half strength and the maximum (85%) was recorded in full strength MS medium supplemented with 2 g L−1 tryptone (Minoo, 2002).