The isolation solution containing 9% mannitol was found necessary for the release and maintenance of viable protoplasts. The isolated protoplasts were round and filled with chloroplasts. Protoplasts of V. planifolia were bigger in size (0.031 mm) than those of V. andamanica (0.022 mm) and could be distinguished by the arrangement of chloroplasts—peripheral in V. planifolia and centrally scattered V. andamanica. The visually distinguishable nature of protoplast can be exploited for the purpose of identifying genetic transformation in these species. When subjected to polyethylene glycol (PEG)-mediated fusion, the protoplasts fused forming a heterokaryon. The fusion product was cultured on MS liquid medium with 0.5 mg L−1 BA, 0.5 mg L−1 IBA supplemented with 3% sucrose and 7% mannitol for 20 days. The cell wall development around the fusion product was observed after 36 h (Minoo et al., 2008). The fusion protoplast technology can be very useful in gene transfer of useful traits to V. planifolia, especially the natural seed set and disease tolerance observed in V. andamanica.
The Future of Vanilla Improvement
The landmarks that have been attained in the form of various technologies can be effectively used for the production of a spectrum of genetic variations in vanilla, thus overcoming a major bottleneck in vanilla breeding and crop improvement programs. The protoplast isolation and fusion technology developed can be used in transfer of useful traits through the production of somatic hybrids, thus making way for genetic manipulations in vanilla. The characterization of Vanilla species, accessions, seedlings, somaclones, and interspecific hybrids, proved the existence and extent of genetic variations that is available and brought by biotechnological tools. The in vitro conservation methods, through synthetic seed, slow growth, and cryopreservation will form an integral and important part of overall conservation strategy in genetic recourses management of vanilla germplasm. Furthermore, the synthetic seed technology forms an ideal means for exchanging disease-free planting material.
References
Agrawal, D.C., Morwal, G.C., and Mascarenhas, A.F. 1992. In vitro propagation and slow growth storage of shoot cultures of Vanilla walkeriae Wight, an endangered orchid. Lindleyana 7 (2): 95–99.
Besse, P., Da Silva, D., Bory, S., Grisoni, M., Le Bellec, F., and Duval, M.-F. 2004. RAPD genetic diversity in cultivated vanilla: Vanilla planifolia, and relationships with V. tahit-ensis and V. pompona. Plant Science 167:379–385.
Besse, P., Da Silva, D., Bory, S., Noirot, M., and Grisoni, M. 2009. COMT intron-size variations in Vanilla species (Orchidaceae). Plant Science 176:452–460.
Bhatia, C.R. 1996. Biotechnology—many application areas. In: Ravi, N. ed. Hindu Survey of Indian Agriculture. The Hindu daily, Chennai, India, 173–175.
Bory, S., Catrice, O., Brown, S., Leitch, I., Gigant, R., Chiroleu, F., Grisoni, M., Duval, M.-F., and Besse, P. 2008a. Natural polyploidy in Vanilla planifolia (Orchidaceae). Genome 51:816–826.
Bory, S., Da Silva, D., Risterucci., A.M., Grisoni, M., Besse, P., and Duval, M.F. 2008b. Development of microsatellite markers in cultivated vanilla: Polymorphism and transferability to other vanilla species. Scientia Horticulturae 115:420–425.
Bory, S., Grisoni, M., Duval, M.-F., and Besse, P. 2008d. Biodiversity and preservation of vanilla: Present state of knowledge. Genetic Resources and Crop Evolution 55:551–571.
Bory, S., Lubinsky, P., Risterucci, A.-M., Noyer, J.-L., Grisoni, M., Duval, M.-F., and Besse, P. 2008c. Patterns of introduction and diversification of Vanilla planifolia (Orchidaceae) in Reunion Island (Indian Ocean). American Journal of Botany 95:805–815.
Bouriquet, G. 1954. (ed.) Le vanillier et la Vanille dans le Monde. Paris: Editions Paul Lechavalier.
Cameron, K.M. (ed.) 2000. 3rd Southern Connection Congress, Canterbury, NZ. January 17–22, 2000.
Cameron, K.M. 2004. Utility of plastid psaB gene sequences for investigating intrafamilial relationships within Orchidaceae. Molecular Phylogenetics and Evolution 31:1157–1180.
Cameron, K.M. 2005. DNA barcoding as a method for vanilla species identification. In: Vanilla 2005 Mexico. The International Symposium on the Vanilla Business, Veracruz, Mexico, November 15–16, 2005.
Cameron, K.M. 2009. On the value of nuclear and mitochondrial gene sequences for reconstructing the phylogeny of vanilloid orchids (Vanilloideae, Orchidaceae) 10.1093/aob/ mcp024. Ann Bot:mcp024.
Cameron, K.M., Chase, M.W., Whitten, W.M., Kores, P.J., Jarrell, D.C., Albert, V.A., Yukawa, T., Hills, H.G., and Goldman, D.H. 1999. A phylogenetic analysis of the Orchidaceae: Evidence from rbcL nucleotide sequences. American Journal of Botany 86:208–224.
Cameron, K.M. and Molina, C. 2006. Photosystem II gene sequences of psbB and psbC clarify the phylogenetic position of Vanilla (Vanilloideae, Orchidaceae). Cladistics 22(3):239–248.
Carlson, P.S. 1973. Methonine sulfoxamine-resistant mutants of tobacco. Science 180: 1366–1368.
Cervera, E. and Madrigal, R. 1981. In vitro propagation (Vanilla planifolia A.) Environmental and Experimental Biology 21:441.
Chawla, H.S. and Wenzel, G. 1987. In vitro selection of barley and wheat for resistance against Helminthosporium sativum. Theoretical and Applied Genetics 74:841–845.
Davidonis, G. and Knorr, D. 1991. Callus formation and shoot regeneration in Vanilla planifolia. Food Biotechnology 5 (1):59–66.
Davidonis, G., Knorr, D., Romagnoli, W., and Lynn, G. 1996. Callus formation Vanilla planifolia. US Patent 5573941.
De Pauw, M.A., Remphrey, W.R., and Palmer, C.E. 1995. The cytokinin preference for in vitro germination and protocorm growth of Cyperidium candidum. Annals of Botany 75:267. Evans, D.A., Sharp, W.R., and Medina-Filho, A.P. 1984. Somaclonal and gametoclonal variation. American Journal of Botany 71:759–774.
Fay, M.F. and Krauss, S.L. 2003. Orchid conservation genetics in the molecular age. In: Dixon, K.W., Kell, S.P., Barrett, R.L., Cribb, P.J. eds. Orchid Conservation. Natural History Publications, Sabah, Malaysia, pp. 91–112.
Ferrão, J.E.M. 1992. A Aventura das Plantas e os Descobrimentos Portugueses. Comissão Nacional para a Comemoração dos Descobrimentos Portugueses, Lisboa, Portugal. Geetha, S.P., Nirmal Babu, K., Rema, J., Ravindran, P.N., and Peter, K.V. 2000. Isolation of protoplasts from cardamom (Elettaria cardamomum Maton.) and ginger (Zingiber officinale Rosc.). Journal of Spices and Aromatic Crops 9 (1):23–30.