Asymbiotic germination of Vanilla planifolia in relation to the timing of seed collection and seed pretreatments

BACKGROUND: Vanilla planifolia is an important tropical orchid for production of natural vanilla flavor. Traditionally, V. planifolia is propagated by stem cuttings, which produces identical genotype that are sensitive to virulent pathogens. However, propagation with seed germination of V. planifolia is intricate and unstable because the seed coat is extremely hard with strong hydrophobic nature. A better understanding of seed development, especially the formation of impermeable seed coat would provide insights into seed propagation and conservation of genetic resources of Vanilla. RESULTS: We found that soaking mature seeds in 4% sodium hypochlorite solution from 75 to 90 min significantly increased germination. For the culture of immature seeds, the seed collection at 45 days after pollination (DAP) had the highest germination percentage. We then investigated the anatomical features during seed development that associated with the effect of seed pretreatment on raising seed germination percentage. The 45-DAP immature seeds have developed globular embryos and the thickened non-lignified cell wall at the outermost layer of the outer seed coat. Seeds at 60 DAP and subsequent stages germinated poorly. As the seed approached maturity, the cell wall of the outermost layer of the outer seed coat became lignified and finally compressed into a thick envelope at maturity. On toluidine blue O staining, the wall of outer seed coat stained greenish blue, indicating the presence of phenolic compounds. As well, on Nile red staining, a cuticular substance was detected in the surface wall of the embryo proper and the innermost wall of the inner seed coat. CONCLUSION: We report a reliable protocol for seed pretreatment of mature seeds and for immature seeds culture based on a defined time schedule of V. plantifolia seed development. The window for successful germination of culturing immature seed was short. The quick accumulation of lignin, phenolics and/or phytomelanins in the seed coat may seriously inhibit seed germination after 45 DAP. As seeds matured, the thickened and lignified seed coat formed an impermeable envelope surrounding the embryo, which may play an important role in inducing dormancy. Further studies covering different maturity of green capsules are required to understand the optimal seed maturity and germination of seeds.