Isolation and fusion of protoplasts in Vanilla species

Abstract

Vanilla, an important flavouring material and spice, is the fermented and cured fruit of the orchid, Vanilla planifolia Andrews (syn. V. fragrans Salisb.). Origin of much of the planting material of vanilla from limited clonal source, coupled with the threat of destruction of its natural habitats, leads to a narrow genetic base being the major bottleneck in crop improvement programmes. Isolation and fusion of protoplasts was attempted to produce hybrids with desirable traits. Protoplasts were successfully isolated from V. planifolia and V. andamanica when incubated in an enzyme solution containing macerozyme R-IO (0.5%) and cellulase Onozuka R-IO (2%) for 8 h at 30°C in dark, with good yield and viability. PEG-mediated protoplast fusion between V. andamanica and V. planifolia was successful and the fusion product (heterokaryon) could easily be identified because the protoplasts of the two species differed in size and arrangement of chlorophyll. This can be helpful in gene transfer for helpful traits, especially the natural seed set and disease tolerance observed in V. andamanica.