A SIMPLE METHOD FOR THE ISOLATION OF PLANT-PROTOPLASTS

A simple protoplast isolation protocol that was designed to recover to tipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevate d temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures, Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO4), and a protectant (CaCl2 2H(2)O), all dissolved in distilled water. The pr otocol has ensured reproducibility, higher yields and is gentle on pro toplasts as the protoplasts obtained were amenable to cell wall regene ration and cell division. Plant regeneration was demonstrated for Nico tiana tabacum cv. Thompson from protoplasts isolated by this method. W all regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non- requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protopla sts isolated and applicability of the protocol to a large number of sp ecies including mucilage-containing plants.