Plant regeneration from protoplasts of Capsicum annuum L. cv. California Wonder.

Plant regeneration from mesophyll protoplasts of pepper, Capsicum annuum L. cv. California Wonder has been demonstrated via shoot organogenesis. Protoplasts isolated from fully expanded leaves of 3-week-old axenic shoots when cultured in TM medium supplemented with 1 mg l-1 NAA, 1 mg l-12,4-D, 0 5 mg l-1 BAP (CM 1) resulted in divisions with a frequency ranging from 20-25 %. Antioxidant ascorbic acid and polyvinylpyrrolidone (PVP) in the medium and incubation in the dark helped overcome browning of protoplasts. Microcalli and macrocalli were formed in TM medium containing 2 mg l-1 NAA and 0·5 mg l-1 BAP (CM Π) and MS gelled medium containing 2 mg 1-1 NAA and 0 5 mg 1-1 BAP (CM III), respectively. Regeneration of plantlets was possible via caulogenesis. Microshoots, 2-5 percallus appeared on MS gelled medium enriched with 0·5 mg l-1 IAA, 2mg l-1 GA and l0mg l-1 BAP (CM IVc). Rooting of microshoots was obtained on half strength gelled medium containing 1 mg l-1 NAA and 0·5mg l-1 BAP. Protoplasts isolated from cotyledons failed to divide and degenerated eventually.

A simple method for the isolation of plant protoplasts.

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described· The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplastreleasing 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 2H2O), all dissolved in distilled water· The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division· Plant regeneration was demonstrated for Nicotiana tabacum cv· Thompson from protoplasts isolated by this method· Wall 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 protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.