Protoplast Isolation and Culture of Aquatic Plant Cryptocoryne wendtii De Wit

Abstract

The optimum conditions for protoplast isolation and culture of Cryptocoryne wendtii De Wit were investigated. Protoplasts were successfully isolated from in vitro four-week-old leaves using an enzyme mixture comprising 2% Cellulase Onozuka R-10, 0.2% Pectolyase Y-23, 0.5 M mannitol, 2.5 mM CaCl₂.2H₂O and 5 mM 2 (N-morpholino)-ethanesulfonic acid (MES), pH 5.6. Approximately 1.04±0.06 × 10⁷ protoplasts per gram fresh weight with 90.79±4.80% viability were obtained after incubating in enzyme solution for 4 hours in the dark and purified with 16 % sucrose gradient centrifugation. Protoplasts were cultured on modified MS medium supplemented with 0.2 mg/l 2,4-dicholorophenoxyacetic acid (2,4-D), 1 mg/l α-naphthalene acetic acid (NAA), 0.5 mg/l zeatin, 0.15 M sucrose and 0.3 M mannitol by agarose-bead with thin layer liquid culture. The protoplasts regenerated cell walls within 24 hours. First cell division was observed after culturing for 2-3 days, and microcolonies were formed within 4 weeks. Enzyme mixture, osmotic solution, incubation time, age of leaves, and sucrose solution concentration were found to influence both yield and viability of protoplasts. Culture media, plant growth regulators and method of culture affected protoplast division.