Carbohydrate and osmotic requirements for high-frequency plant regeneration from protoplast-derived colonies of indica and japonica rice varieties

The effects were studied of various carbohydrates and osmotic stress, created by high agarose or carbohydrate concentrations, on the regeneration of fertile plants from protoplast-derived colonies of several indies (IR43, Jaya, Pusa Basmati 1) and japonica (Taipei 309) rice varieties. Observations of the cultures developed on media containing one of these carbohydrates (cellobiose, fructose, glucose, lactose, maltose, mannitol, sorbitol or sucrose), each at 88 mM, indicated that maltose was the preferential carbon source for the proliferation of embryogenic callus and shoot regeneration. Maltose-containing medium induced shoot formation in 24-66% of the protoplast-derived tissues, depending upon the rice variety, compared to shoot regeneration from 4-32% of the tissues in sucrose-supplemented medium. Media containing 288 mM maltose or an equimolar combination of 88 mM maltose and 200 mM mannitol, caused water loss from calli and promoted the growth of embryogenic calli. These calli formed shoots with greater frequencies when subsequently transferred to shoot regeneration medium with 88 mM maltose. A medium containing 88 mM maltose and semi-solidified with 1.0% (w/v) instead of 0.5% (w/v) agarose had a similar beneficial effect on the growth of embryogenic calli and simultaneously supported high-frequency (48-55%) shoot formation. The optimum shoot regeneration frequencies (60-78%) were obtained when protoplast-derived colonies were serially cultured on to shoot regeneration medium containing 1.0% (w/v) agarose for 4 weeks, followed by a 2-week culture period on the same medium with 0.5% (w/v) agarose. Plants regenerated on medium containing maltose and/or 1.0% (w/v) agarose were phenotypically normal and fertile.