PLANT-REGENERATION FROM PROTOPLASTS OF EU STOMA-GRANDIFLORUM (GRISEB)SCHINNERS

Optimal conditions to regenerate plants from Eustoma grandiflorum 'Bic olor Purple' protoplasts investigated in this study led to an efficien t plant regeneration methood. Protoplasts were isolated enzymatically from young leaves of Eustoma grandiflorum seedlings cultured aseptical ly on MS medium for 50 days. The enzyme solution contains 2.0% Cellula se Onozuka R10, 0.2% Macerozyme R10, 0.1% CaCl2 . 2H(2)O and 9.0% mann itol (pH 5.5). Isolated protoplasts were cultured in a modified MS (20 0 mg . liter(-1) NH4NO3) medium containing 1.0% sucrose, 9.0% mannitol , 1.0 mg . liter(-1) NAA and 0.1 mg . liter(-1) BA at the density of 1 similar to 2x10(4) . ml(-1). Cell division occurred after 4 similar t o 5 days in culture. A fresh medium was added weekly to promote colony formation. Colonies (0.5 similar to 1.0 mm in diameter) were transfer red to a modified MS agar medium containing 0.1 mg . liter(-1) BA and 1.0 mg . liter(-1) NAA for callus proliferation. Shoots regenerated ra pidly upon transferring the calli to MS medium containing 0.3 mg . lit er(-1) BA. The regenerated shoots were propagated on MS agar medium co ntaining 0.1 mg . liter(-1) BA and 1.0 mg . liter(-1) GA(3). Hyperhydr ation of shoots was prevented by ventilating the culture bottles throu gh PTFE membrane (18 mm diam.) attached to the cap. Shoots which elong ated more than 1 cm were treated with Oxyberon powder (0.5% IBA) and t hen transplanted to rock wool cubes. The IBA-treated shoots formed the roots and grew into normal plants. But the percentage of bicolor flow ers in protoplast-derived Eustoma grandiflorum plants was significantl y lower than the usual means of obtaining plants from seeds.