Plant Regeneration from Protoplasts of Eustoma grandiflorum (Griseb.) Schinners

Abstract

Optimal conditions to regenerate plants from Eustoma grandiflorum 'Bicolor Purple' protoplasts investigated in this study led to an efficient plant regeneration methood.
Protoplasts were isolated enzymatically from young leaves of Eustoma grandiflorum seedlings cultured aseptically on MS medium for 50 days. The enzyme solution contains 2.0% Cellulase Onozuka R10, 0.2% Macerozyme R10, 0.1% CaCl₂•2H₂O and 9.0% mannitol (pH 5.5). Isolated protoplasts were cultured in a modified MS (200 mg•liter^-1 NH₄NO₃) medium containing 1.0% sucrose, 9.0% mannitol, 1.0 mg•liter^-1 NAA and 0.1mg•liter^-1 BA at the density of 12×10⁴•ml^-1.
Cell division occurred after 45 days in culture. A fresh medium was added weekly to promote colony formation. Colonies (0.51.0 mm in diameter) were transferred 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 rapidly upon transferring the calli to MS mediumcontaining 0.3 mg•liter^-1 BA.
The regenerated shoots were propagated on MS agar medium containing 0.1 mg•liter^-1 BA and 1.0 mg•liter^-1 GA₃. Hyperhydration of shoots was prevented by ventilating the culture bottles through PTFE membrane (18 mm diam.) attached to the cap. Shoots which elongated more than 1 cm were treated with Oxyberon powder (0.5% IBA) and then transplanted to rock wool cubes. The IBA-treated shoots formed the roots and grew into normal plants. But the percentage of bicolor flowers in protoplast-derived Eustoma grandiflorum plants was significantly lower than the usual means of obtaining plants fromseeds.