TISSUE AND PROTOPLAST CULTURE OF RICE BEAN [VIGNA-UMBELLATA (THUNB) OHWI AND OHASHI]

Plant regeneration by organogenesis was obtained from cultivar CUR 8505 of rice bean. Green nodular callus was initiated when epicotyl segments were cultured on 0.8% w/v agar-solidified medium containing MS salts, B5 organic components, 3.0% w/v sucrose and supplemented with NAA (0.1-0.5 mg l-1), BAP (0.5 mg l-1), kinetin (0.5 mg l-1) and zeatin (0.5-1.0 mg l-1). Shoot buds developed after 2-3 sub-cultures on this medium, each of 20-25 days' duration. Shoots elongated when the callus was cultured on 0.6% w/v agar solidified B5 medium with 3.0% w/v sucrose, IBA (0.005 mg l-1) and BAP (0.2 mg l-1). Elongated shoots developed root systems following transfer to 0.8% w/v agar solidified hormone-free MS medium with 3.0% w/v sucrose. Viable protoplasts were isolated from epicotyls of axenic seedlings using an enzyme mixture consisting of Rhozyme HP150 (0.4% w/v), Cellulase R10 (0.2% w/v) and Pectolyase Y23 (0.1% w/v) dissolved in a solution containing CPW salts and 9.0% w/v mannitol. Protoplast yields were dependent upon seedling age, with maximum yields being obtained from those aged five days. Protoplasts regenerated cell walls and underwent sustained division when cultured in agarose-solidified KM8P medium (modified after Kao and Michayluk). The plating density affected protoplast division frequencies and plating efficiencies, both being optimal at an initial density of 2.0 x 10(4) ml-1. Dividing protoplasts developed into microcalli, which produced compact, nodular green calli. These results are discussed in relation to the genetic improvement of this pulse crop.