A reproducible procedure for plant regeneration from seedling hypocotyl protoplasts of Vigna sublobata L.

Viable protoplasts of Vigna sublobata L. were isolated enzymatically from hypocotyls of axenic seedlings. Protoplast yields were dependent upon seedling age, with maximum yields (2.25 ± 0.35 × 10(6) g fwt(-1)) from seedlings aged 6 d. Protoplasts regenerated cell walls and underwent sustained divisions when cultured in either agarose-solidified or liquid K8P medium. The plating density affected the division frequency and plating efficiency; the division frequency (68 ±0 6.0%) was maximum at 4.0 × 10(4) ml(-1) while plating efficiency was maximum (1.3 ± 0.1%) at 5.0 × 10(4) ml(-1). Dividing protoplasts developed into microcalli, which produced glossy green compact nodular calli on transfer to 8.0 gl(-1) w/v agar-solidified medium containing MS salts, B5 organic components, 30 g l(-1) sucrose, NAA (0.2-0.5 mg l(-1)), zeatin riboside (0.5-2.0 mg l(-1)) and GA3 (0.5-1.0 mg l(-1)). These calli, after sub-culture on the same medium, produced shoot buds which underwent elongation following transfer of tissues to 6.0 g l(-1) agar-solidified B5 medium containing 30g l(-1) sucrose, IBA (0.01 mg l(-1)) and BAP (1.0 mg l(-1)). Elongated shoots developed roots after transfer to 8.0g l(-1) agar-solidified, hormone-free MS medium with 30 g l(-1) sucrose.