Isolation, callus formation and plantlet regeneration from mesophyll protoplasts of Tylophora indica (Burm. f.) Merrill: an important medicinal plant

Protoplast culture and plant regeneration of an important medicinal plant Tylophora indica were achieved through callus regeneration. Protoplasts were isolated from leaf mesophyll cells and cultured at a density of 5 x 10(5) protoplasts per gram fresh weight, which is required for the highest frequency of protoplast division (33.7%) and plating efficiency (9.3%). The first division was observed 2 d after plating and the second division after 4 d. Culture medium consists of Murashige and Skoog (MS) liquid medium with 4 mu M 2,4-D, 0.4 M mannitol and 3% (w/v) sucrose with pH adjusted to 5.8. After 45 d of culture at 25A degrees C in the dark, protoplasts formed colonies consisting of about 100 cells. The protoplast-derived microcalli were visible to the naked eye within 60 d of culture and reached a size of 0.2-0.4 mm in diameter after 90 d. Calli of 0.2-0.4-mm size were transferred to MS medium supplemented with 2,4-D (4 A mu M), 3% (w/v) sucrose and 0.8% (w/v) agar, formed friable organogenic calli (7-8 mm size) after 8 wk under incubation in normal light period supplemented with 200 A mu mol m(-2) S-1 of day light fluorescent illumination. The calli were transferred to MS medium supplemented with thidiazuron (TDZ) (1-7 mu M) and naphthalene acetic acid (NAA) (0.2-0.4 mu M) for regeneration. The calli developed shoot buds after 3-4 wk, and the frequencies of calli-forming shoots varied from 5% to 44%. Optimum shoot regeneration occurred on MS medium supplemented with 5 mu M TDZ and 0.4 mu M NAA. On this medium, 44% cultures responded with an average number of 12 shoots per callus. Whole plants were recovered following rooting of shoots in 1/2 MS medium supplemented with 3 mu M indole 3-butyric acid.