Isolation,callus formation and plantlet regeneration from mesophyll protoplasts of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. f.) Merrill: an important medicinal plant |
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Authors: | T Dennis Thomas |
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Institution: | (1) Postgraduate and Research Department of Botany, St. Thomas College, Arunapuram (P.O.), Pala, 686 574, Kottayam (Dt.), Kerala, India |
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Abstract: | 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 × 105 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 μM 2,4-D, 0.4 M mannitol and 3% (w/v) sucrose with pH adjusted to 5.8. After 45 d of culture at 25°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 μM), 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 μmol m−2 S−1 of day light fluorescent illumination. The calli were transferred to MS medium supplemented with thidiazuron (TDZ) (1–7 μM)
and naphthalene acetic acid (NAA) (0.2–0.4 μM) 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 μM TDZ
and 0.4 μM 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 μM indole 3-butyric acid. |
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