Improved shoot regeneration from nodules of Charybdis numidica in a temporary immersion system

A procedure for improved shoot regeneration from meristematic nodules of Charybdis numidica in a-temporary immersion culture system was developed by optimizing immersion frequencies, volume of the nutrient medium, and alternating application of growth regulators. Modified liquid MS medium with 3% sucrose, 20 muM BAP and 5 muM NAA (shoot induction medium) was used to induce microshoot formation on 15 g of nodules in 1000 ml bottles. Volumes of medium (250 or 500 ml) and immersion frequency (5 min every 12 or 24 h) did not significantly influence shoot regeneration rates. Shoot induction medium additionally supplemented with 5 muM paclobutrazol in most cases led to less shoots but this effect was not significant, either. Microshoots formed under these conditions were severely hyperhydrated. Nearly complete elimination of hyperhydricity and enhanced formation of properly elongated shoots were achieved by running a shoot induction step with induction medium containing paclobutrazol followed by an elongation step with a medium supplemented only with 5 muM gibberellic acid GA(3). This two-step procedure yielded about 900 healthy shoots per bottle after a two-month cultivation period. Root induction was performed ex vitro during acclimatization and the plantlets could be established in the greenhouse with good success.