Callus induction and plant regeneration of Iris dichotoma Pall. in endangered species

Iris dichotoma Pall. is an important endangered plant belonging to the family Iridaceae. A method was developed for the rapid micropropagation of I. dichotoma through plant regeneration from leaf, rhizome, and root explant-derived calli. Leaf, rhizome, and root segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0-3.0 mgㆍL-¹) for callus induction. Callus production was highest at 1.0 mgㆍL-¹ 2,4-D, where 73.8% and 45.5% of cultured rhizome and root cuttings, respectively, produced calli. The viable calli were maintained at an induced concentration of 2,4-D (3.0 mgㆍL-¹). They were then transferred to MS medium supplemented with various concentrations of 2,4-D (0-3.0 mgㆍL-¹) in combination with 6-benzyladenine (BA: 0, 1.0 and 3.0 mgㆍL-¹) for adventitious shoot regeneration. The addition of a low concentration of 2,4-D into BA-containing medium significantly increased the frequency of shoot regeneration in leaf, rhizome, and root-derived calli. The highest number of adventitious shoots (26.4 per callus) formed at 0.5 mgㆍL-¹ 2,4-D and 1.0 mg/l BA. For rooting of the shoots, half- strength MS medium supplemented with different concentrations of indole 3-butyric acid (IBA) 0-3.0 mgㆍL-¹ was tested. The optimal results were observed using half-strength MS medium supplemented with 1.0 mgㆍL-¹ IBA, on which 98% of the regenerated shoots developed roots with an average of 3.5 roots per shoot within 45 days. The plantlets raised in vitro were acclimatized and transferred to soil with 95% success. This in vitro propagation protocol will be useful for conservation and mass propagation of this endangered plant.