Effect of Urea-Type Cytokinins on the Adventitious Shoots Regeneration from Cotyledonary Node Explant in the Common Ice Plant, Mesembryanthemum crystallinum

摘要

Mesembryanthemum crystallinum (common ice plant) was used as a model plant to study the regulatory properties of Crassulacean acid metabolism (CAM) and tolerance to abiotic stresses. Although transformation is a useful genetic approach, it has not been established in this species due to recalcitrancy for regeneration. To establish an efficient procedure for regeneration of M. crystallinum, we examined the effects of urea-type cytokinins, thidiazuron (TDZ) and forchlorofenuron (CPPU) on the adventitious shoot induction.Adventitious shoots were generated only from explants obtained from the cotyledonary node, not from explants obtained from the cotyledon, hypocotyl and roots. Urea-type cytokinins, TDZ and CPPU were more effective for the induction and the morphogenesis of adventitious shoots than adenine-type cytokinin, 6-benzyladenopurine (BA). We have found that the 2.5 mg L-1 TDZ induced the largest number of multiple shoots and the highest frequency of adventitious shoot induction from single explant. In addition, fewer hyperhydric shoots were produced on the medium containing TDZ than in that containing BA and CPPU in the presence of 1.0 mg L-1 NAA. The regenerated shoots rooted on the MS medium within one month, and the rooting was promoted by replacing the agar medium with vermiculite or Florialite. The fertile plant with normal morphological properties was harvested for four months after sowing. Using the improved regeneration procedure with TDZ, we successfully introduced a kanamycin-resistant gene (nptII-HPH) into the cotyledonary node mediated by Agrobacterium tumefaciens. These results indicated that this regeneration procedure using cotyledonary node explants and TDZ could be useful for the genetic engineering of M. crystallinum.