An Efficient Procedure for Regeneration from Leaf-derived Calluses of Lonicera macranthoides 'Jincuilei', an Important Medicinal Plant

'Jincuilei' is a mutant selected from Lonicera macranthoides Hand.-Mazz. It produces abundant flowers that never open with a chlorogenic acid (CGA) content up to 6.0%. Propagation through rooting or grafting has only a 30% survival rate. This study was undertaken to establish an efficient protocol for rapidly regenerating this mutant. Leaf explants were inoculated on Gamborg's B 5 medium supplemented with different concentrations of 6-benzyladenine (BA) and 2,4-dichlorophenozyacetic acid (2,4-D). The optimal combination for callus induction was 4.4 μM BA with 2.26 μm 2,4-D, which resulted in 86.7% of leaf explants producing calluses in 4 weeks. Calluses produced from this optimal medium were cultured on B 5 medium containing different concentrations of kinetin (KT) and α-naphthalene acetic acid (NAA). The best formulation for shoot induction was B 5 medium containing 0.9 μM KT and 5.4 μM NAA in which 73.4% of cultured calluses produced shoots in 8 weeks, and shoot numbers ranged from three to six per callus piece (1 cm 3 ). Adventitious shoots were cut and rooted in half-strength Murashige and Skoog medium supplemented with 14.8 μM 3-indolebutyric acid. Roots initiated 10 d after culture, and rooting percentages ranged from 98% to 100%. Plantlets grown in a container substrate in a shaded greenhouse had over a 95% survival rate. During the last 6 years, over four million plantlets were regenerated using this established procedure, and there was no somaclonal variation. Fresh and dry weights of 1000 flowers, CGA contents, and dry flower yields of the regenerated plants were not significantly different from those of the stock 'Jincuilei' propagated by cutting, indicating that plants regenerated from this established procedure were stable. This established in vitro culture method has led to rapid commercial production of this medicinal plant on more than 1500 ha of production field.