Micropropagation of Embelia ribes Burm f. through proliferation of adult plant axillary shoots

Micropropagation of Embelia ribes was achieved through proliferation of axillary shoots obtained from mature plants. Nodal shoot segments, collected March-May, exhibited high-frequency (75%) shoot initiation when cultured on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ) at 1.13 µM and indole-3-butyric acid (IBA) at 0.49 µM. Subculture of sprouted shoots from the original explants on medium containing TDZ (1.13 and 0.45 µM) during the first and second subcultures was found essential for further shoot proliferation, while inhibition of shoot elongation by TDZ could be overcome by transferring shoot cultures onto MS medium containing 6-benzylaminopurine (BAP; 11.10 µM) for the third subculture. Treating the explants with an antioxidant mixture of 568 µM ascorbic acid, 119 µM citric acid, and 307 µM glutathione prior to inoculation, coupled with subculture at 2-wk intervals onto fresh medium, both helped to reduce browning of the explants and facilitated production of five to six shoots/explant. MS medium supplemented with BAP (4.44 µM) and IBA (0.49 µM) induced shoot multiplication, producing five to six shoots/explant with a shoot length of 3 to 4 cm over a 4-wk culture period. Shoots of 3 to 4 cm in length exhibited 100% rooting within 4 wk after transfer to media containing half the nutrient salt concentration of MS medium with 3.69 µM IBA. Ex vitro rooting in the greenhouse from the in vitro shoots treated with 4.93 µM IBA for 30 min exhibited 95% rooting in soilrite™ medium in a 4-wk period. About 85% of micropropagated plants were established successfully in root trainers. Three-month-old, hardened plants could further be successfully established in the field. In 1 yr, by using the above protocol, 3,200 plants could be produced from a single shoot and 2,700 could be established in the field.