Repetitive somatic embryogenesis and plant recovery in white clover

Breeding and selection was used to generate a population of white clover (Trifolium repens L.) from cultivar Osceola with a high embryogenic capacity. Somatic embryos were obtained from immature cotyledons of white clover placed onto EC6 basal medium containing 40 mg L(-1) of 2,4-D and 6% sucrose. The effects of 2,4-D at 20 and 40 mg L(-1) and of the carbohydrates, sucrose and maltose, were evaluated for their influence in the establishment of repetitive somatic embryogenesis. To determine the optimal protocol for plant recovery from somatic embryos, the effects of MS vs. EC6 basal salts, sucrose vs. maltose, B5 vitamins vs. yeast extract, and inclusion or exclusion of activated charcoal were evaluated. Repeated subculture of white clover somatic embryos on EC6 basal medium containing 6% sucrose with 2,4-D at 20 or 40 mg L(-1) effectively maintains repetitive embryogenesis. Medium containing MS salts with 6% maltose as the carbohydrate source was the most efficient for plant recovery.