IN-VITRO REGENERATION OF COMMERCIAL CULTIVARS OF SUBTERRANEAN CLOVER

Regeneration of subterranean clover (Trifolium subterraneum L.) was ac hieved by both shoot organogenesis and somatic embryogenesis. Shoots d erived via organogenesis were initiated from the hypocotyls of mature imbibed seed. The hypocotyl, including the emerging radicle, was slice d longitudinally into two halves and cultured on shoot induction mediu m. After 30 days, adventitious shoots were formed from the hypocotyl r egion while the radicle showed no development. Shoots were then subcul tured onto shoot multiplication medium and finally onto a root initiat ion medium. Histological studies revealed that shoots arose de novo an d did not originate from pre-existing meristems. In the second regener ation protocol, shoot apical meristems from young seedlings were induc ed to form callus. Following four to six weeks culture in the dark, so matic embryos appeared spontaneously on the calli. A majority of embry os had a well-defined root pole, two cotyledonary lobes, and were capa ble of germination, albeit at a low frequency. Regenerated plants obta ined from both protocols appeared phenotypically normal.