Root hair protoplasts ofLotus corniculatus L. (birdsfoot trefoil) express their totipotency

Treatment of the roots of 24–48 h old seedlings of the forage legumeLotus corniculatus with 1.0% Cellulase YC, and 0.1% Pectolyase Y-23 in 4.2% mannitol solution released protoplasts from the tips of root hairs within 30–40 sec of enzyme incubation. Roots from approximately 1000 seedlings yielded 1.7×10⁵ protoplasts. Ten percent of protoplasts divided to form cell colonies when cultured at 1.0×10⁵ ml^–1 in droplets of KM8P medium with 0.6% Sea Plaque agarose. Colonies formed callus on UM agar medium; protoplast-derived tissues produced shoots on B5 medium containing 0.05 mg 1^–1 of BAP. Regenerated plants were phenotypically and cytologically normal (2n=2x=24±2), and produced nitrogen fixing root nodules following inoculation withRhizobium. These results confirm the totipotency of protoplasts isolated from specialised epidermal cells of seedling roots ofLotus corniculatus.