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 1.7×10(5) protoplasts. Ten percent of protoplasts divided to form cell colonies when cultured at 1.0×10(5) 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.
Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, University Park, NG7 2RD, Nottingham, UK.
J H
JH
Rasheed
M K
MK
Al-Mallah
E C
EC
Cocking
M R
MR
Davey