EFFECT OF NURSE CULTURE AND CONDITIONED MEDIUM ON COLONY FORMATION AND PLANT-REGENERATION FROM LOLIUM-PERENNE PROTOPLASTS

An efficient nurse culture system was established for perennial ryegra ss protoplast culture which allowed up to 59 000 protoplast-derived gr een plants to be regenerated from 1 mi of sedimented suspension cells. Nurse cultures improved plating efficiency, vitality of the protoplas t-derived colonies and the frequency of green plants (number of green plants/100 plants), while regeneration frequency (number of plants/100 colonies) was determined mainly by the protoplast donor suspension. C onditioned medium taken from the embryogenic protoplast-donor suspensi on (self-conditioning) increased the plating efficiency and frequency of green plants, while conditioned media harvested from 6 other suspen sions on average had a negative effect on colony formation and colony vitality. Concentrated self-conditioned medium resulted in a higher pl ating efficiency than self-conditioned medium diluted 1:1 with protopl ast culture medium, Nurse culture was more efficient than conditioned medium at normal protoplast density and was also superior for compensa ting suboptimal protoplast density. There is evidence that the conditi oning effect was reduced to 50% when the conditioned medium was stored for 2 days, suggesting involvement of unstable chemical factors and t hat the superiority of nurse cultures may be due to a continuous suppl y of conditioning compounds.