Effect of nurse culture and conditioned medium on colony formation and plant regeneration from Lolium perenne protoplasts

Abstract

An efficient nurse culture system was established for perennial ryegrass protoplast culture which allowed up to 59 000 protoplast-derived green plants to be regenerated from 1 ml of sedimented suspension cells. Nurse cultures improved plating efficiency, vitality of the protoplast-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. Conditioned medium taken from the embryogenic protoplast-donor suspension (self-conditioning) increased the plating efficiency and frequency of green plants, while conditioned media harvested from 6 other suspensions on average had a negative effect on colony formation and colony vitality. Concentrated self-conditioned medium resulted in a higher plating efficiency than self-conditioned medium diluted 1:1 with protoplast culture medium. Nurse culture was more efficient than conditioned medium at normal protoplast density and was also superior for compensating suboptimal protoplast density. There is evidence that the conditioning effect was reduced to 50% when the conditioned medium was stored for 2 days, suggesting involvement of unstable chemical factors and that the superiority of nurse cultures may be due to a continuous supply of conditioning compounds.