Electrofusion of Protoplasts and Heterokaryon Survival in the Genus Medicago

To develop a system of clectrofusion for the genus Medicago, protoplast yields from 2 genotypes of M, sativa and 7 wild type1 accessions were determined. Protoplast extraction protocols from mesophyll cells, from suspension culture and from calls were evaluated. An alternating current of 10 volts an a frequency of 150 mhz was applied to tht1 protoplasts Ho align them in a pearl chain formation. A direct current pulse of 150 volts applied for 0.25 usec gave the highest number of protoplast fusions for material from mesophyll cells and suspension culture for both M. sativa and wild species. Over 20% of the protoplasts which occupied places in the “pearl chains” yielded fusion products. An average of 25% of the protoplasts treated with an electrical fusion pulse survived, started, to regenerate cell walls and to divide after a 12 days interval. This system can be used to produce new hybrids which, previously, could not be made in the genus Medicago.