Development of procedures towards the somatic hybridization of alfalfa and Medicago marina L.

Abstract

Protoplasts were isolated from mesophyll tissue, callus and seedling cotyledons of Medicago sativa L. cv. Regen S and the halophyte M. marina L. Cotyledon protoplasts of Regen S were cultured in protoplast and cell culture media used previously for alfalfa protoplast culture and in media that had been simplified. There were no differences in the plating efficiencies of protoplasts cultured in the simple and complex media, but cells produced in the latter were greener and they colonized sooner. Protoplasts of M. marina grew at one-half the rate of Regen S protoplasts. Etiolated cotyledon protoplasts of Regen S were fused at 31°C using a solution containing PEG, DMSO and calcium at high pH. The frequency of fusion was 16% of the surviving protoplasts. These methods for protoplast isolation, culture and fusion should be useful in the somatic hybridization of alfalfa and M. marina.