Protoplast Fusion in the Genus Medicago and Isoenzyme Analysis of Parental and Somatic Hybrid Cell Lines

Leaf mesophyll protoplasts of Medicago arborea (2n = 32) were electrofused with cell suspension and/or callus protoplasts of Medicago sativa. Heterokaryons were identified in agarose beds by their dual fluorescein isothiocianate—chlorophyll fluorescence and their coordinates were recorded. Hybrid minicalli were manually picked up and grown first in nurse culture and then in callus induction medium. Hybrid nature of the selected calli was confirmed by isoenzyme analyses. In order to verify whether morphogenesis of somatic hybrid calli was affected by cell incompatibility, mesophyll and cell suspension protoplasts, derived from the same plant of M. sativa with high embryogenic capacity, were fused. Only callus tissues derived from mesophyll protoplasts retained the highly embryogenic character of the M. sativa genotype, while hybrid cell lines were non-morphogenic and showed isoenzyme patterns similar to tissues derived from cell suspension protoplasts. The achievement of somatic hybrid plants in the genus Medicago is discussed.