Asymmetric somatic hybridization between alfalfa (Medicago sativa L.) cultivar and CMS line

Abstract

In order to develop methods for the transfer of organelle genetic information in alfalfa (Medicago sativa L.) from one species to another, we investigated asymmetric somatic hybridization and succeeded in developing a stable method for cybrid callus formation. Protoplasts of a cultivar (Spredor II) were inactivated with iodoacetamide (IOA) and protoplasts of the cytoplasmic male sterile (CMS) line L-2905 were treated with X-rays. Both types of protoplasts were fused electrically. Hetero-fusion frequency could be increased by the increase of the ratio of CMS protoplasts to cultivar protoplasts in a fusion chamber. Inactivation of cultivar protoplast could be almost achieved by treatment with 6mM IOA when the agarose block method was used. A higher X-ray dose was needed to inactivate CMS protoplasts compared with other plants. Analysis of restriction fragment length polymorphism (RFLP) of mitochondrial DNAs (mtDNAs) of the parents using the Southern blot hybridization method revealed that the digestion of total DNA with Xho I and hybridization of atpA enabled to differentiate mtDNAs between the cultivar and CMS. Analysis of calli from asymmetric fusion product by using a combination of Xho I and atpA confirmed that hybrid type calli showing the bands corresponding to those of both parents could be obtained. Based on the investigations on the conditions of I0A concentration and X-ray dose, it was observed that treatment with 6 mM IOA was suitable for decreasing cultivar type escape callus formation. Isozyme analysis revealed that the malate dehydrogenase isozyme pattern of some of the hybrid type calli was different from that of CMS control, suggesting the presence of cybrid clones. Although mtDNA of regenerated plants was analyzed, RFLP pattern of all of them was the same as that of cultivar control.