Somatic hybridization between M. sativa and annual Medicago.

Yuko Mizukami*, T. Takamizo**, M. Kato*, S. Inami*, M. Kanbe* and I. Houmura*

* Aichi-Pref. Agri. Res. Ctr., Nagakute, Aichi 480-1193, Japan

** MAFF. Research Secretariat, Chiyoda, Tokyo 100-5850, Japan

Some annual Medicago Species have been reported to be the source of useful agronomic traits, for examples resistance to pests and/or diseases. Though they are sexually incompatible with alfalfa, somatic hybridization can produce symmetric and asymmetric hybrids among them. Three hybridizing combinations were examined, M. sativa (2n=32, cv. Rangelander) and M. scutellata(2n=32, cv.Robinson)/ M. rugosa (2n=32, cv.Paragosa)/ M. polymorpha(2n=16, Japanese ecotype). Isolated protoplasts from embryogenic calli of M. sativa and cotyledons of annual Medicago were fused using electrofusion setup (SSH-10,Shimadzu,Japan). Selection of the fused hybrid protoplasts was carried out by means of IOA treatment to M. sativa. After fusion, protoplasts were cultured on KM8P medium using agarose beads method with nurse cells. Individual colonies were proliferated on UM medium and regenerated on half strength SH medium. Regenerated plants were grown in a green house.

More than 9 putative hybrids were obtained from each hybridizing combination. They are all perennial type and their plant morphology was similar to M. sativa, although differentiation were observed among hybrids and hybridizing combinations. Their chromosome number were ranged from 43 to 56. From the results of Southern blotting analysis by ribosomal DNA (rDNA), all putative hybrids had both the specific bands of M. sativa and those of annual Medicago. Using mitochondorial DNA (mtDNA), a few hybrids had the both bands of parental species and others had those of M. sativa only. In case of chloroplast DNA (cpDNA), the specific bands of annual Medicago were not observed in all hybrids. Therefore, obtained plants through electric fusion were asymmetric somatic hybrids and their organelle composition were the same as a parental form or additive pattern of them. Most hybrids had very low pollen fertility. There were also abnormal morphology of flower.

During four years cultivation, clonal propagation by stem cuttings were carried out. As years passed, their chromosome were deleted and those number became to 32. Some hybrids recovered their fertility. RAPD and RFLP analysis revealed that the hybridity were lost in most of hybrids. It suggested that chimeric or phenotypic unstable plants were caused. It might be given from the chromosome variations ; e. g. deletion, translocation between parental chromosomes and so on.

Further analysis about the chromosomal composition, relative genomic contribution of parents and genomic stability will be needed to stable somatic hybrids/cybrids. In this experiment, only a few hybrids still have kept their hybridity and set some seeds. The analysis using these progeny will be helpful to elucidate those probrem.