Theor Appl Genet (1996) 93:183-189 Springer-Verlag 1996 E. Nenz F. Pupilli F. Damiani S. Arcioni Somatic hybrid plants between the forage legumes Medicago sativa L. and Medicago arborea L. Received: 15 August 1995 / Accepted: 22 December 1995 Abstract Interspecific somatic hybrid plants were ob- tained by symmetrical electrofusion of mesophyll proto- plasts of Medicago sativa with callus protoplasts of Med- icago arborea. Somatic hybrid calli were picked manually from semi-solid culture medium after they were identified by their dual color in fluorescent light. Twelve putative hy- brid calli were selected and one of them regenerated plants. The morphogenesis of the somatic hybrid calli was induced by the synthetic growth regulator 1,2 benzisoxazole-3- acetic acid. Somatic hybrid plants showed intensive ge- nome rearrangements, as evidenced by isozyme and RFLP analysis. The morphology of somatic hybrid plants was in general intermediate between the parents. The production of hybrids by protoplast fusion between sexually incom- patible Medicago species is related to the in vitro respon- siveness of the parental protoplasts. The possibility of us- ing somatic hybrid plants in alfalfa breeding is discussed. Key words M. sativa M. arborea Protoplast electro- fusion - Somatic hybrids Genome rearrangements Introduction Medicago sativa (2n=4x=32; alfalfa, lucerne) is the world's most important forage crop (Michaud et al. 1988) and, as a perennial growing in different environments, its forage production is affected by biotic and abiotic stresses. Between annuals and perennials (Quiros and Bauchan 1988) the genus Medicago comprises more than 60 differ- ent species, which contain most of the genetic resources necessary to overcome the stresses faced by cultivated alfalfa (Isaac 1957; McEvans 1966; Mariani et al. 1978). Communicated by R Salamini E. Nenz (~) R Pupilli E Damiani S. Arcioni Istituto di Ricerche sut Miglioramento Genetico delle Piante Foraggere del C.N.R., Via della Madonna Alta 130, 06128 Perugia, Italy However, the possibility of transferring useful traits into M. sativa by sexual crosses is restricted to strains with the same ploidy level (Mariani 1968) and belonging to the M. sativa complex; consequently, the utilization of wild Medicago species as germ-plasm sources for the genetic improvement of alfalfa requires the overcoming of sexual barriers through somatic hybridization. This method could also be used for exploiting the production of tetraploid plants by somatic fusion of two unrelated, highly hetero- zygous and genetically improved diploid plants (Dumbier and Bingham 1975). In any cultivar of M. sativa it is pos- sible to find genotypes able to regenerate from different plant organs (Arcioni et al. 1990) and a number of cases of protoplast fusion with other Medicago species have been reported (Arcioni et al. 1994). But hybrid plants able to reach maturity have only been obtained with the combina- tions M. sativa+M.falcata (2n=4x=32) (T6ou16 1983; Mendis et al. 1991) andM. sativa+M, coerulea (2n=2x=16) (Pupilli et al. 1992); in other words, between species of the same Medicago sativa-falcata-coerulea complex (Quiros and Bauchan 1988) which, though having different ploidy levels, can be made sexually compatible by the use of nn- reduced gametes (Bingham 1979). The present paper re- ports somatic hybridization between M. sativa and M, ar- borea (2n=4x=32), two sexually incompatible and phylo- genetically distant species. M. arborea is the oldest spe- cies of the genus Medicago and the only one with a woody growth habit. It is unlikely to have been involved in the origin of the present herbaceous Medicagos (Lesins and Lesins 1979). The main features of agronomic interest in M. arborea are drought resistance and the absence of sum- mer and winter dormancy. It is used for pasture (Corleto et al. 1980) in dry lands, like the Mediterranean countries, where it can ensure an appreciable forage production in winter and in summer when the perennial Medicago spe- cies are dormant and the annual ones have already com- pleted their biological cycle. In addition, M. arborea is re- sistant to bacterial wilt (Co~nebacterium insidiosum; Renfro and Sprangue 1959) and to both races of anthrach- nose (Elgin and Ostazeky 1982). Up to now the useful traits present in the wild Medicago species, and suitable for the