Interspecific hybridization of perennial Medicago species using ovule-embryo culture

New interspecific hybrids between alfalfa (Medicago sativa L.) and several perennial Medicago species were obtained by embryo rescue techniques. The methodology, designated ovule-embryo culture, involved preculturing the fertilized ovule (10 to 20 days post-pollination) for a period of six to 12 days followed by excision and direct culture of the embryo. Placement of the hybrid embryo directly onto culture medium without the interim ovule culture was unsuccessful. Ovule culture to germination without removing the embryo also was unsuccessful. Ovule-embryo culture was essential for recovering interspecific hybrids between diploid alfalfa (2n=2x=16) and the following diploid (2n=2x=16) species: M. hybrida Traut., M. marina L., M. papillosa Boiss., M. rhodopea Velen. and M. rupestris M.B. In addition, trispecies hybrids between M. sativa x M. dzhawakhetica Bordz. F1 hybrids (2n=3x=24) and either M. cancellata M.B. (2n=6x=48) or M. saxatilis M.B. (2n=6x=48) were obtained from ovuleembryo culture. Media manipulations using M. sativa x M. rupestris F1 and first backcross generation embryos demonstrated the optimum concentration of 12.5 mM NH4 (+) for successful embryo rescue; ammonium salt formulation (whether chloride, nitrate or sulfate) was not critical. From a few thousand crosses, hybrids between M. sativa and either M. rhodopea or M. rupestris were recovered relatively efficiently with 157 and 66 hybrids, respectively. However, only 13 hybrids between M. sativa and M. papillosa were obtained from more than 2,000 crosses, and just two hybrids each have been recovered from the combinations M. sativa x M. hybrida and M. sativa x M. marina from 2,000 to 3,000 crosses. The predominant chromosome number between diploid alfalfa and the other diploid perennial species was 2n=2x=16. Morphology of the hybrids was generally intermediate. Electrophoretic analysis of the F1 hybrids and parental clones on uniform or gradient polyacrylamide gels demonstrated that peroxidase phenotypes could be used to confirm hybridity. For all interspecific combinations there was at least one peroxidase isozyme unique to the wild species that was present in the F1 interspecific hybrid.