Interspecific hybridization of Medicago sativa L. and M. rupestris M. B. using ovule–embryo culture

An ovule–embryo culture method was used to produce the first interspecific hybrids between alfalfa (Medicago sativa L.) and Medicago rupestris M. B. Culture of fertilized ovules from the cross diploid (2n = 2x = 16) M. sativa (jpjp) × diploid (2n = 2x = 16) M. rupestris began 14 days after pollination. After 5 days in culture, the interspecific hybrid embryo was removed and transferred to fresh medium, where development into a plant occurred. Forty-six M. sativa – M. rupestris F1 hybrids have been recovered using this technique. All but one of the 46 F1 hybrids were diploid (2n = 2x = 16); the only exception was tetraploid (2n = 4x = 32). The most frequent meiotic configurations observed in the F1 hybrid plants were eight bivalents or seven bivalents and two univalents, indicating significant homology between M. sativa and M. rupestris genomes. However, pollen stainability (0–12%) and pollen germination (0–6%) were extremely low. Similar to the production of the F1, no first backcross (BC1) plants were obtained from seed; however, the ovule–embryo culture method was found to be a very effective method for recovering BC1 plants and hundreds of BC1 plants have been produced. The BC1 plants from crossing the F1 with diploid M. sativa were predominantly diploid. Medicago rupestris can now be considered a potential germplasm source for alfalfa improvement. The ovule–embryo culture method represents the first successful recovery of Medicago interspecific hybrids via some form of embryo rescue. Importantly, it appears this technique can be applied to other interspecific hybrid combinations in the Medicago genus.Key words: Medicago, alfalfa, embryo culture, interspecific hybrid.