Abstract
Protoplast fusion was investigated as a means of obtaining somatic hybrids between alfalfa, Medicago sativa L., and two sexually incompatible annual species, M. intertexta L. (Mill.) and M. scutellata L. (Mill.). A selection scheme was developed using genetically stable transgenic fusion partners, each carrying a different antibiotic resistance gene. A kanamycin-resistant transgenic alfalfa plant (RS-K2A5051a) that was phenotypically and karyotypically normal was used as a source of mesophyll protoplasts. Because M. intertexta and M. scutellata do not regenerate to plants from culture, A. rhizogenes was used to produce root cultures of each that were resistant to hygromycin B. The root cultures exhibited chromosome stability and were used for protoplast isolation. Double antibiotic selection for somatic hybrid callus was completed 3–5 weeks after polyethylene glycol fusion. Southern blot analysis with a ribosomal DNA probe confirmed that the selected calli were nuclear hybrids. The regenerability of alfalfa from culture was expressed only in the alfalfa (X) M. intertexta hybrid calli, where embryos and several plantlets were obtained.
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