Expression of a Chicken Ovalbumin Gene in Three Lucerne Cultivars

Routine procedures have been developed for the transformation of lucerne (Medicago sativa cv. Rangelander) with foreign genes using the Agrobacterium tumefaciens binary vector system and for the regeneration of transgenic plants from tissue culture, via somatic embryogenesis. Lucerne transformation was carried out with a gene encoding neomycin phosphotransferase (npt), which conferred resistance to the antibiotic kanamycin, together with a cDNA clone encoding chicken ovalbumin which was modified for expression in plant cells. The ovalbumin cDNA protein coding sequence was combined with the cauliflower mosaic virus 35S promoter and the nopaline synthase 3' flanking sequence to make a chimeric ovalbumin gene. A DNA construct containing both these genes was transferred to lucerne, and ovalbumin was detected in leaves of regenerated plants using protein immunoblots. Pulse-chase labelling experiments and analysis of leaves from the top to bottom of the transformed plants indicated that ovalbumin, once formed, was stable in the leaves of transgenic lucerne. A wide variation in ovalbumin level was frequently observed in plants regenerated from multiple embryos on a single transformed callus. This variation correlated with changes in the restriction enzyme digestion pattern of the ovalbumin DNA from the transgenic plants. These results indicate that each transformed callus may have arisen from more than one transformation event. An alternative interpretation is that the callus may have arisen from a single transformed cell but during cell proliferation the DNA in some cells may have undergone rearrangement prior to embryogenesis. Transformation and regeneration procedures were also developed for two Australian commercial cultivars of lucerne. Although the frequency of recovery of transformed plants was lower than with cv. Rangelander, these protocols open the way for a relatively rapid