GENETIC-TRANSFORMATION OF LOTUS-CORNICULATUS WITH AGROBACTERIUM-TUMEFACIENS AND THE ANALYSIS OF THE INHERITANCE OF TRANSGENES IN THE T-1 GENERATION

The herbage legume, Lotus corniculatus (bird's-foot trefoil), was tran sformed using the disarmed Agrobacterium tumefaciens strain LBA4404 (p AL4404) carrying a binary construct, pJit73. This plasmid carries two antibiotic resistance genes, aphIV and nptII encoding resistance to hy gromycin and kanamycin respectively, and the easily detectable reporte r gene, uidA encoding the enzyme beta-glucuronidase (GUS). Transgenic plants were regenerated from two separate co-cultivations of leaves wi th A. tumefaciens either with or without an acetosyringone pretreatmen t. A total of 110 putative transformants were regenerated, 52 (47%) of which grew on selection media containing hygromycin. Twenty-five plan ts were analysed further for morphological variation and presence of t ransgenes and were used to study the inheritance of expression of the transgenes in the T-1 generation. Expression patterns of the transgene s in the T-1 progeny generated from these 25 plants differed. In the m ajority of plant lines aphIV and uidA transgenes segregated together, but the apparent number of copies of the transgenes varied. No express ion of either transgene was detected in the progeny from three plants, while the progeny from six other plants were resistant to hygromycin but had no GUS expression. Progeny of all of the remaining 16 plants h ad GUS activity. For three plants, inheritance data were consistent wi th more than one dose of uidA and aphIV; another two plants yielded da ta expected for exactly one dose of both transgenes. In the progeny of the remaining 11 plants, the percentage of seedlings expressing both uidA and aphIV was lower than expected.