TRANSFORMATION OF A GRAIN LEGUME (LUPINUS-ANGUSTIFOLIUS L.) VIA AGROBACTERIUM-TUMEFACIENS-MEDIATED GENE-TRANSFER TO SHOOT APICES

Transgenic plants of Lupinus angustifolius L. (cvs. Unicrop and Merrit ) were routinely generated using Agrobacterium-mediated gene transfer to shoot apices. The bar gene for resistance to phosphinothricin (PPT, the active ingredient of the herbicide Basta) was used as the selecta ble marker. After co-cultivation, the shoot apex explants were transfe rred onto a PPT-free regeneration medium and their tops were thoroughl y wetted with PPT solution (2 mg/ml). The multiple axillary shoots dev eloping from the shoot apices were excised onto a medium containing 20 mg/l PPT. The surviving shoots were transferred every second week ont o fresh medium containing 20 mg/l PPT. At each transfer, the number of surviving shoots decreased, until it stabilized. Indeed, some of thes e chimeric shoots surviving the PPT selection, eventually produced new green healthier axillary shoots which could be transferred to soil. T his whole process took from 5 to 9 months after co-cultivation. Averag e transformation frequencies of 2.8% for cv. Unicrop and of 0.4% for t he commercial cultivar Merrit were achieved. Molecular analysis of T-0 , T-1, and T-2 generations demonstrated stable integration of the fore ign gene into the plant genome and expression of the integrated gene. Transformed plants of the T-1 and T-2 generations were resistant in gl asshouse trials where the herbicide Basta (0.1 mg/ml) was sprayed onto whole plants. These results demonstrate that Agrobacterium-mediated g ene transfer to preorganised meristematic tissue combined with axillar y regeneration can form the basis of a routine transformation system f or legume crop species which are difficult to regenerate from other ex plants.