Application of linked and unlinked co-transformation to generate triple stack, marker-free, transgenic white clover (Trifolium repensL.)

Key message The generation of marker-free white clover plants with three transgenes in a single T-DNA by cotransformation in one step is reported here. Both linked and un-linked co-transformation can be used to overcome limitations of methods, such as re-transformation or sexual crossing of transgenic plants, to enable transfer of multiple genes to a single plant. Un-linked co-transformation can also facilitate the production of selectable marker-free transgenic plants. In this study, transgenic white clover plants were generated by Agrobacterium-mediated linked co-transformation using a single T-DNA of 9803 bp expressing: anisopentenyl transferase(IPT) gene for delayed leaf senescence under the control of an organ specificMYB32promoter from Arabidopsis, a white clovernodule enhanced malate dehydrogenase(neMDH) gene for aluminium tolerance controlled by the endogenousPhosphate Transporter 1(PT1) promoter, and thecoat proteingene from Alfalfa Mosaic Virus (CP-AMV) controlled by the35Spromoter from Cauliflower Mosaic Virus. The selectable marker gene encoding hygromycin phosphotransferase (hph) was borne on a separate T-DNA. Forty independent transgenic events carrying the triple stack were generated, with estimated co-transformation efficiencies of 0.22 to 0.23%. Forty three percent of the events generated had a single insertion, while two events were selectable marker-free. Transcript abundance studies of the three transgenes of interest demonstrated the transcriptional competence of the inserted T-DNA. This study illustrates the feasibility of transferring multiple genes in a large single T-DNA into white clover by Agrobacterium-mediated co-transformation. Furthermore, observations of consistently delayed leaf senescence, statistically significant increases in TrneMDHtranscript, and presence ofCP-AMV transcript, support further analysis of these events for delayed leaf senescence under drought conditions, aluminium tolerance, and resistance to AMV.