02904naa a2200313 a 450000100080000000500110000800800410001902200140006002400360007410000170011024501660012726000090029350000690030252018910037165300420226265300280230465300140233265300100234670000160235670000150237270000140238770000150240170000140241670000130243070000170244370000140246070000200247477300960249410612492020-12-03       2020    bl uuuu     u00u1 u    #d  a0167-68577 a10.1007/s11240-020-01891-62DOI1 aNARANCIO, R.  aApplication of linked and unlinked co transformation to generate triple stack, marker free, transgenic white clover (Trifolium repens L.).h[electronic resource]  c2020  aArticle history: Received: 19 May 2020 / Accepted: 15 July 2020.  aABSTRACT. 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: an isopentenyl transferase (IPT) gene for delayed leaf senescence under the control of an organ specific MYB32 promoter from Arabidopsis, a white clover nodule enhanced malate dehydrogenase (neMDH) gene for aluminium tolerance controlled by the endogenous Phosphate Transporter 1 (PT1) promoter, and the coat protein gene from Alfalfa Mosaic Virus (CP-AMV) controlled by the 35S promoter 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 TrneMDH transcript, and presence of CP-AMV transcript, support further analysis of these events for delayed leaf senescence under drought conditions, aluminium tolerance, and resistance to AMV.  © 2020, Springer Nature B.V.  aAgrobacterium-mediated transformation  aDelayed leaf senescence  aMultigene  aT-DNA1 aDING, Y.-L.1 aLIN, Y.-H.1 aSAHAB, S.1 aPANTER, S.1 aHAYES, M.1 aJOHN, U.1 aANDERSON, H.1 aMASON, J.1 aSPANGENBERG, G.  tPlant Cell, Tissue and Organ Culture, 2020. DOI: https://doi.org/10.1007/s11240-020-01891-6