03007naa a2200301 a 450000100080000000500110000800800410001902400360006010000170009624502950011326000090040850000690041752018910048665300420237765300280241965300140244765300100246170000160247170000150248770000140250270000150251670000140253170000130254570000170255870000140257570000200258977300960260910615392020-12-03       2020    bl uuuu     u00u1 u    #d7 a10.1007/s11240-020-01891-62DOI1 aNARANCIO, R.  aCorrection tobApplication of linked and unlinked co‑transformation to generate triple stack, marker‑free, transgenic white clover (Trifolium repens L.) (Plant Cell, Tissue and Organ Culture (PCTOC), (2020), 142, 3, (635-646), 10.1007/s11240-020-01891-6).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