Generation of selection marker-free transgenic plants by cotransformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain

Abstract

Cotransformation of Nicotiana tabacum was done using a single Agrobacterium tumefaciens strain harbouring a cointegrate vector (one copy per cell) and a binary vector (ten to 15 copies per cell). The T-DNAs of the cointegrate plasmid, pGV2260::pSSJ1 and the binary plasmid, pGA472 carried hph and nptII as plant selection markers, respectively. When the binary T-DNA marker (nptII) was used for selection, the non-selected cointegrate T-DNA with hph cotransformed at 36% frequency. However, upon using the cointegrate T-DNA with hph for selection, cotransformation of binary T-DNA with nptII was much higher (56-74%). Segregation of the T-DNA markers hph and nptII was followed in the T₁ generation to screen for the elimination of the selection marker. The elimination of nptII of the multicopy binary vector was found in the progeny of only one of the three T₀ plants and at a low frequency of 3%. However, elimination of hph of the single-copy cointegrate vector was found at 16-18% frequency in the progeny of both the T₀ plants analysed. The use of the T-DNA of low copy number cointegrate vector for initial selection improves the cotransformation frequency of non-selected T-DNA of the multicopy binary vector. This strategy also increases the frequency of generation of selection marker-free transgenic plants in the T₁ generation.