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Generation of Marker- And/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation

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Generation of Marker- And/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation

Gen-Ping Wang et al. Front Plant Sci.

Abstract

Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of "clean" GM wheat containing only the foreign genes of agronomic importance.

Keywords: agrobacterium-mediated transformation; dual binary vector; durum wheat (triticum turgidum L. var. durum cv stewart 63); genetically modified (Gm) wheat; marker- and backbone-free transgenic line.

Figures

Figure 1
Figure 1
Five sets of T-DNA vectors developed and tested in this study. (A) The constructs of five different dual binary combinations; (B) Vector elements. The vector elements were cited from Thole et al. (2007) with minor modifications. Five different dual binary vectors were introduced into Agrobacterium strain AGL1, respectively. pCS167-1B and pAL154 have the pSa-Rep ori to provide replication functions in trans for the pCG181-1G, pCG185-1G, pCG185-2G, pCG185-3G, pCG185-4G vector series, respectively. In addition, pCS167-1B has a T-DNA harboring a bar expression cassette. Arrows show the location of the primer sets for detection of the integration of backbone sequences in T0 plants. The presence of NPTI, LB, and RB in the T0 plants derived from each vectors were detected by the indicated three primer sets.
Figure 2
Figure 2
Southern blot analyses of T0 wheat plants. (A) Schematic show of the gusA gene expression cassettes, BglII restriction sites and T-DNA borders. (B) 1G7B vector. (C) 5G7B vector. (D) 5TBTG154 vector. (E) Effect of different additional vir genes combinations on transgene structure in T0 wheat plants which derived from 1G7B, 5G7B, or 5TBTG154 containing the same gusA cassette in same T-DNA but with different vir gene(s). Plant genomic DNA was digested using restriction enzyme BglII which cuts in the middle of the T-DNA in the gusA expression unit. Membranes were probed with the gusA gene. Average copy number was estimated using the number of hybridizing fragments.
Figure 3
Figure 3
Segregation of gusA and bar transgenes in T1 progenies of E02-2-4 T0 independent line derived from 5G7B vector combination. (A) Schematic show of the gusA and bar gene expression cassettes, the location of BglII and SacI restriction sites, and T-DNA borders. (B) PCR detection of gusA and bar transgenes in T1 progenies of E02-2-4 T0 plant. (C) GUS expression assay. (D) PAT assay. The presence of BAR is able to assimilate ammonium when treated with phosphinothricin acetyltransferase (PAT), led to the absence of ammonium ions in medium, thus yield a pale yellow reaction. In contrast, non-transgenic plants without the bar gene are not able to assimilate ammonium and result in a jewelry blue or dark blue color. (E) Southern blot analysis of gusA gene. (F) Southern blot analysis of bar gene. M, λ-HindIII ladder. Lanes 1–10, T1 progenies. Genomic DNA and plasmid were digested with both SacI and BglII, respectively, which produced 3103 bp gusA fragment and 1631 bp bar fragment, hybridized with a 1051 bp gusA probe and 444 bp bar probe, respectively. The data presented in (B–D) was obtained by analyzing the same set of 21 plants. Line 22, non-transgenic control.

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