Abstract
SummaryGenotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker‐free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium‐mediated method, with efficiency of up to 37.7%, as confirmed by the use of Quickstix strips, histochemical staining, PCR analysis and Southern blotting. Of particular interest, marker‐free transgenic wheat plants from various commercial Chinese varieties and their F1 hybrids were successfully obtained for the first time, with a frequency of 4.3%, using a plasmid harbouring two independent T‐DNA regions. The average co‐integration frequency of the gus and the bar genes located on the two independent T‐DNA regions was 49.0% in T0 plants. We further found that the efficiency of generating marker‐free plants was related to the number of bar gene copies integrated in the genome. Marker‐free transgenic wheat plants were identified in the progeny of three transgenic lines that had only one or two bar gene copies. Moreover, silencing of the bar gene was detected in 30.7% of T1 positive plants, but the gus gene was never found to be silenced in T1 plants. Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in the transgenic plants.
Highlights
Hexaploid wheat is an important worldwide food crop that contributes as much as 35% of the calories consumed by the global population (Godfray et al, 2010; Shewry, 2009)
The gus (b-glucuronidase) reporter gene was inserted into the BamHI and SacI sites in the multiple cloning site (MCS) of pWMB122 to form the vector pWMB123 (Figure 1), which was used for all of the transformation experiments in this study
All of the commercial Chinese wheat cultivars tested showed strong gus expression in the infected immature embryos (Figure S2), which suggests that the cultivars used in the present investigation were amenable to Agrobacterium infection
Summary
Hexaploid wheat is an important worldwide food crop that contributes as much as 35% of the calories consumed by the global population (Godfray et al, 2010; Shewry, 2009). Genetic transformation techniques have been used successfully in the improvement of some major crop species, including soya bean, maize and cotton. New varieties of these plants that have been developed by transgenic methods are planted widely in many countries, offering great benefits to farmers and helping to protect the environment (James, 2016). Marker-free transgenic Fielder and Gladius wheat plants were obtained with an efficiency of 3% without imposing selection pressure during tissue culture step. This dramatic progress will almost certainly promote the development and eventual commercial introduction of transgenic wheat varieties
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