A biolistic method for high-throughput production of transgenic wheat plants with single gene insertions

Abstract

BackgroundThe relatively low efficiency of biolistic transformation and subsequent integration of multiple copies of the introduced gene/s significantly complicate the genetic modification of wheat (Triticum aestivum) and other plant species. One of the key factors contributing to the reproducibility of this method is the uniformity of the DNA/gold suspension, which is dependent on the coating procedure employed. It was also shown recently that the relative frequency of single copy transgene inserts could be increased through the use of nanogram quantities of the DNA during coating.ResultsA simplified DNA/gold coating method was developed to produce fertile transgenic plants, via microprojectile bombardment of callus cultures induced from immature embryos. In this method, polyethyleneglycol (PEG) and magnesium salt solutions were utilized in place of the spermidine and calcium chloride of the standard coating method, to precipitate the DNA onto gold microparticles. The prepared microparticles were used to generate transgenics from callus cultures of commercial bread wheat cv. Gladius resulting in an average transformation frequency of 9.9%. To increase the occurrence of low transgene copy number events, nanogram amounts of the minimal expression cassettes containing the gene of interest and the hpt gene were used for co-transformation. A total of 1538 transgenic wheat events were generated from 15,496 embryos across 19 independent experiments. The variation of single copy insert frequencies ranged from 16.1 to 73.5% in the transgenic wheat plants, which compares favourably to published results.ConclusionsThe DNA/gold coating procedure presented here allows efficient, large scale transformation of wheat. The use of nanogram amounts of vector DNA improves the frequency of single copy transgene inserts in transgenic wheat plants.

Wheat wounding-responsive HD-Zip IV transcription factor GL7 is predominantly expressed in grain and activates genes encoding defensins

“…For stable transformation of wheat (T. aestivum L. cv. Bobwhite), the pTdGL7 construct was cotransformed together with a plant selectable marker cassette (Ubi-hpt-nos) into wheat using microprojectile bombardment as described in (Kovalchuk et al 2009;Ismagul et al 2018). The integration of promoter-GUS fusions in transgenic plants was confirmed by PCR using primers derived from promoter and GUS (uidA) sequences.…”

A competence of embryo-derived tissues of tetraploid cultivated wheat species Triticum dicoccum and Triticum timopheevii for efficient and stable transgenesis mediated by particle inflow gun

“…There is general agreement that the transgene segregation could be more predictable in the case of a single insert or a low number of insertions. We believe that some methodological approaches, such as the use of dephosphorylated gene cassette [ 41 ] or linear form of the construct [ 48 ], and the PEG/Mg2+ coating procedure [ 49 ] will be useful for combination with the method presented here to increase the number of transgenic events with simple integration patterns.…”

Genome Editing in Agriculture: Technical and Practical Considerations

“…However, the quality of the integration events is usually lower than Agrobacterium-mediated transformation and rarely results in clean single-copy integration of the DNA construct [104,105]. Both circular and linear DNA can be introduced into plant cells by particle bombardment, and the co-delivery of different DNA fragments or plasmids is quite efficient because they can be coated onto the same microparticles [121,122]. A recent study in wheat demonstrated the ZFN-mediated precise editing of the three homoeologous ALS genes following bombardment with the ZFN construct and a short dsDNA donor with compatible overhangs [76].…”