High Efficiency Wheat Transformation Mediated by Agrobacterium tumefaciens

Abstract

Wheat is one of the most important cereals for humans but quite recalcitrant in transformation. We have thoroughly examined every aspect of the wheat transformation protocols mediated by Agrobacterium tumefaciens and we were able to identify and optimize the key factors. Immature embryos isolated from healthy plants grown in a greenhouse were pre-treated with centrifuging and co-cultivated with A. tumefaciens . The frequency of transformation (independent transgenics/ explant) was between 50 % and 60 % were routinely observed and higher than 90 % were recorded in the best cases. Not surprisingly, the key factors did not differ much from those in other cereal plants such as rice and maize. Both bar and hpt genes were good as selection markers. Fielder, a spring wheat cultivar, constantly showed high effi ciency of transformation by our protocol. We have been able to obtain transgenic plants from the embryos harvested from the greenhouses throughout the year. Most of the transformed plants were normal in morphology and fully fertile. More than 40 % of the transformants had a single copy of the transgenes, which were inherited in a Mendelian fashion in most of the lines analyzed. Transgenic wheat has been generated at high frequency by several research groups by our protocol by now. Therefore, wheat has fi nally joined the list of cereals that can be effi ciently transformed.

Identification and characterization of Sr13 , a tetraploid wheat gene that confers resistance to the Ug99 stem rust race group

“…We cloned a genomic fragment of 8,055 bp from Langdon BAC 1181K4 into a Hind III-HF/ Spe I linearized binary vector pLC41Hm ( 49 ). This fragment included the complete coding region and introns (3,513 bp) plus 2,108 bp upstream of the start codon (including the 538 bp 5′ UTR) and 2,434 bp downstream of the stop codon (including the 2,187 bp 3′ UTR).…”

A GRF–GIF chimeric protein improves the regeneration efficiency of transgenic plants

“…High wheat regeneration efficiencies have been reported before using the proprietary Japan Tobacco method in the variety Fielder 29 , 30 , 31 . However, the company warns that these high values require the optimization of multiple factors with narrow optimal windows and that “those values can drop drastically when one of the factors become suboptimal” 29 ( Supplementary Table 5 ).…”

The Enhancement of Plant Disease Resistance Using CRISPR/Cas9 Technology

“…Regeneration of rice protoplasts is still very challenging, but important optimization efforts may render it feasible in the near future. In wheat, although very high Agrobacterium -mediated transformation efficiencies of up to 90% have been reported for specific wheat genetic backgrounds ( Ishida et al, 2015a , b ), particle bombardment has been more widely accepted as the standard method in wheat genetic transformation ( Hakam et al, 2015 ; Wang et al, 2018 ). Remarkable success has been achieved by particle bombardment of both immature embryos and callus cells to obtain transient expression of the CRISPR/Cas9 DNA, and transgene-free homozygous mutant T0 plants have been generated in the absence of any selection ( Zhang et al, 2016 ).…”