An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.)

Abstract

BackgroundDespite wheat being a worldwide staple, it is still considered the most difficult to transform out of the main cereal crops. Therefore, for the wheat research community, a freely available and effective wheat transformation system is still greatly needed.ResultsWe have developed and optimised a reproducible Agrobacterium-mediated transformation system for the spring wheat cv ‘Fielder’ that yields transformation efficiencies of up to 25%. We report on some of the important factors that influence transformation efficiencies. In particular, these include donor plant health, stage of the donor material, pre-treatment by centrifugation, vector type and selection cassette. Transgene copy number data for independent plants regenerated from the same original immature embryo suggests that multiple transgenic events arise from single immature embryos, therefore, actual efficiencies might be even higher than those reported.ConclusionWe reported here a high-throughput, highly efficient and repeatable transformation system for wheat and this system has been used successfully to introduce genes of interest, for RNAi, over-expression and for CRISPR–Cas9 based genome editing.

A roadmap for gene functional characterisation in wheat

“…Transformation by overexpression of transcription factors such as maize Baby Boom and Wuschel2 has also yielded improved transformation efficiencies in monocots (Lowe et al, 2016), although there are no formal reports yet in wheat. Recently, an open-access wheat transformation system with transformation efficiencies of up to 25% was published (Hayta et al, 2019), albeit for a single cultivar.…”

“…For CRISPR/Cas9 and other non-transient transgenic approaches several varieties may be used, although relatively few wheat varieties have been shown to display high enough transformation efficiencies to be practical. This means that traditionally most transgenic studies in wheat have been limited to a few varieties, such as 'Fielder ', Cadenza, 'Bobwhite', 'Kenong 199' and Kronos (Li et al, 2012;Richardson et al, 2014;Hayta et al, 2019). This is now changing thanks to work by groups at NIAB (UK), CAAS (China) and CSIRO (Australia) who have successfully transformed 39 (Wallington, 2015), 15 (Wang et al, 2017) and six (Richardson et al, 2014) varieties, respectively.…”

TRA1: a locus responsible for controllingAgrobacterium-mediated transformability in barley

“…This is because no other cultivar has been found to give a similar or higher 31 transformation efficiency (Bartlett et al, 2008;Harwood et al, 2009;Harwood, 2012). Wheat, like 32 barley, is also transformable using Agrobacterium but despite successful attempts to increase 33 transformation efficiency (Hayta et al, 2019), the process is still cultivar dependant: cv Fielder being 34 one of the most responsive cultivars. Efficiencies as high as 86.7% can be obtained with barley cv 35…”

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“…Recently, an open-access wheat transformation system with transformation efficiencies of up to 25% was published (Hayta et al, 2019), albeit for a single cultivar.…”

“…For CRISPR/Cas9 and other non-transient transgenic approaches several varieties may be used, although relatively few wheat varieties have been shown to display high enough transformation efficiencies to be practical. This means that traditionally most transgenic studies in wheat have been limited to a few varieties, such as 'Fielder', Cadenza, 'Bobwhite', 'Kenong 199' and Kronos (Li et al, 2012;Richardson et al, 2014;Hayta et al, 2019). This is now changing thanks to work by groups at NIAB (UK), CAAS (China) and CSIRO (Australia) who have successfully transformed 39 (Wallington, 2015), 15 and six (Richardson et al, 2014) varieties, respectively.…”