Genetic Transformation of Common Wheat (Triticum aestivum L.) Using Biolistics

CRISPR/Cas technology has recently become the molecular tool of choice for gene function studies in plants as well as crop improvement. Wheat is a globally important staple crop with a well annotated genome and there is plenty of scope for improving its agriculturally important traits using genome editing technologies, such as CRISPR/Cas. As part of this study we targeted three different genes in hexaploid wheat Triticum aestivum: TaBAK1-2 in the spring cultivar Cadenza as well as Ta-eIF4E and Ta-eIF(iso)4E in winter cultivars Cezanne, Goncourt and Prevert. Primary transgenic lines carrying CRISPR/Cas-induced indels were successfully generated for all targeted genes. While BAK1 is an important regulator of plant immunity and development, Ta-eIF4E and Ta-eIF(iso)4E act as susceptibility (S) factors required for plant viruses from the Potyviridae family to complete their life cycle. We anticipate the resultant homozygous tabak1-2 mutant lines will facilitate studies on the involvement of BAK1 in immune responses in wheat, while ta-eif4e and ta-eif(iso)4e mutant lines have the potential to become a source of resistance to wheat spindle streak mosaic virus (WSSMV) and wheat yellow mosaic virus (WYMV), both of which are important pathogens of wheat. As winter wheat varieties are generally less amenable to genetic transformation, the successful experimental methodology for transformation and genome editing in winter wheat presented in this study will be of interest to the research community working with this crop.

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“…Wheat embryos of all varieties were transformed via particle bombardment essentially as previously described [ 21 ].…”

Section: Methodsmentioning

confidence: 99%

“…Donor plants were grown as above for 10–12 weeks to provide immature embryos which were isolated at 12–16 days post anthesis (dpa). The shoot/root axis was removed and the immature scutella were plated ~30 per plate on the induction medium [ 21 ], and used as target tissue, giving one day pre-culture at 22 °C, dark, prior to bombardment.…”

Section: Methodsmentioning

confidence: 99%

Efficient CRISPR/Cas-Mediated Targeted Mutagenesis in Spring and Winter Wheat Varieties

Hahn

Loures

Sparks

et al. 2021

Plants

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“…Wheat embryos of all varieties were transformed via particle bombardment essentially as previously described (Sparks and Doherty, 2020).…”

Section: Transformationmentioning

confidence: 99%

“…Donor plants were grown as above for 10-12 weeks to provide immature embryos which were isolated at 12-16 days post anthesis (dpa). The shoot/root axis was removed and the immature scutella were plated ~ 30 per plate on the induction medium (Sparks and Doherty, 2020), and used as target tissue, giving one day pre-culture at 22 o C, dark, prior to bombardment. 0.6µm gold particles (BioRad Laboratories Ltd, UK) were coated with plasmid DNAs as specified below and co-bombarded into tissues of the relevant wheat varieties using a rupture pressure of 650psi and 28.5" Hg vacuum.…”

Section: Transformationmentioning

confidence: 99%

Efficient CRISPR/Cas-mediated Targeted Mutagenesis in Spring and Winter Wheat Varieties

Hahn

Loures

Sparks

et al. 2021

Preprint

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The CRISPR/Cas technology has recently become a molecular tool of choice for gene function studies in plants as well as crop improvement. Wheat is a globally important staple crop with a well annotated genome and there is plenty of scope for improving its agriculturally important traits using genome editing technologies, such as CRISPR/Cas. As part of this study we targeted three different genes in hexaploid wheat Triticum aestivum: TaBAK1-2 in the spring cultivar Cadenza as well as Ta-eIF4E and Ta-eIF(iso)4E in winter cultivars Cezanne, Goncourt and Prevert. The primary transgenic lines carrying CRISPR/Cas-induced indels were successfully generated for all targeted genes. As winter wheat varieties are generally less amenable to genetic transformation, the successful experimental methodology for transformation and genome editing in winter wheat presented in this study will be of interest to the research community working with this crop.

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“…All three sgRNA plasmids were co-delivered along with pCas9- GFP (Zhang et al, 2019) encoding the wheat codonoptimised Cas9 fused in frame at the C-terminus with Green Fluorescent Protein (GFP), and pRRes1.111 (Alotaibi et al, 2018) which directs expression of the bar selectable marker gene into immature wheat embryos of wheat cv. Cadenza using biolistics essentially as described in Sparks and Doherty (2020). CRISPR/Cas9 mutagenized T 0 lines were genotyped for indels using the PCR band-shift assay (Nekrasov et al, 2017).…”

Section: Generation Of Wheat Genetic Materialsmentioning

confidence: 99%

Remarkable recent changes in genetic diversity of the avirulence geneAvrStb6in global populations of the wheat pathogenZymoseptoria tritici

Stephens¹,

Ölmez²,

Blyth³

et al. 2020

Preprint

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Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is one of the most economically important diseases of wheat. Recently, both factors of a gene-for-gene interaction between Z. tritici and wheat, the wheat receptor-like kinase Stb6 and the Z. tritici secreted effector protein AvrStb6, have been identified. Previous analyses revealed a high diversity of AvrStb6 alleles present in historic Z. tritici isolate collections, with up to ~ 18% of analysed isolates possessing the avirulence isoform of AvrStb6 identical to that originally identified in the reference isolate IPO323. With Stb6 present in many commercial wheat cultivars globally, we aimed to assess potential changes in AvrStb6 genetic diversity and the incidence of alleles allowing evasion of Stb6-mediated resistance in more recent Z. tritici populations. Here we show, using targeted re-sequencing of AvrStb6, that this gene is universally present in field isolates sampled from major wheat-growing regions of the world between 2013-2017. However, in contrast to the data from studies of historic isolates, our study revealed a complete absence of the originally described avirulence isoform of AvrStb6 amongst modern Z. tritici isolates. Moreover, a remarkably small number of alleles, each encoding AvrStb6 protein isoforms conditioning virulence on Stb6-containing wheat, were found to predominate among modern Z. tritici isolates. A single virulence isoform of AvrStb6 was found to be particularly abundant throughout the global population. These findings indicate that, despite the ability of Z. tritici to sexually reproduce on resistant hosts, AvrStb6 avirulence alleles tend to be eliminated in subsequent populations.

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Genetic Transformation of Common Wheat (Triticum aestivum L.) Using Biolistics

Cited by 8 publications

References 6 publications

Efficient CRISPR/Cas-Mediated Targeted Mutagenesis in Spring and Winter Wheat Varieties

Efficient CRISPR/Cas-Mediated Targeted Mutagenesis in Spring and Winter Wheat Varieties

Efficient CRISPR/Cas-mediated Targeted Mutagenesis in Spring and Winter Wheat Varieties

Remarkable recent changes in genetic diversity of the avirulence geneAvrStb6in global populations of the wheat pathogenZymoseptoria tritici

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