Genome editing in potato plants by agrobacterium-mediated transient expression of transcription activator-like effector nucleases
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Genome editing (also known as targeted mutation) has promise for molecular breeding. Compared with the CRISPR/Cas9 system, the transcription activator-like effector nucleases (TALENs) have likely a lesser off-target rate in genome editing. Both a rapid test system for the functionality of designed TALENs and an effective delivery system for introducing the TALENs to plants are critical for successful target mutation. TALENs have usually been tested in protoplasts or introduced to plants with viral vectors. However, plant regeneration from protoplast culture can generate extensive somatic variation. Viral vectors are not always available, and plants edited by these vectors usually require virus elimination. Here, we used a non-viral, Agrobacterium-mediated transient expression approach, to serve both rapid test and effective delivery of TALENs into two vegetatively propagated potato cultivars, Solanum tuberosum Russet Burbank and Shepody. Two TALENs with different molecular weights (22 and 27 aa-repeat modules) were expressed to target two endogenous genes (starch branching enzyme and an acid invertase) by Agrobacterium-mediated infiltration (agroinfiltration) into leaves of potato plants. The infiltrated leaf DNA was analyzed using restriction site loss assay and subsequent DNA sequencing. Deep sequencing of these tetraploid cultivars was also conducted to determine the zygosity at the targeted chromosomal loci. TALENs, with different molecular weights, successfully agroinfiltrated and induced mutations at both targeted loci.
KeywordsNon-transgenic Agroinfiltration Site-specific mutagenesis Polyploid plants Allele specificity Vegetatively propagated plants Somatic genome manipulation Molecular breeding
We thank Muhammad Haroon, Agriculture and Agri-Food Canada, for his general support in the laboratory and his technical support in plant and DNA preparation. The research funding was from Agriculture and Agri-Food Canada A-base, the Natural Sciences and Engineering Research Council of Canada (NSERC), and the deep sequencing was supported by the New Brunswick Agricultural Innovation Program.
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Conflict of interest
The authors declare that they have no conflict of interest.
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