First‐generation genome editing in potato using hairy root transformation

Abstract

Genome editing and cis-gene breeding have rapidly accelerated crop improvement efforts, but their impacts are limited by the number of species capable of being genetically transformed. Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens-based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating transgenic material but has been limited to analysis of hairy root clones. In this study, strains of A. rhizogenes were tested in the wild diploid potato relative Solanum chacoense, which is recalcitrant to infection by Agrobacterium tumefaciens. One strain of A. rhizogenes MSU440 emerged as being capable of delivering a T-DNA carrying the GUS marker and generating transgenic hairy root clones capable of GUS expression and regeneration to whole plants. CRISPR/Cas9 reagents targeting the potato PHYTOENE DESATURASE (StPDS) gene were expressed in hairy root clones and regenerated. We found that 64%-98% of transgenic hairy root clones expressing CRISPR/Cas9 reagents carried targeted mutations, while only 14%-30% of mutations were chimeric. The mutations were maintained in regenerated lines as stable mutations at rates averaging at 38% and were capable of germ-line transmission to progeny. This novel approach broadens the numbers of genotypes amenable to Agrobacterium-mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials.

Overexpression of phosphatidylserine synthase IbPSS1 affords cellular Na+ homeostasis and salt tolerance by activating plasma membrane Na+/H+ antiport activity in sweet potato roots

“…Thus, our root transgenic system serves as a fast, convenient, and reliable tool for functional genomics in sweet potato. Given that each single TR represents an independent transformation event, this system can be a useful tool for testing the gene-editing efficiency among different CRISPR-Cas9/Cas12a/Cas12b variants or for developing regenerated and genome-edited germplasms of this autohexaploid crop species [34][35][36] .…”

The Establishment of Two Efficient Transformation Systems in Quinoa

“…Hairy root is originated from a single cell and usually non-chimeric, which can be chosen as explant to regenerate stable transgenic plantlets (Chen et al 2018). Up to date, some species have regenerated successfully from hairy root cultures, such as tobacco (Gurusamy et al 2017), alfalfa (Jin et al 2003), maize (Xu et al 2006), potato (Butler et al 2020). Especially for spinach, which is closely related to quinoa, the transgenic shoots have been regenerated from the hairy roots (Ishizaki et al 2002).…”

Prospects for Genome Editing of Potato

“…CRISPR/Cas9 technology was successfully employed for targeting the potato phytoene desaturase (StPDS) gene, expressed in hairy root clones and regenerated. Targeted mutation was expressed in 64-98% of the transformed hairy root clones and this broadens the potato genotypes amenable to Agrobacterium-mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials [69].…”