In planta Genome Editing in Commercial Wheat Varieties.

Yuelin Liu,Naoaki Taoka,Kanako Kawaura,Weifeng Luo,Yoko Kamiya,Seiichi Toki,Ryozo Imai,Qianyan Linghu,Yozo Nagira,Masaki Endo,Fumitaka Abe,Kazuhiro Sato,Haruyasu Hamada,Kazumitsu Onishi,Hiroshi Hisano

doi:10.3389/fpls.2021.648841

Abstract

Limitations for the application of genome editing technologies on elite wheat (Triticum aestivum L.) varieties are mainly due to the dependency on in vitro culture and regeneration capabilities. Recently, we developed an in planta particle bombardment (iPB) method which has increased process efficiency since no culture steps are required to create stably genome-edited wheat plants. Here, we report the application of the iPB method to commercially relevant Japanese elite wheat varieties. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaQsd1 were bombarded into the embryos of imbibed seeds with their shoot apical meristem (SAM) exposed. Mutations in the target gene were subsequently analyzed within flag leaf tissue by using cleaved amplified polymorphic sequence (CAPS) analysis. A total of 9/358 (2.51%) of the bombarded plants (cv. “Haruyokoi,” spring type) carried mutant alleles in the tissue. Due to the chimeric nature of the T0 plants, only six of them were inherited to the next (T1) generation. Genotypic analysis of the T2 plants revealed a single triple-recessive homozygous mutant of the TaQsd1 gene. Compared to wild type, the homozygous mutant exhibited a 7 days delay in the time required for 50% seed germination. The iPB method was also applied to two elite winter cultivars, “Yumechikara” and “Kitanokaori,” which resulted in successful genome editing at slightly lower efficiencies as compared to “Haruyokoi.” Taken together, this report demonstrates that the in planta genome editing method through SAM bombardment can be applicable to elite wheat varieties that are otherwise reluctant to callus culture.

Highlights

Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein9 (Cas9) nuclease is a recent development which can greatly accelerate breeding efforts with precise modifications in traits of interest (Miao et al, 2013) and has already been applied to a variety of crop plants (Miao et al, 2013; Shan et al, 2014; Svitashev et al, 2015)
Detection of CRISPR/Cas9-Mediated Genome Editing in T0 Plants
A part of the cleaved amplified polymorphic sequence (CAPS) analysis is shown in Supplementary Figure 2; three T0 plants (H1, H2, and H3) showed undigested bands after PstI digestion, suggesting that mutations had occurred at the PstI target site (Supplementary Figure 1)

Summary

Introduction

Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas9) nuclease is a recent development which can greatly accelerate breeding efforts with precise modifications in traits of interest (Miao et al, 2013) and has already been applied to a variety of crop plants (Miao et al, 2013; Shan et al, 2014; Svitashev et al, 2015). These methods generally require callus culture and regeneration processes that are lengthy, costly and laborintensive These conventional culture-based transformation methods are only applicable to genotypes that are amenable for cell culture and regeneration, which significantly limits the application of genome editing to commercial varieties in major crops such as wheat (Triticum aestivum L.), maize (Zea mays L.), soybean (Glycine max L.). To overcome these issues, the in planta particle bombardment (iPB) method which utilizes shoot apical meristem (SAM) as a target tissue for transformation was developed in wheat (Hamada et al, 2018). Application of this method to divergent varieties, including elite commercial cultivars, has not been tested until now

Methods

Results

Conclusion