Abstract
Recalcitrance to tissue culture and genetic transformation is the major bottleneck for gene manipulation in crops. In barley, immature embryos of Golden Promise have typically been used as explants for transformation. However, the genotype dependence of this approach limits the genetic modification of commercial varieties. Here, we developed an anther culture-based system that permits the effective creation of transgenic and gene-edited plants from commercial barley varieties. The protocol was tested in Golden Promise and four Australian varieties, which differed in phenology, callus induction, and green plant regeneration responses. Agrobacterium-mediated transformation was performed on microspore-derived callus to target the HvPDS gene, and T0 albinos with targeted mutations were successfully obtained from commercial varieties. Further editing of three targets was achieved with an average mutation rate of 53% in the five varieties. In 51 analyzed T0 individuals, Cas9 induced a large proportion (69%) of single-base indels and two-base deletions in the target sites, with variable mutation rates among targets and varieties. Both on-target and off-target activities were detected in T1 progenies. Compared with immature embryo protocols, this genotype-independent platform can deliver a high editing efficiency and more regenerant plants within a similar time frame. It shows promise for functional genomics and the application of CRISPR technologies for the precise improvement of commercial varieties.
Highlights
Developed gene-targeting and genome-editing techniques facilitate the accurate manipulation of specific genomic sequences, allowing reverse genetics, genome engineering, and targeted transgene integration experiments to be conducted in an efficient and precise manner (Bortesi and Fischer, 2015)
The typical barley anther culture protocol for doubled haploid (DH) production consists of seven phases: (1) sowing F1 seeds; (2) F1 genotyping for heterozygosity with molecular markers (i.e., KASP); (3) microspore staging to identify the mid- to late-uninucleate stage; (4) anther dissection and mannitol pretreatment; (5) callus/embryo induction; (6) plant regeneration; and (7) growing regenerants to maturity (Figure 1; Broughton et al, 2014)
After the induction phase, the culture was interrupted after the induction of a mixture of embryos and calli, and only microspore-derived embryogenic calli were transformed with Agrobacterium that carried a binary vector for barley clustered regularly interspaced short palindromic repeats (CRISPR)/ Cas9 gene editing (Supplemental Figure 1)
Summary
Developed gene-targeting and genome-editing techniques facilitate the accurate manipulation of specific genomic sequences, allowing reverse genetics, genome engineering, and targeted transgene integration experiments to be conducted in an efficient and precise manner (Bortesi and Fischer, 2015). Owing to its simplicity of programming and robustness, the CRISPR/Cas system is a breakthrough in genome editing, especially for creating targeted mutations to eliminate genes that negatively affect food quality, confer susceptibility to pathogens, or divert metabolic flux away from valuable end products. With regard to the regulation and commercialization of CRISPR-edited products, the US Department of Agriculture and the Australian Office of the Gene Technology Regulator have determined that edited crops without foreign DNA are exempt from regulation as genetically modified organisms (Waltz, 2018; Mallapaty, 2019). This decision will enable and promote the use of gene editing
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