Highly efficient and genotype-independent barley gene editing based on anther culture  

作　　者：Yong Han Sue Broughton Li Liu Xiao-Qi Zhang Jianbin Zeng Xiaoyan He Chengdao Li 

机构地区：[1]Western Barley Genetics Alliance,College of Science,Health,Engineering and Education,Murdoch University,Murdoch,WA 6150,Australia [2]Department of Primary Industries and Regional Development,3 Baron-Hay Court,South Perth,WA 6151,Australia [3]Western Australian State Agricultural Biotechnology Centre,Murdoch University,Murdoch,WA 6150,Australia [4]College of Agronomy,Qingdao Agricultural University,Qingdao,Shandong 266109,China

出　　处：《Plant Communications》2021年第2期89-101,共13页植物通讯（英文）

基　　金：supported by the Western Australian Department of Primary Industries and Regional Development;the Western Australian State Agricultural Biotechnology Center,Murdoch University.

摘　　要：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 ontarget and off-target activities were detected in T1 progenies.Compared with immature embryo protocols,this genotype-independent platformcan 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.

关 键 词：AGROBACTERIUM CRISPR genetic transformation Hordeum vulgare off-target targeted mutation 

分 类 号：S512.3[农业科学—作物学]