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.

组织培养和遗传转化的顽抗是作物基因操作的主要瓶颈。在大麦中，金色承诺的未成熟胚通常被用作转化的外植体。然而，这种方法的基因型依赖性限制了商业品种的基因改造。在这里，我们开发了一种基于花药培养的系统，可以从商业大麦品种中有效地创建转基因和基因编辑植物。该方案在 Golden Promise 和四个澳大利亚品种中进行了测试，这些品种在物候、愈伤组织诱导和绿色植物再生反应方面有所不同。对小孢子来源的愈伤组织进行农杆菌介导的转化，以靶向HvPDS基因，并从商业品种中成功获得具有靶向突变的T0白化病。实现了三个靶点的进一步编辑，五个品种的平均突变率为53%。在 51 个分析的 T0 个体中，Cas9 在目标位点诱导了很大比例 (69%) 的单碱基插入缺失和两碱基缺失，目标和品种之间的突变率存在差异。在 T1 后代中检测到了靶向和脱靶活性。与未成熟胚胎方案相比，这种独立于基因型的平台可以在相似的时间内提供高编辑效率和更多的再生植物。它显示了功能基因组学和应用 CRISPR 技术精确改良商业品种的前景。