Plant Commun|基于花药培养的高效非基因型大麦基因编辑
对组织培养和遗传转化的抗性是作物基因操作的主要瓶颈。在大麦中，金色希望（Golden Promise）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遗传修饰。本研究，我们开发了一个基于花药培养的系统，允许从商业大麦品种中有效地创造转基因和基因编辑植株。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株再生反应方面存在差异。利用农杆菌介导的方法对小孢子愈伤组织进行HvPDS基因的转化，成功地从商品品种中获得了T0白化菌。进一步编辑了3个目标，5个品种的平均突变率为53%。在所分析的51个T0个体中，Cas9诱导了大量（69%）的单碱基indels和靶位点的双碱基缺失，在靶点和品种之间具有不同的突变率。在T1代后代中检测到靶向和非靶向活性。与幼胚处理相比，该平台具有较高的编辑效率，在相似的时间范围内获得更多的再生植株。它显示了功能基因组学和CRISPR技术在商业品种精确改良中的应用前景。
大麦花药培养基因编辑流程图
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.
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