Duan Ke, Zhao Ying-Jie, Li Zi-Yi, Zou Xiao-Hua, Yang Jing, Guo Cheng-Lin, Chen Si-Yu, Yang Xiu-Rong, Gao Qing-Hua
Shanghai Key Laboratory of Protected Horticultural Technology, Forestry and Fruit Tree Research Institute, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201403, China.
Lanzhou New Area Academy of Modern Agricultural Sciences, Lanzhou 730300, China.
Plants (Basel). 2021 Oct 20;10(11):2229. doi: 10.3390/plants10112229.
Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. 'Shanghai Angel' by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from harboring a sugar transporter gene driven by a fruit-specific promoter. Transformed strawberry was developed through an -mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of 'Shanghai Angel' and other cultivars to improve agronomic traits.
基因内起源是一种用于作物改良的全原生工程技术。采用基因内策略将野生种的基因导入栽培草莓可扩大遗传变异性。通过优化噻苯隆剂量并确定最合适的外植体，为草莓品种“上海天使”建立了一种高效的再生体系。表达盒由携带一个由果实特异性启动子驱动的糖转运蛋白基因的所有DNA片段组装而成。通过农杆菌介导的策略培育转化草莓，不使用任何选择标记。除了PCR筛选外，还进行了基于探针的双链液滴数字PCR（ddPCR）以确定T-DNA插入情况。获得了4个独立的转化芽，最高效率为5.3%。两条株系被确认为嵌合体，另外两条为完全转化体，分别含有6个和11个基因内拷贝。这些转化体中T-DNA之外存在载体骨架，表明未获得基因内草莓。目前的工作优化了不进行抗生素筛选生产转化草莓的程序，并首次通过ddPCR准确测定了草莓基因组中的插入拷贝数。这些策略可能有望用于“上海天使”和其他品种的工程改造，以改善农艺性状。
