CRISPR/Cas9介导的高效四倍体马铃薯试管薯基因编辑体系的建立

Establishment of CRISPR/Cas9-mediated Highly Efficient Gene Editing System in Microtubers of Potatoes

【目的】基因编辑技术的发展使得马铃薯实现精准分子育种成为了可能,试管薯作为遗传转化的理想材料,但其诱导和转化具有基因型依赖性,建立高效且普遍适用的试管薯基因编辑技术体系可为其精准分子育种提供技术支撑。【方法】以四倍体栽培马铃薯品种青薯9号和并薯6号为材料,对试管薯诱导及其遗传转化体系进行摸索;同时,转化CRISPR/Cas9基因编辑载体进行基因组编辑。另外,利用筛选的条件对其他3个马铃薯品种进行测试。【结果】全黑暗条件下,采用2叶/段的扩繁方式,在含有10%蔗糖和5 mg/L激动素的培养基上,5个马铃薯品种均可成功诱导出试管薯,但诱导效果存在差异。青薯9号的最佳分化激素配方为0.5 mg/L 6-苄氨基腺嘌呤、0.2 mg/L吲哚-3-乙酸、0.2 mg/L赤霉素、2 mg/L玉米素,再生效率、转化频率和基因编辑效率分别为41.5%、51.9%和82.1%。利用上述筛选的激素配方,在其他4个四倍体马铃薯品种中均可高效地实现试管薯的遗传转化再生,其中并薯6号、Desiree和晋薯16号的编辑效率分别为63.2%、33.3%和10%。【结论】建立了5个不同基因型四倍体马铃薯高效的试管薯遗传转化再生体系,其中的4份材料成功地实现了基因组编辑。

【Objective】The development of gene editing technology has made it possible to achieve precise molecular breeding in potato.Microtubers are ideal materials for genetic transformation,but its induction and genetic transformation are of genotype-dependence.Thus,establishing an efficient and widely used gene editing technology may provide technical support for precise molecular breeding for potatoes in future.【Method】Cultivated tetraploid potato variety Qingshu No.9 and Bingshu No.6 were used to explore microtuber induction and genetic transformation.Concurrently,the CRISPR/Cas9 gene editing vector was transformed for genome editing.In addition,the screening system was tested in another three potato varieties.【Result】The efficient tuberizations of five potato varieties were obtained using stem segment with two leaves on solid media including 10%sucrose and 5 mg/L kinetin under dark condition,while induction effect varied.The optimal ratio of hormones for the regeneration of Qingshu No.9 was 0.5 mg/L 6-benzylaminopurine,0.2 mg/L indole-3-acetic acid,0.2 mg/L gibberellin,and 2 mg/L zeatin,under which the regeneration rate,the transformation frequency,genome editing rate was 41.5%,51.9%,and 82.1%,respectively.Using the above screened the formula of hormones,genetic transformation and regeneration was efficiently achieved in another four potato varieties,and the genome editing rate was 63.2%,33.3%,and 10%for Bingshu No.6,Desiree,and Jingshu No.16,respectively.【Conclusion】We established the highly efficient genetic transformation and regeneration system for five potato varieties,and genome editing can be completed for four potato varieties.