Kaniganti Sirisha, Palakolanu Sudhakar Reddy, Thiombiano Benjamin, Damarasingh Jagadeesh, Bommineni Pradeep Reddy, Che Ping, Sharma Kiran Kumar, Jones Todd, Bouwmeester Harro, Bhatnagar-Mathur Pooja
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Hyderabad, Telangana, 502324, India.
Department of Biotechnology, Osmania University, Hyderabad, Telangana, 500 007, India.
Plant Cell Rep. 2025 Mar 27;44(4):90. doi: 10.1007/s00299-025-03474-1.
High transformation and gene editing efficiencies in sorghum-produced, transgene-free SDN1-edited plants exhibit precise mutations, reduced germination stimulants, and enhanced resistance to Striga infection. Sorghum (Sorghum bicolor L.) is a primary food staple grain for millions in Sub-Saharan Africa (SSA). It is mainly constrained by the parasitic weed Striga, which causes up to 100% yield losses and affects over 60% of cultivable farmlands and livelihoods. In this study, CRISPR/Cas9 technology is utilized to induce mutations in core strigolactone (SL) biosynthetic genes, i.e., CCD7, CCD8, MAX1, in addition to an uncharacterized gene (DUF) in the fine-mapped 400 kb lgs1 region in sorghum to develop durable Striga resistance. Two sorghum cultivars were delivered with the expression cassettes through immature embryo-based Agrobacterium-mediated transformation. Our study demonstrated transformation and gene editing efficiencies of ~ 70 and up to 17.5% (calculated based on the numuber of established plants), respectively, in two sorghum genotypes. Subsequent analysis of homozygous E lines in the E generation confirmed stable integration of mutations for all targeted genes. Loss-of-function mutations in the CCD7, CCD8, MAX1, and DUF genes led to a significant downregulation of the expression of associated genes in the SL biosynthetic pathway. The phenotypic analysis of edited lines revealed changes in phenotypic patterns compared to wild-type plants. Analysis of root exudates showed significant reductions in SL production in edited lines compared to wild-type plants. Striga infection experiments demonstrated delayed or reduced emergence rates of Striga in edited lines with lower SL production, highlighting the potential for genetically altering SL production to control Striga infestations. This study provides insights into the functional roles of CCD7, CCD8, MAX1, and DUF genes in sorghum towards reduced and/or altered SL production and improved resistance to Striga infestations.
在高粱中产生的无转基因SDN1编辑植物具有高转化和基因编辑效率，表现出精确的突变、减少的萌发刺激物以及增强的对独脚金感染的抗性。高粱（Sorghum bicolor L.）是撒哈拉以南非洲（SSA）数百万人的主要主食谷物。它主要受到寄生杂草独脚金的制约，独脚金会导致高达100%的产量损失，并影响超过60%的可耕地和生计。在本研究中，利用CRISPR/Cas9技术在高粱精细定位的400 kb lgs1区域中，除了一个未表征基因（DUF）外，诱导核心独脚金内酯（SL）生物合成基因，即CCD7、CCD8、MAX1发生突变，以培育持久的独脚金抗性。通过基于未成熟胚的农杆菌介导转化，将表达盒导入两个高粱品种。我们的研究表明，在两种高粱基因型中，转化效率和基因编辑效率分别约为70%和高达17.5%（基于已建立植株的数量计算）。随后对E代纯合E系的分析证实了所有靶向基因的突变稳定整合。CCD7、CCD8、MAX1和DUF基因的功能丧失突变导致SL生物合成途径中相关基因的表达显著下调。编辑系的表型分析显示，与野生型植物相比，表型模式发生了变化。根系分泌物分析表明，与野生型植物相比，编辑系中SL的产生显著减少。独脚金感染实验表明，SL产量较低的编辑系中独脚金的出苗率延迟或降低，突出了通过基因改变SL产量来控制独脚金侵染的潜力。本研究深入了解了CCD7、CCD8、MAX1和DUF基因在高粱中对减少和/或改变SL产量以及提高对独脚金侵染抗性方面的功能作用。
