Zhang Manyu, Zhou Lili, Afridi Mahideen, Guo Huiming, Cheng Hongmei
School of Agricultural Sciences, Zhengzhou University, Zhengzhou, 450001, China.
National Key Laboratory of Agricultural Microbiology, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
Funct Integr Genomics. 2025 Jun 2;25(1):116. doi: 10.1007/s10142-025-01627-9.
The hairy root induction system has been widely applied in studying gene expression and function in plant species due to its rapidity and efficiency. The hairy root system is an efficient tool for evaluating the activities of CRISPR/Cas systems. Cotton hairy roots were primarily induced through cotton tissue culture under aseptic conditions and by injecting cotton stem tips under non-aseptic conditions. However, both methods are lab-intensive and time-consuming. In this study, an efficient cotton hairy root induction procedure was established via infecting cotton hypocotyls with Agrobacterium rhizogenes under non-sterile conditions. Cotton seedlings with expanded cotyledons were decapitated with a slanted cut, and the residual hypocotyl (maintained 1 cm apical portion) was inoculated with A. rhizogenes. Over 90% of the infected explants from all three tested varieties could produce hairy roots after 8 days of inoculation. The effictiveness of the method was tested by overexpressing two reporter genes (eGFP and GUS). The transformation efficiency of the GUS and eGFP were ranged from50-68.18% and 40.9-68.18%. In addition, the editing efficiency of target sites in different CRISPR/Cas systems were also tested in hairy root. This method provided technical support for screening suitable target sites for cotton gene editing.
发根诱导系统因其快速性和高效性，已在植物物种的基因表达和功能研究中得到广泛应用。发根系统是评估CRISPR/Cas系统活性的有效工具。棉花发根主要通过无菌条件下的棉花组织培养以及非无菌条件下注射棉花茎尖来诱导。然而，这两种方法都需要大量实验室操作且耗时。在本研究中，通过在非无菌条件下用发根农杆菌感染棉花下胚轴，建立了一种高效的棉花发根诱导程序。将子叶展开的棉花幼苗斜切去顶，然后用发根农杆菌对接种剩余的下胚轴（保留1厘米顶端部分）。接种8天后，所有三个测试品种中超过90%的受感染外植体能够产生发根。通过过表达两个报告基因（eGFP和GUS）来测试该方法的有效性。GUS和eGFP的转化效率分别为50 - 68.18%和40.9 - 68.18%。此外，还在发根中测试了不同CRISPR/Cas系统中靶位点的编辑效率。该方法为筛选棉花基因编辑的合适靶位点提供了技术支持。
