Nguyen Doai Van, Hoang Trang Thi-Huyen, Le Ngoc Thu, Tran Huyen Thi, Nguyen Cuong Xuan, Moon Yong-Hwan, Chu Ha Hoang, Do Phat Tien
Laboratory of Plant Cell Biotechnology, Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam.
Department of Integrated Biological Science, Pusan National University, Busan, South Korea.
Front Plant Sci. 2022 Feb 11;12:770062. doi: 10.3389/fpls.2021.770062. eCollection 2021.
Hairy root induction system has been applied in various plant species as an effective method to study gene expression and function due to its fast-growing and high genetic stability. Recently, these systems have shown to be an effective tool to evaluate activities of CRISPR/Cas9 systems for genome editing. In this study, mediated hairy root induction was optimized to provide an effective tool for validation of plant transformation vector, CRISPR/Cas9 construct activities as well as selection of targeted gRNAs for gene editing in cucumber ( L.). Under the optimized conditions including OD at 0.4 for infection and 5 days of co-cultivation, the highest hairy root induction frequency reached 100% for the cucumber variety Choka F1. This procedure was successfully utilized to overexpress a reporter gene () and induce mutations in two homolog genes and using CRISPR/Cas9 system. For induced mutation, about 78% of transgenic hairy roots exhibited mutant phenotypes including sparse root hair and root hair-less. The targeted mutations were obtained in individual , , or both and genes by heteroduplex analysis and sequencing. The hairy root transformation system established in this study is sufficient and potential for further research in genome editing of cucumber as well as other plants.
发根诱导系统因其生长迅速和遗传稳定性高，已被应用于多种植物物种，作为研究基因表达和功能的有效方法。最近，这些系统已被证明是评估用于基因组编辑的CRISPR/Cas9系统活性的有效工具。在本研究中，对发根农杆菌介导的发根诱导进行了优化，以提供一种有效的工具，用于验证黄瓜（Cucumis sativus L.）的植物转化载体、CRISPR/Cas9构建体活性以及用于基因编辑的靶向gRNA的选择。在包括感染时OD为0.4和共培养5天的优化条件下，黄瓜品种Choka F1的最高发根诱导频率达到100%。该方法成功用于过表达一个报告基因（GUS），并使用CRISPR/Cas9系统在两个同源基因CsCER1和CsCER2中诱导突变。对于诱导突变，约78%的转基因发根表现出突变表型，包括根毛稀疏和无根毛。通过异源双链分析和测序，在单个CsCER1、CsCER2或CsCER1和CsCER2两个基因中获得了靶向突变。本研究建立的发根转化系统足以并有可能用于黄瓜以及其他葫芦科植物基因组编辑的进一步研究。
