Streamlined Agrobacterium rhizogenes-mediated hairy root transformation for efficient CRISPR/Cas9-based gene editing evaluation in diverse Citrullus cultivars  

作　　者：Yong Zhao Hongju Zhu Xuqiang Lu Muhammad Anees Nan He Dongdong Yang Zihao Chen Zonglie Hong Junhong Zhang Wenge Liu 

机构地区：[1]National Key Laboratory for Germplasm Innovation&Utilization of Horticultural Crops,Zhengzhou Fruit Research Institute,Chinese Academy of Agricultural Sciences,Zhengzhou,Henan 450009,China [2]National Key Laboratory for Germplasm Innovation&Utilization of Horticultural Crops,Huazhong Agricultural University,Wuhan,Hubei 430070,China [3]Department of Plant Sciences,University of Idaho,Moscow,ID 83844,USA [4]Zhongyuan Research Center,Chinese Academy of Agricultural Sciences,Xinxiang,Henan 453003,China

出　　处：《Horticultural Plant Journal》2025年第2期816-826,共11页园艺学报(英文版)

基　　金：supported by the Basic Research Funds of Chinese Academy of Agricultural Sciences(Grant No.1610192023201);the National Key Research and Development Program of China(Grant No.SQ2023YFE0201239);China Agriculture Research System of MOF and MARA(Grant No.CARS-25-03);the Major Science and Technology Projects of Henan Province(Grant No.221100110400);the Funding of Joint Research on Agricultural Variety Improvement of Henan Province(Grant No.2022010503);the Agricultural Science and Technology Innovation Program(Grant No.CAAS-ASTIP-2021-ZFRI)。

摘　　要：The CRISPR/Cas9 genome-editing system serves as a pivotal tool for enhancing crop genetics.Within this system,single guide RNAs(sgRNAs)are instrumental in the precise cleavage of DNA double strands.However,the efficiency of gene editing varies among sgRNAs,emphasizing the need to meticulously select target sites,especially in the context of Citrullus lanatus,a species notorious for its challenging and inefficient generation of transgenic plants through stable transformation.This study employed an Agrobacterium rhizogenes-mediated hairy root method to assess effective target sites for gene editing of ClCIPK17 across various Citrullus species.Hairy roots were successfully induced in different plant tissues at diverse growth stages of Citrullus lanatus,Citrullus mucosospermus,and Citrullus amarus.Employing a vector with two sgRNAs(sgRNA1 and sgRNA5)positioned within conserved regions of exon 1 and exon 5 of ClCIPK17 in the CRISPR/Cas9 system,targeted mutations were detected in 90.9%of accessions across the four Citrullus species.Notably,73.94%of all examined hairy roots exhibited mutations at the sgRNA1 site,while the sgRNA5 site showed no mutations.Among the 31 different mutation types identified at the sgRNA1 site,base deletion was the most prevalent.Using the sgRNA1 site of ClCIPK17,stable transgenic watermelon buds were obtained from explants,and the targeted mutations of the sgRNA1 site were confirmed.These findings underscore the viability of the hairy root transformation system in assessing the editing efficiency of sgRNA targets in diverse Citrullus species.

关 键 词：Citrullus lanatus sgRNAs Agrobacterium rhizogenes Hairy root transformation CRISPR/Cas9 

分 类 号：S651[农业科学—果树学]