Establishment of Agrobacterium rhizogenes-mediated Transformation System in Melon (Cucumis melo) and Rapid Detection of CRISPR/Cas9 Target Sites
SHU Yao1,2, YANG Song-Han2,3, KONG Ke-Xing1,2, LYU Ruo-Han1,2, LYU Gui-Yun3, ZHANG Chun-Qiu2*, SONG Shi-Qing1*
1 College of Horticultural Science & Technology, Hebei Normal University of Science & Technology/Hebei Key Laboratory of Horticultural Germplasm Excavation and Innovative Utilization/Hebei Higher Institute Application Technology Research and Development Center of Horticultural Plant Biological Breed, Qinhuangdao 066004, China; 2 Beijing Vegetables Research Center, Beijing Academy of Agriculture and Forestry Sciences/State Key Laboratory of Vegetable Biobreeding/National Engineering Research Center for Vegetables/Beijing Key Laboratory of Vegetable Germplasms Improvement/ Key Laboratory of Biology and Genetics Improvement of Horticultural Crops (North China)/Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture and Rural Affairs, Beijing 100097, China; 3 College of Horticulture, Hebei Agricultural University, Baoding 071000, China
Abstract：The utilization of the CRISPR/Cas9 system for gene editing is an effective method for investigating gene functions and pursuing genetic improvement in melons (Cucumis melo). However, there lacks a method that rapidly detects the target sites of CRISPR/Cas9 vectors prior to genetic transformation. In this study, the melon phytoene desaturase (PDS) gene CmPDS was used as target gene, two sgRNAs were designed and introduced together into the CRISPR/Cas9 vector. Agrobacterium rhizogenes K599 was used to infect cotyledon explants of the thick-skinned melon 'K7-2' and the thin-skinned melon 'LB'. The induced adventitious roots were firstly identified using Bar test strips, and then the target regions were sequenced to see if the regions were edited and clarified the types of mutation. The PCR products sequencing results showed that the 2 target sites were all edited. Further sequencing of the individual PCR products demonstrated an editing efficiency of 100% for target 1 and 76.5% for target 2, with multiple mutation types observed, including insertions and deletions of single or multiple bases, as well as large deletions between 2 target sites. This study successfully established a genetic transformation system for melons that was mediated by the A. rhizogenes, and achieved rapid detection target sites of CRISPR/Cas9 vector, and also validated the effectiveness of using the CRISPR/Cas9 system in melon gene editing. This study provides an important technical support for genetic functional studies and genetic improvements of melons.
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