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.
SHU Yao,YANG Song-Han,KONG Ke-Xing, et al. Establishment of Agrobacterium rhizogenes-mediated Transformation System in Melon (Cucumis melo) and Rapid Detection of CRISPR/Cas9 Target Sites[J]. 农业生物技术学报, 2025, 33(4): 924-932.
[1] 蒋悦, 罗倩, 杨琴, 等. 2021. 烟草NtMYB4a基因CRISPR/Cas9敲除载体构建与遗传转化[J]. 农业生物技术学报, 29(9): 1698-1709.(Jiang Y, Luo Q, Yang Q, et al.2021. Construction and genetic transformation of tobacco (Nicotiana tabacum) NtMYB4a gene CRISPR/Cas9 knockout vector[J]. Journal of Agricultural Biotechnology, 29(9): 1698-1709.) [2] 任如意, 薛巨坤, 国会艳, 等. 2017. 北玄参毛状根诱导及其植株再生[J]. 植物学报, 52(06): 783-787.(Ren R Y, Xue J K, Guo H Y, et al.2017. Induction of hairy roots of Scrophularia buergeriana and its plant regeneration[J]. Chinese Bulletin of Botany, 52(06): 783-787.) [3] 施和平, 王蓓, 杨树楠, 等. 2016. 五寸石竹毛状根诱导及其植株再生[J]. 植物学报, 51(03): 363-368.(Shi H P, Wang B, Yang S N, et al.2016. Induction of hairy roots of Dianthus chinensis and its plant regeneration[J]. Chinese Bulletin of Botany, 51(03): 363-368.) [4] 唐伶俐, 徐龙兰, 徐永阳, 等. 2024. 农杆菌介导的厚皮甜瓜遗传转化体系的建立[J]. 果树学报, 2024, 41(03): 533-542.(Tang L L, Xu L L, Xu Y Y, et al.2024. Establishment of genetic transformation system mediated by Agrobacterium in muskmelon[J]. Journal of Fruit Science, 2024, 41(03): 533-542.) [5] 王平勇, 徐永阳, 赵光伟, 等. 2019. 发根农杆菌介导甜瓜转基因过表达体系的建立[J]. 中国瓜菜, 32(12): 15-18, 30.(Wang P Y, Xu Y Y, Zhao G W, et al. 2019. Establishment of Agrobacterium rhizogenes-mediated gene overexpression system in melon[J]. Chinese Cucurbits and Vegetables, 32(12): 15-18, 30.) [6] 王燕燕, 王丹, 崔馨文, 等. 2023. 植物毛状根应用的研究[J]. 食品与发酵工业, 49(21): 293-302.(Wang Y Y, Wang D, Cui X W, et al.2023. Advances on the application of hairy roots in plants[J]. Food and Fermentation Industries, 49(21): 293-302.) [7] 徐正贤, 江洁, 罗灶霞, 等. 2021. 温郁金组织培养高效再生及毛状根诱导体系初探[J]. 杭州师范大学学报(自然科学版), 20(02): 125-129+142.(Xu Z X, Jiang J, Luo Z X, et al. 2021. Efficient plant regeneration via tissue culture and primary study on the hairy root induction of Curcuma wenyujin[J]. Journal of Hangzhou Normal University (Natural Science Edition), 20(02): 125-129+142.) [8] 张春秋, 李斯贝, 胡紫玉, 等. 2022. 不同类型甜瓜高效再生体系的建立[J]. 中国瓜菜, 35(1): 5.(Zhang C Q, Li S B, Hu Z Y, et al. 2022. Establishment of regeneration system for different varieties of melons[J]. Chinese Cucurbits and Vegetables, 35(1): 5.) [9] 周雪婷, 武凯凯, 崔蓝芳, 等. 2024. 苹果发根农杆菌介导转化体系构建与优化[J/OL]. 果树学报, 1-14.(Zhou X T, Wu K K, Cui L F, et al.2024. Construction and optimization of transformation system mediated by Agrobacterium rhizogenes in apple[J/OL]. Journal of Fruit Science, 1-14.) [10] Deng H, Pei Y, Xu X, et al.2024. Ethylene-MPK8-ERF.C1-PR module confers resistance against Botrytis cinerea in tomato fruit without compromising ripening[J]. New Phytologist, 242(2): 592-609. [11] Huang P, Lu M, Li X, et al.2022. An efficient Agrobacterium rhizogenes-mediated hairy root transformation method in a soybean root biology study[J]. International Journal of Molecular Sciences, 23(20): 12261. [12] Kaul T, Thangaraj A, Jain R, et al.2024. CRISPR/Cas9-mediated homology donor repair base editing system to confer herbicide resistance in maize (Zea mays L.)[J]. Plant Physiology and Biochemistry, 207: 108374. [13] Ma X, Zhang Q, Zhu Q, et al.2015. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants[J]. Molecular Plant, 8(8): 1274-1284. [14] Nguyen D, Hoang T, Le N, et al.2022. An efficient hairy root system for validation of plant transformation vector and CRISPR/Cas construct activities in cucumber (Cucumis sativus L.)[J]. Frontiers in Plant Science, 12: 770062. [15] Ran F, Hsu P, Wright J, et al.2013. Genome engineering using the CRISPR-Cas9 system[J]. Nature Protocols, 8, 2281-2308. [16] Ren X, Yang Z, Xu J, et al.2014. Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila[J]. Cell Reports, 9(3): 1151-1162. [17] Wan D Y, Guo Y, Cheng Y, et al.2020. CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera)[J]. Horticulture Research, 7, 116. [18] Wang T, Wei J J, Sabatini D M, et al.2014. Genetic screens in human cells using the CRISPR-Cas9 system[J]. Science, 343(6166): 80-84. [19] Xing H L, Dong L, Wang Z P.et al.2014. A CRISPR/Cas9 toolkit for multiplex genome editing in plants[J]. BMC Plant Biology, 14: 327. [20] Xu X, Wang W, Du Y, et al.2024. A single-nucleotide substitution in the leucine-rich-repeat-only gene CsLRR1 confers powdery mildew resistance in cucumber[J]. Plant Communications, 5(3): 100774. [21] Zhang R, Chen S, Meng X, et al.2021. Generating broad-spectrum tolerance to ALS-inhibiting herbicides in rice by base editing[J]. Science China (Life Sciences), 64(10): 1624-1633 [22] Zhou L, Wang Y, Wang P, et al.2022. Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene editing analysis in cotton[J]. Frontiers in Plant Science, 13: 1059404.
