An efficient and universal protoplast-based transient gene expression system for genome editing in Brassica crops

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外文摘要：Protoplast-based transient gene expression system has been widely used in plant genome editing because of its simple operation and less time-consuming.In order to establish a universal protoplast-based transient transfection system for verifying activities of genome editing vectors containing targets in Brassica,we systematically optimized factors affecting protoplast isolation and transient gene expression.We established an efficient protoplast-based transient gene expression system(PTGE)in Chinese cabbage,achieving high protoplast yield of 4.9 × 105·g-1FW,viability over 95％,and transfection efficiency of 76％.We showed for the first time that pretreatment of protoplasts with a hypotonic MMG could significantly enhance the transfection efficiency.Furthermore,protoplasts incubated at 37 ℃ for 6 min improved the transfection efficiency to 86％.We also demonstrated that PTGE worked well(more than 50％transfection efficiency)in multiple Brassica species including cabbage,Pak Choi,Chinese kale,and turnip.Finally,PTGE was used for validating the activities of CRISPR/Cas9 vectors containing targets in Chinese cabbage,cabbage,and pak choi,demonstrating the broad applicability of the established PTGE for genome editing in Brassica crops.

外文关键词：

Chinese cabbagePTGETransfection efficiencyCRISPR/Cas9

作者：

Xiaoxiao Yu、Jintai Yu、Yin Lu、Wenjing Li、Guanzhong Huo、Jun Zhang、Yan Li、Jianjun Zhao、Jun Li

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作者单位：

State Key Laboratory of North China Crop Improvement and Regulation,College of Life Sciences,Hebei Agricultural University,Baoding,Hebei 071001,China

Hebei Key Laboratory of Plant Physiology and Molecular Pathology,Hebei Agricultural University,Baoding,Hebei 071001,China

College of Modern Science and Technology,Hebei Agricultural University,Baoding,Hebei 071001,China

Key Laboratory of Vegetable Germplasm Innovation and Utilization of Hebei,Collaborative Innovation Center of Vegetable Industry in Hebei,College of Horticulture,Hebei Agricultural University,Baoding,Hebei 071001,China

Human Resource Department,Hebei Agricultural University,Baoding,Hebei 071001,China

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基金：

Key project of National Natural Science Foundation of ChinaInnovative Research Group Project of Hebei Natural Science FoundationS & T Program of HebeiS & T Program of HebeiNatural Science Foundation of HebeiStarting Grant from Hebei Agricultural University

项目编号：

32330096C202420424621372901D23567601HC2023204119YJ201958

出版年：

2024

DOI：

10.1016/j.hpj.2024.06.001

园艺学报(英文版)

CSTPCD

ISSN：

年,卷(期)：2024.10(4)

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