Abstract
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, Rhizobium rhizogenes 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 (Cucumis sativus L.). Under the optimized conditions including OD650 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 (gus) and induce mutations in two Lotus japonicus ROOTHAIRLESS1 homolog genes CsbHLH66 and CsbHLH82 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 CsbHLH66, CsbHLH82, or both CsbHLH66 and CsbHLH82 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 cucumis plants.
Highlights
Originating in the Indian subcontinent, cucumber (Cucumis sativus L.) has become one of the top consumed vegetables widely grown in the world (Paris et al, 2012; Taha et al, 2020)
The targeted mutations including heterozygous, homozygous, and biallelic indels in two targeted genes CsbHLH66 and CsbHLH82 was obtained at primary and propagated transgenic hairy roots. These results provide an effective tool for gene expression, gene function, and genome editing studies in cucumber
Cotyledon pieces from 2-day-old seedlings of ChokaF1 cultivar were first inoculated with R. rhizogenes K599 strain to evaluate the effects of co-cultivation duration on induced hairy root efficacy
Summary
Originating in the Indian subcontinent, cucumber (Cucumis sativus L.) has become one of the top consumed vegetables widely grown in the world (Paris et al, 2012; Taha et al, 2020). Cucumber Hairy Root Transformation System are various approaches used to improve cucumber production such as optimizing cultivation conditions (Taha et al, 2020), plant grafting (Elsheery et al, 2020), and breeding. The genome editing boosted by CRISPR/ Cas systems has been widely applied on various crops as a powerful tool to improve plant traits and gene function studies (Zhang et al, 2021). Applications of genome editing in cucumber have been challenged by less effective transformation procedures that are time consuming and labor extensive processes (Hu et al, 2017). The improvement of procedures and the development of supporting tools are necessary to accelerate genetic engineering in cucumber
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