An Efficient Hairy Root System for Validation of Plant Transformation Vector and CRISPR/Cas Construct Activities in Cucumber (Cucumis sativus L.)

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.

Peat-based hairy root transformation using Rhizobium rhizogenes as a rapid and efficient tool for easily exploring potential genes related to root-knot nematode parasitism and host response

“…1 c). Transgenic hairy roots enable the accomplishment of nematode parasitic cycle [ 26 , 41 , 43 ] and the functional analysis of the promoter for the target gene [ 44 , 45 ], inferring the capacity of the PCI method to explore candidate genes associated with nematode parasitism.…”

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene editing analysis in cotton

“…Therefore, it is essential to select efficient sgRNAs to reduce the workload. Hairy root system has been used for genome editing of various plant species, such as Medicago truncatula ( Zhang et al., 2020 ), cucumber ( Nguyen et al., 2021 ), oilseed rape ( Jedličková et al., 2022 ). For cotton, the hairy root system has been applied for studying promoter activity ( Kim et al., 2011 ), secondary metabolites production ( Verma et al., 2009 ; Kim et al., 2011 ), plant generation ( Cui et al., 2020 ), cotton-nematode interaction ( Wubben et al., 2009 ), however, it has not been used for genome editing.…”

Research on the function of CsMYB36 based on an effective hair root transformation system

“…Seeds of inbred CU2 cucumber ( Cucumis sativus L.) (Provided by Hunan Academy of Agricultural Sciences) were water-bathed at 55°C for 30 min after removing seed coats, surface sterilized in 75% alcohol for 30 s and then in 7.5% sodium hypochlorite for 15 min, then washed 4–5 times in sterile water 5 (Extended Data Figure S1A). Unwounded seeds were placed on MS30 medium ( Table 1 ) for 1 day in the dark ( Figure 1(a) and Extended Data Figure S1B), and then grown at 25 ± 1°C and 16 h light/8 h dark for 4 days to obtain the true leaf unfolding explants ( Figure 1(b) and Extended Data Figure S1C).…”

“…Hairy root transformation systems have been established in cucumber to study salt stress and root hair development. 5 , 6 However, the transformation frequency of the in vitro cotyledon transformation protocols require 34–37 days. 6 Although the in vivo puncture hypocotyl transformation method is simple and rapid for obtaining the transgenic hair roots, it does not enable subculturing preservation and propagation of transgenic root materials.…”