A fast, simple, high efficient and one-step generation of composite cucumber plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation

Fan, Yinglun; Xu, Fenglin; Zhou, Huizhen; Liu, Xinxin; Yang, Xin-Yue; Weng, Kaixia; Sun, Xinlu; Lyu, Shanhua

doi:10.1007/s11240-020-01781-x

Cited by 21 publications

(25 citation statements)

References 43 publications

(63 reference statements)

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“…The maps of all constructs and sequences are shown in Figure S5. The binary vector pGSE401-KtRfg1 ( Figure S5a), pCAMBIA1305 ( Figure S5b) and pYGUS1305 ( Figure S5c) were introduced into A. rhizogenes strain K599 [34,35] by electroporation, respectively. K599 strain was cultured as described previously [35].…”

Section: Plant Materials and Growth Conditionsmentioning

confidence: 99%

“…The binary vector pGSE401-KtRfg1 ( Figure S5a), pCAMBIA1305 ( Figure S5b) and pYGUS1305 ( Figure S5c) were introduced into A. rhizogenes strain K599 [34,35] by electroporation, respectively. K599 strain was cultured as described previously [35]. To make the inoculation medium, fresh culture K599 bacteria (HGCI) grown on the plate was resuspended using 0.25× Gamborg B-5 basal medium to OD600 ≈ 0.6-1.0.…”

Section: Plant Materials and Growth Conditionsmentioning

confidence: 99%

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One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation

et al. 2020

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Background: Agrobacterium rhizogenes-mediated (ARM) transformation is a highly efficient technique for generating composite plants composed of transgenic roots and wild-type shoot, providing a powerful tool for studying root biology. The ARM transformation has been established in many plant species, including soybean. However, traditional transformation of soybean, transformation efficiency is low. Additionally, the hairy roots were induced in a medium, and then the generated composite plants were transplanted into another medium for growth. This two-step operation is not only time-consuming, but aggravates contamination risk in the study of plant-microbe interactions. Results: Here, we report a one-step ARM transformation method with higher transformation efficiency for generating composite soybean plants. Both the induction of hairy roots and continuous growth of the composite plants were conducted in a single growth medium. The primary root of a 7-day-old seedling was decapitated with a slanted cut, the residual hypocotyl (maintained 0.7-1 cm apical portion) was inoculated with A. rhizogenes harboring the gene construct of interest. Subsequently, the infected seedling was planted into a pot with wet sterile vermiculite. Almost 100% of the infected seedlings could produce transgenic positive roots 16 days post-inoculation in 7 tested genotypes. Importantly, the transgenic hairy roots in each composite plant are about three times more than those of the traditional ARM transformation, indicating that the one-step method is simpler in operation and higher efficiency in transformation. The reliability of the one-step method was verified by CRISPR/Cas9 system to knockout the soybean Rfg1, which restricts nodulation in Williams 82 (Nod-) by Sinorhizobium fredii USDA193. Furthermore, we applied this method to analyze the function of Arabidopsis YAO promoter in soybean. The activity of YAO promoter was detected in whole roots and stronger in the root tips. We also extended the protocol to tomato. Conclusions: We established a one-step ARM transformation method, which is more convenient in operation and higher efficiency (almost 100%) in transformation for generating composite soybean plants. This method has been validated in promoter functional analysis and rhizobia-legume interactions. We anticipate a broad application of this method to analyze root-related events in tomato and other plant species besides soybean.

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Section: Plant Materials and Growth Conditionsmentioning

confidence: 99%

Section: Plant Materials and Growth Conditionsmentioning

confidence: 99%

One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation

et al. 2020

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“…Therefore, A . tumefaciens -mediated transformation is time-consuming, recalcitrant nature and not efficient enough to allow the high-throughput for functional genomic research [ 23 ]. Besides A .…”

Section: Discussionmentioning

confidence: 99%

Agrobacterium rhizogenes-mediated hairy root transformation as an efficient system for gene function analysis in Litchi chinensis

Qin

Wang

et al. 2021

Plant Methods

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Background Litchi chinensis Sonn. is an economically important fruit tree in tropical and subtropical regions. However, litchi functional genomics is severely hindered due to its recalcitrance to regeneration and stable transformation. Agrobacterium rhizogenes-mediated hairy root transgenic system provide an alternative to study functional genomics in woody plants. However, the hairy root transgenic system has not been established in litchi. Results In this study, we report a rapid and highly efficient A. rhizogenes-mediated co-transformation system in L. chinensis using Green Fluorescent Protein (GFP) gene as a marker. Both leaf discs and stem segments of L. chinensis cv. ‘Fenhongguiwei’ seedlings were able to induce transgenic hairy roots. The optimal procedure involved the use of stem segments as explants, infection by A. rhizogenes strain MSU440 at optical density (OD600) of 0.7 for 10 min and co-cultivation for 3 days, with a co-transformation efficiency of 9.33%. Furthermore, the hairy root transgenic system was successfully used to validate the function of the key anthocyanin regulatory gene LcMYB1 in litchi. Over-expression of LcMYB1 produced red hairy roots, which accumulated higher contents of anthocyanins, proanthocyanins, and flavonols. Additionally, the genes involving in the flavonoid pathway were strongly activated in the red hairy roots. Conclusion We first established a rapid and efficient transformation system for the study of gene function in hairy roots of litchi using A. rhizogenes strain MSU440 by optimizing parameters. This hairy root transgenic system was effective for gene function analysis in litchi using the key anthocyanin regulator gene LcMYB1 as an example.

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“…Recently, the hairy root transformation has been widely utilized to validate and optimize induced mutagenesis by the CRISPR/Cas9 system (Michno et al, 2015;Du et al, 2016;Jacobs and Martin, 2016;Li et al, 2019;Le et al, 2020). Both in vitro and in vivo cucumber hairy root cultures have been established for different research purposes (Shi and Lindemann, 2006;Anuar et al, 2011;Fan et al, 2020). However, the application of cucumber hairy root transformation for genome editing using CRISPR/Cas systems has not been reported yet.…”

Section: Introductionmentioning

confidence: 99%

“…However, the application of cucumber hairy root transformation for genome editing using CRISPR/Cas systems has not been reported yet. Moreover, it has been reported that hairy root induction frequency is affected by cucumber varieties, bacterial strains, and transformation methods (JingLong et al, 2017;Fan et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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

Nguyen

Hoang

et al. 2022

Front. Plant Sci.

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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.

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A fast, simple, high efficient and one-step generation of composite cucumber plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation

Cited by 21 publications

References 43 publications

One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation

One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation

Agrobacterium rhizogenes-mediated hairy root transformation as an efficient system for gene function analysis in Litchi chinensis

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

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