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Related Experiment Video

Updated: Apr 4, 2026

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots
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High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

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Optimised Protocol for Efficient Generation, Confirmation, Transformation, and CRISPR Editing of Grapevine Hairy

Sasha M Tietz1, Kyle Brenner1, Tumelo Moyo1

  • 1South African Grape and Wine Research Institute, Stellenbosch University, JH Neethling building, Victoria street, Stellenbosch, 7600, South Africa.

Journal of Experimental Botany
|April 3, 2026
PubMed
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A high-density integrated map for grapevine based on three mapping populations genotyped by the Vitis18K SNP chip.

TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik·2022
Same author

Grapevine Plasticity in Response to an Altered Microclimate: Sauvignon Blanc Modulates Specific Metabolites in Response to Increased Berry Exposure.

Plant physiology·2015
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We developed a standardized method for hairy root cultures (HRCs) in grapevines, improving their generation and maintenance. This optimized protocol enables efficient genetic modification, advancing grapevine biotechnology and functional genomics.

Area of Science:

  • Plant Biotechnology
  • Genomics
  • Molecular Biology

Background:

  • Hairy root cultures (HRCs) are valuable in plant biotechnology but suffer from inconsistent establishment.
  • Variability in HRC generation limits their widespread application in grapevine research and development.

Purpose of the Study:

  • To establish a standardized and optimized reference methodology for the routine generation, multiplication, and maintenance of HRCs across diverse grapevine genotypes.
  • To develop and validate molecular tools for efficient transformation and selection of grapevine HRCs.
  • To demonstrate the utility of the developed HRC system for gene function studies and biotechnological applications.

Main Methods:

  • Evaluation of multiple Rhizobium strains, grapevine cultivars, explant types, infection protocols, and growth media.
Keywords:
Rhizobium rhizogenesVitisCRISPRGrapevineHairy rootsUniversal multiplex primers

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  • Development of molecular tools including universal multiplex primers, antibiotic resistance markers (kanamycin, hygromycin), and fluorescent reporters (DsRed, eyGFPuv).
  • Application of the HRC system for overexpression of VviMYBA1 and CRISPR/Cas9 gene editing of VviPUB19.

    • Main Results:

    • A robust protocol for HRC generation was established, effective across various grapevine genotypes and adaptable to other plant species (Sutherlandia frutescens, Aspalathus linearis).
    • DsRed was identified as a versatile fluorescent reporter for HRC transformation.
    • Successful VviMYBA1 overexpression led to increased anthocyanin accumulation, and CRISPR/Cas9 editing of VviPUB19 was achieved in grapevine HRCs.

    Conclusions:

    • The standardized HRC methodology provides a reliable platform for grapevine research.
    • The developed molecular tools and HRC system facilitate rapid gene function studies and biotechnological advancements.
    • CRISPR-edited grapevine HRCs offer an efficient alternative to whole-plant transformations for functional genomics.