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<p>Hairy roots induced by <i>Agrobacterium</i> spp. are widely used in plant biotechnology. Composite plants, consisting of a wild-type shoot and a mix of transformed hairy roots and non-transformed roots, offer a versatile platform for investigating gene function, plant-microbe interactions, and systemic signaling. However, existing protocols for composite plant generation in the model <i>Arabidopsis thaliana</i> remain underdeveloped and inefficient limiting their broader application in basic research. Here, we optimized an <i>Agrobacterium</i> strain K599-mediated hairy root transformation protocol for <i>A. thaliana</i> Columbia-0 using a fractional factorial design to systematically evaluate key transformation parameters. These included bacterial growth medium, inoculation strategy, acetosyringone supplementation, and photoperiod. The optimized protocol, combining ATGN medium and a cut-and-coat inoculation strategy, consistently achieved high average transformation efficiencies (~80%). To improve hairy root identification, we engineered a K599 strain with the mCherry reporter integrated into the Ri plasmid T-DNA. This strain ensures that mCherry expressing roots contain the Ri plasmid T-DNA, eliminating ambiguity from the use of binary vector-based reporters for hairy root identification. The engineered strain showed high transformation efficiency comparable with wild-type strain and provides a robust tool for accurate identification of hairy roots. Our optimized workflow enables the use of hairy roots for basic research in composite <i>A. thaliana</i> plants and provides an <i>Agrobacterium</i> toolkit to support future hairy root studies.</p>

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A highly efficient platform for Arabidopsis hairy root induction using Agrobacterium strain K599

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月10日

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  • Savio D. Rodrigues,
  • Dries Delmotte,
  • Gianni De Coster,
  • Ona Sweron,
  • Eveline Van den Steen,
  • Jonas De Saeger,
  • Laurens Pauwels,
  • Barbara De Coninck

摘要

Hairy roots induced by Agrobacterium spp. are widely used in plant biotechnology. Composite plants, consisting of a wild-type shoot and a mix of transformed hairy roots and non-transformed roots, offer a versatile platform for investigating gene function, plant-microbe interactions, and systemic signaling. However, existing protocols for composite plant generation in the model Arabidopsis thaliana remain underdeveloped and inefficient limiting their broader application in basic research. Here, we optimized an Agrobacterium strain K599-mediated hairy root transformation protocol for A. thaliana Columbia-0 using a fractional factorial design to systematically evaluate key transformation parameters. These included bacterial growth medium, inoculation strategy, acetosyringone supplementation, and photoperiod. The optimized protocol, combining ATGN medium and a cut-and-coat inoculation strategy, consistently achieved high average transformation efficiencies (~80%). To improve hairy root identification, we engineered a K599 strain with the mCherry reporter integrated into the Ri plasmid T-DNA. This strain ensures that mCherry expressing roots contain the Ri plasmid T-DNA, eliminating ambiguity from the use of binary vector-based reporters for hairy root identification. The engineered strain showed high transformation efficiency comparable with wild-type strain and provides a robust tool for accurate identification of hairy roots. Our optimized workflow enables the use of hairy roots for basic research in composite A. thaliana plants and provides an Agrobacterium toolkit to support future hairy root studies.