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CRISPR-Cas9-mediated genome editing has revolutionized functional genomics and crop improvement. However, to maximize the adoption of the CRISPR-Cas9 technology, an efficient method to transform and regenerate genetically edited plants is necessary. In this protocol, we describe a detailed method to generate a CRISPR-Cas9 construct based on the Golden Gate cloning system and a simple and efficient method to transform and regenerate tomato plants from cotyledons co-cultured with Agrobacterium. Our protocol allows the production of at least ten Cas-positive independent lines from one hundred cotyledons. This protocol is routinely used in our laboratory to obtain tomato mutant lines and has been proven effective across several genotypes.

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A Simple and Efficient Protocol to Transform and Regenerate CRISPR-Cas9-Mediated Genome-Edited Tomato Plants

  • 实验方案

  • 发表日期：

    2025年

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  • Eduardo Martínez-Estrada,
  • Daniela de la Mora-Franco,
  • Stefan de Folter

摘要

CRISPR-Cas9-mediated genome editing has revolutionized functional genomics and crop improvement. However, to maximize the adoption of the CRISPR-Cas9 technology, an efficient method to transform and regenerate genetically edited plants is necessary. In this protocol, we describe a detailed method to generate a CRISPR-Cas9 construct based on the Golden Gate cloning system and a simple and efficient method to transform and regenerate tomato plants from cotyledons co-cultured with Agrobacterium. Our protocol allows the production of at least ten Cas-positive independent lines from one hundred cotyledons. This protocol is routinely used in our laboratory to obtain tomato mutant lines and has been proven effective across several genotypes.