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<p>Conventional breeding of pure line plants is a lengthy process, often requiring multiple generations of self-fertilization. Doubled haploid (DH) technology offers an accelerated alternative by enabling the production of completely homozygous lines within a single generation. Targeted mutation of the <i>Domain of Unknown Function 679 membrane protein</i> (<i>DMP</i>) gene has recently emerged as an effective tool for in vivo haploid induction. In this study, the CRISPR/Cas9 system was employed to edit the tomato <i>SlDMP</i> gene, achieving transformation and editing efficiencies of 25.86% and 7.33%, respectively. A selected homozygous line, DMPT1-233, carrying a 1-bp cytosine deletion that resulted in a truncated DUF679 domain, was evaluated for its haploid induction ability. While pollen viability in the edited line remained comparable to the wild type (76% vs. 74%), DMPT1-233 successfully induced haploids across four different maternal backgrounds. Haploid induction rates (HIR) ranged from 0.16 to 1.12%, with the highest efficiency (1.12%) observed in the TM-3 maternal line. Haploid plants were confirmed by flow cytometry and displayed characteristic phenotypes, including significantly reduced plant stature, narrower leaves, and complete male sterility. Quantitative analysis showed that haploid stomatal guard cells were significantly smaller than diploids, measuring approximately 74% in length and 80% in width. Genotypic analysis confirmed the maternal origin of all induced haploids. These findings demonstrate that CRISPR/Cas9-mediated <i>DMP</i> mutations in tomato generate functional haploid inducers, providing a promising approach for the rapid development of inbred lines to accelerate tomato breeding.</p>

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Application of CRISPR/Cas9 genome editing for maternal haploid production in tomato (Solanum lycopersicum L.)

  • 期刊论文

  • 发表日期：

    2026年3月25日

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  • Phatcharee Buathong,
  • Roypim Sukkasem,
  • Saengtong Pongjaroenkit,
  • Yuppayao Kophimai,
  • Jutamas Kumchai,
  • Supachai Vuttipongchaikij,
  • Chotipa Sakulsingharoj

摘要

Conventional breeding of pure line plants is a lengthy process, often requiring multiple generations of self-fertilization. Doubled haploid (DH) technology offers an accelerated alternative by enabling the production of completely homozygous lines within a single generation. Targeted mutation of the Domain of Unknown Function 679 membrane protein (DMP) gene has recently emerged as an effective tool for in vivo haploid induction. In this study, the CRISPR/Cas9 system was employed to edit the tomato SlDMP gene, achieving transformation and editing efficiencies of 25.86% and 7.33%, respectively. A selected homozygous line, DMPT1-233, carrying a 1-bp cytosine deletion that resulted in a truncated DUF679 domain, was evaluated for its haploid induction ability. While pollen viability in the edited line remained comparable to the wild type (76% vs. 74%), DMPT1-233 successfully induced haploids across four different maternal backgrounds. Haploid induction rates (HIR) ranged from 0.16 to 1.12%, with the highest efficiency (1.12%) observed in the TM-3 maternal line. Haploid plants were confirmed by flow cytometry and displayed characteristic phenotypes, including significantly reduced plant stature, narrower leaves, and complete male sterility. Quantitative analysis showed that haploid stomatal guard cells were significantly smaller than diploids, measuring approximately 74% in length and 80% in width. Genotypic analysis confirmed the maternal origin of all induced haploids. These findings demonstrate that CRISPR/Cas9-mediated DMP mutations in tomato generate functional haploid inducers, providing a promising approach for the rapid development of inbred lines to accelerate tomato breeding.