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<p>With the complete sequencing of the Tartary buckwheat (TB) genome, Studies are increasingly focusing on genes related to flavonoid metabolism and environmental adaptability. However, the absence of a mature TB genetic transformation system has limited studies on TB at the molecular level. There is a pressing need to establish effective transient expression systems for TB. This study utilized TB cotyledons and seeds as explants, and optimized transformation conditions by applying GUS driven by the CaMV35S promoter as a reporter for the transformation efficiency. Results indicated high transformation efficiency under specific conditions: an OD<sub>600</sub> of Agrobacterium at 0.9 with 3 days of co-cultivation for cotyledons, and an OD<sub>600</sub> of Agrobacterium at 1.5 with 2–3 days of co-cultivation during the middle filling stage for seeds. Furthermore, this system was used to demonstrate significant activation of the <i>FtFLS1</i> promoter by environmental stresses such as cold, UV-B and drought stress and a significant response of the <i>FtRDE2</i> promoter to cold stress, validating its utility for promoter analysis. Additionally, the system facilitated not only subcellular localization of characterized proteins including FtMYB6 and FtANX7, but also functional validation of key genes like <i>FtMYB6</i> and <i>FtAGPS1</i>. Conclusively, the established TB cotyledon and seed transient expression systems provide robust tools for studying promoter activity, subcellular localization, and gene function, offering new and effective means for advancing TB molecular biology research.</p>

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Establishment and application of novel transient cotyledon and seed transformation systems in Tartary buckwheat

  • 期刊论文

  • 发表日期：

    2025年2月10日

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  • Huala Wu,
  • Hui Li,
  • Jiayi Lin,
  • Yujun Ouyang,
  • Chenglei Li,
  • Hongyou Li,
  • Tao Wang,
  • Qi Wu,
  • Haixia Zhao

摘要

With the complete sequencing of the Tartary buckwheat (TB) genome, Studies are increasingly focusing on genes related to flavonoid metabolism and environmental adaptability. However, the absence of a mature TB genetic transformation system has limited studies on TB at the molecular level. There is a pressing need to establish effective transient expression systems for TB. This study utilized TB cotyledons and seeds as explants, and optimized transformation conditions by applying GUS driven by the CaMV35S promoter as a reporter for the transformation efficiency. Results indicated high transformation efficiency under specific conditions: an OD600 of Agrobacterium at 0.9 with 3 days of co-cultivation for cotyledons, and an OD600 of Agrobacterium at 1.5 with 2–3 days of co-cultivation during the middle filling stage for seeds. Furthermore, this system was used to demonstrate significant activation of the FtFLS1 promoter by environmental stresses such as cold, UV-B and drought stress and a significant response of the FtRDE2 promoter to cold stress, validating its utility for promoter analysis. Additionally, the system facilitated not only subcellular localization of characterized proteins including FtMYB6 and FtANX7, but also functional validation of key genes like FtMYB6 and FtAGPS1. Conclusively, the established TB cotyledon and seed transient expression systems provide robust tools for studying promoter activity, subcellular localization, and gene function, offering new and effective means for advancing TB molecular biology research.