Aliu Ephraim, Ji Qing, Wlazlo Anna, Grosic Sehiza, Azanu Mercy K, Wang Kan, Lee Keunsub
Department of Agronomy, Iowa State University, Ames, IA, United States.
Crop Bioengineering Center, Iowa State University, Ames, IA, United States.
Front Plant Sci. 2024 Jul 23;15:1429353. doi: 10.3389/fpls.2024.1429353. eCollection 2024.
-mediated transformation is an essential tool for functional genomics studies and crop improvements. Recently developed ternary vector systems, which consist of a T-DNA vector and a compatible virulence () gene helper plasmid (ternary helper), demonstrated that including an additional gene helper plasmid into disarmed strains significantly improves T-DNA delivery efficiency, enhancing plant transformation. Here, we report the development of a new ternary helper and thymidine auxotrophic strains to boost -mediated plant transformation efficiency. Auxotrophic strains are useful in reducing overgrowth after the co-cultivation period because they can be easily removed from the explants due to their dependence on essential nutrient supplementation. We generated thymidine auxotrophic strains from public strains EHA101, EHA105, EHA105D, and LBA4404. These strains exhibited thymidine-dependent growth in the bacterial medium, and transient expression assay using Arabidopsis seedlings showed that they retain similar T-DNA transfer capability as their original strains. Auxotrophic strains EHA105Thy- and LBA4404T1 were tested for maize B104 immature embryo transformation using our rapid transformation method, and both strains demonstrated comparable transformation frequencies to the control strain LBA4404Thy-. In addition, our new ternary helper pKL2299A, which carries the gene from pTiBo542 in addition to other gene operons (, , , , , and ), demonstrated consistently improved maize B104 immature embryo transformation frequencies compared to the original version of pKL2299 (33.3% vs 25.6%, respectively). Therefore, our improved system, including auxotrophic disarmed strains and a new ternary helper plasmid, can be useful for enhancing plant transformation and genome editing applications.
介导的转化是功能基因组学研究和作物改良的重要工具。最近开发的三元载体系统由一个T-DNA载体和一个兼容的毒力（）基因辅助质粒（三元辅助质粒）组成，该系统表明，在解除武装的菌株中加入额外的基因辅助质粒可显著提高T-DNA传递效率，增强植物转化。在此，我们报告了一种新型三元辅助质粒和胸苷营养缺陷型菌株的开发，以提高介导的植物转化效率。营养缺陷型菌株在共培养期后减少过度生长方面很有用，因为它们依赖必需营养补充，所以很容易从外植体中去除。我们从公共菌株EHA101、EHA105、EHA105D和LBA4404中产生了胸苷营养缺陷型菌株。这些菌株在细菌培养基中表现出依赖胸苷的生长，并且使用拟南芥幼苗进行的瞬时表达分析表明，它们保留了与原始菌株相似的T-DNA转移能力。使用我们的快速转化方法，对营养缺陷型菌株EHA105Thy-和LBA4404T1进行了玉米B1突变体未成熟胚转化测试，两种菌株的转化频率与对照菌株LBA4404Thy-相当。此外，我们的新型三元辅助质粒pKL2299A除了其他基因操纵子（、、、、和）外，还携带来自pTiBo542的基因，与原始版本的pKL2299相比，其玉米B1突变体未成熟胚转化频率持续提高（分别为33.3%和25.6%）。因此，我们改进的系统，包括营养缺陷型解除武装菌株和新型三元辅助质粒，可用于增强植物转化和基因组编辑应用。
