Use of GRF-GIF chimeras and a ternary vector system to improve maize (Zea mays L.) transformation frequency,The Plant Journal

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Use of GRF-GIF chimeras and a ternary vector system to improve maize (Zea mays L.) transformation frequency
The Plant Journal ( IF 6.2 ) Pub Date : 2024-06-23 , DOI: 10.1111/tpj.16880
Wout Vandeputte _{1,

2} , Griet Coussens _{1,

2} , Stijn Aesaert _{1,

2} , Jari Haeghebaert _{1,

2} , Lennert Impens _{1,

2} , Mansour Karimi _{1,

2} , Juan M Debernardi ₃ , Laurens Pauwels _{1,

2}

Affiliation

Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium-mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene-editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1-VIR2 virulence helper plasmid to improve T-DNA delivery, and/or expressing a fusion protein between a GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1-VIR2 helper vector with no effect on event quality regarding T-DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5- to 6.5-fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR-/Cas9-mediated gene editing. Our results demonstrate how a GRF-GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene-editing applications and molecular biology studies in maize.

中文翻译：

使用 GRF-GIF 嵌合体和三元载体系统提高玉米 (Zea mays L.) 转化频率

玉米（ Zea mays L.）是一种重要作物，其农艺和工业应用已被广泛研究，是遗传研究的主要经典模式生物之一。农杆菌介导的未成熟玉米胚胎的转化是引入转基因的常用方法，但低转化频率仍然是许多基因编辑应用的瓶颈。以前增强转化的方法包括改进组织培养基和使用形态发生调节剂，例如BABY BOOM和WUSCHEL2 。在这里，我们表明，使用 pVS1-VIR2 毒力辅助质粒可以提高频率，以改善 T-DNA 递送，和/或表达生长调节因子( GRF ) 和GRF 相互作用因子( GIF ) 蛋白之间的融合蛋白以改善再生。使用潮霉素作为避免逃逸的选择剂，使用 pVS1-VIR2 辅助载体时，玉米自交系 B104 的转化频率从 2.3% 显着提高至 8.1%，且对 T-DNA 拷贝数的事件质量没有影响。与 ZmGRF1 和 ZmGIF1 之间的新型融合蛋白相结合，转化频率进一步提高了 3.5 至 6.5 倍，且对植物生长没有明显影响，同时允许高效的 CRISPR-/Cas9 介导的基因编辑。我们的结果证明了 GRF-GIF 嵌合体与三元载体系统的结合如何有可能进一步提高玉米基因编辑应用和分子生物学研究的效率。

更新日期：2024-06-23

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