Leite Dias Sara, Rizzo Paride, D'Auria John Charles, Kochevenko Andriy
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, 06466 Seeland, Germany.
Int J Mol Sci. 2025 May 6;26(9):4425. doi: 10.3390/ijms26094425.
Buckwheat is a promising crop with grains that are rich in nutrients and bioactive compounds. Genome sequence data for common and Tartary buckwheat have recently become available. Currently, there is a critical need for the development of a simple and reliable transient gene expression protocol, as well as a stable genetic transformation method, to facilitate metabolic engineering of bioactive compounds, functional analysis of genes, targeted editing, and, in a long-term perspective, to accelerate the breeding process in buckwheat. In this paper, we report optimized methods for -mediated transient and stable transformation of and . . Leaf and cotyledon tissues were infiltrated with an . -bearing construct containing and reporter genes. Histochemical staining and Western blotting were used to confirm the expression of reporter proteins. We also demonstrate the usefulness of the developed method for engineering the gramine biosynthetic pathway in buckwheat. and genes were transiently expressed in buckwheat leaves, and the de novo production of gramine was confirmed by LC-MS. Moreover, in planta genetic transformation of common and Tartary buckwheat with a reporter gene () and selectable marker gene () was achieved by -mediated vacuum infiltration. Genomic integration of the construct was confirmed by polymerase chain reaction (PCR), whereas the production of eGFP was confirmed by fluorescence microscopy.
荞麦是一种很有前景的作物，其籽粒富含营养物质和生物活性化合物。普通荞麦和苦荞麦的基因组序列数据最近已经可得。目前，迫切需要开发一种简单可靠的瞬时基因表达方案以及稳定的遗传转化方法，以促进生物活性化合物的代谢工程、基因功能分析、靶向编辑，并从长远来看，加速荞麦的育种进程。在本文中，我们报告了用于[具体物种]介导的[具体物种]瞬时和稳定转化的优化方法。用含有[具体基因]和[报告基因]的[载体名称]载体浸润叶片和子叶组织。采用组织化学染色和蛋白质免疫印迹法来确认报告蛋白的表达。我们还证明了所开发的方法在荞麦禾本科生物碱生物合成途径工程中的实用性。[具体基因]在荞麦叶片中瞬时表达，并通过液相色谱 - 质谱法确认了禾本科生物碱的从头合成。此外，通过[具体物种]介导的真空浸润实现了普通荞麦和苦荞麦携带报告基因（[具体基因]）和选择标记基因（[具体基因]）的植物体内遗传转化。通过聚合酶链反应（PCR）确认构建体的基因组整合，而通过荧光显微镜确认增强型绿色荧光蛋白（eGFP）的产生。
