The development of genetic transformation methods is critical for enabling the thorough characterization of an organism and is a key step in exploiting any species as a platform for synthetic biology and metabolic engineering approaches. In this work we describe the development of an Agrobacterium rhizogenes-mediated hairy root transformation protocol for the crop and medicinal legume fenugreek (Trigonella foenum-graecum). Fenugreek has a rich and diverse content in bioactive specialised metabolites, notably diosgenin, which is a common precursor for synthetic human hormone production. This makes fenugreek a prime target for identification and engineering of specific biosynthetic pathways for the production of triterpene and steroidal saponins, phenolics, and galactomanans. Through this transformation protocol, we identified a suitable promoter for robust transgene expression in fenugreek. Finally, we establish the proof of principle for the utility of the fenugreek system for metabolic engineering programs, by heterologous expression of known triterpene saponin biosynthesis regulators from the related legume Medicago truncatula in fenugreek hairy roots.

遗传转化方法的开发对于实现生物的全面表征至关重要，并且是开发任何物种作为合成生物学和代谢工程方法平台的关键步骤。在这项工作中，我们描述了发根农杆菌介导的农作物和药用豆类胡芦巴（Trigonella foenum-graecum）毛状根转化方案的开发）。胡芦巴在具有生物活性的专门代谢产物中含量丰富且多样化，尤其是薯os皂甙元，薯di皂甙元是合成人激素生产的常见前体。这使胡芦巴成为鉴定和工程化生产三萜和甾体皂苷，酚类和半乳聚糖的特定生物合成途径的主要目标。通过此转化协议，我们确定了胡芦巴中强健的转基因表达的合适启动子。最后，我们通过胡芦巴毛状根中相关豆科植物紫花苜蓿的已知三萜皂苷生物合成调节剂的异源表达，建立了胡芦巴系统用于代谢工程程序的原理性证明。