Guimaraes Larissa Arrais, Pereira Bruna Medeiros, Araujo Ana Claudia Guerra, Guimaraes Patricia Messenberg, Brasileiro Ana Cristina Miranda
Parque Estação Biológica, Embrapa Recursos Genéticos e Biotecnologia, CP 02372, Final W5 Norte, Brasília, DF Brazil.
Universidade de Brasília, Campus Darcy Ribeiro, Brasília, DF Brazil.
Plant Methods. 2017 Apr 11;13:25. doi: 10.1186/s13007-017-0176-4. eCollection 2017.
Peanut () production is largely affected by a variety of abiotic and biotic stresses, including the root-knot nematode (RKN) that causes yield losses worldwide. Transcriptome studies of wild species, which harbor resistance to a number of pests and diseases, disclosed several candidate genes for resistance. Peanut is recalcitrant to genetic transformation, so the use of -derived hairy roots emerged as an alternative for in-root functional characterization of these candidate genes.
The present report describes an ex vitro methodology for hairy root induction in detached leaves based on the well-known ability of peanut to produce roots spontaneously from its petiole, which can be maintained for extended periods under high-humidity conditions. Thirty days after infection with the 'K599' strain, 90% of the detached leaves developed transgenic hairy roots with 5 cm of length in average, which were then inoculated with . For improved results, plant transformation, and nematode inoculation parameters were adjusted, such as bacterial cell density and growth stage; moist chamber conditions and nematode inoculum concentration. Using this methodology, a candidate gene for nematode resistance, was successfully overexpressed in hairy roots of the nematode-susceptible peanut cultivar 'Runner', resulting in 98% reduction in the number of galls and egg masses compared to the control, 60 days after infection.
This methodology proved to be more practical and cost-effective for functional validation of peanut candidate genes than in vitro and composite plant approaches, as it requires less space, reduces analysis costs and displays high transformation efficiency. The reduction in the number of RKN galls and egg masses in peanut hairy roots overexpressing corroborated the use of this strategy for functional characterization of root expressing candidate genes. This approach could be applicable not only for peanut-nematode interaction studies but also to other peanut root diseases, such as those caused by fungi and bacteria, being also potentially extended to other crop species displaying similar petiole-rooting competence.
花生生产受到多种非生物和生物胁迫的很大影响，包括根结线虫，其在全球范围内导致产量损失。对具有多种病虫害抗性的野生花生品种进行转录组研究，揭示了几个抗线虫候选基因。花生对遗传转化具有抗性，因此利用花生来源的毛状根成为对这些候选基因进行根内功能表征的一种替代方法。
本报告描述了一种基于花生从叶柄自发生根的已知能力，在离体叶片中诱导毛状根的体外方法，该方法可以在高湿度条件下长期维持。用 “K599” 菌株感染30天后，90% 的离体叶片长出平均长度为5厘米的转基因毛状根，然后接种线虫。为了获得更好的结果，对植物转化和线虫接种参数进行了调整，如细菌细胞密度和生长阶段；保湿室条件和线虫接种物浓度。使用这种方法，线虫抗性候选基因在感线虫花生品种 “Runner” 的毛状根中成功过表达，与对照相比，感染线虫60天后，虫瘿和卵块数量减少了98%。
与体外和复合植物方法相比，该方法在花生候选基因功能验证方面被证明更实用且具有成本效益，因为它需要的空间更小，降低了分析成本并显示出高转化效率。在过表达线虫抗性候选基因的花生毛状根中，根结线虫虫瘿和卵块数量的减少证实了该策略可用于根表达候选基因的功能表征。这种方法不仅适用于花生 - 线虫相互作用研究，也适用于其他花生根病，如由真菌和细菌引起的病害，还可能扩展到具有类似叶柄生根能力的其他作物品种。
