Aggarwal Pooja Rani, Nag Papri, Choudhary Pooja, Chakraborty Niranjan, Chakraborty Subhra
National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067 India.
Plant Methods. 2018 Jul 6;14:55. doi: 10.1186/s13007-018-0315-6. eCollection 2018.
Chickpea ( L.), an important legume crop is one of the major source of dietary protein. Developing an efficient and reproducible transformation method is imperative to expedite functional genomics studies in this crop. Here, we present an optimized and detailed procedure for -mediated root transformation of chickpea.
Transformation positive roots were obtained on selection medium after two weeks of inoculation. Expression of green fluorescent protein further confirmed the success of transformation. We demonstrate that our method adequately transforms chickpea roots at early developmental stage with high efficiency. In addition, root transformation was found to be genotype-independent and the efficacy of our protocol was highest in two (Annigiri and JG-62) of the seven tested chickpea genotypes. Next, we present the functional analysis of chickpea hairy roots by expressing () gene involved in proanthocyanidins biosynthesis. Overexpression of enhanced the level of proanthocyanidins in hairy roots that led to the decreased colonization of fungal pathogen, . Furthermore, the induction of transgenic roots does not affect functional studies involving infection of roots by fungal pathogen.
Transgenic roots expressing genes of interest will be useful in downstream functional characterization using reverse genetics studies. It requires 1 day to perform the root transformation protocol described in this study and the roots expressing transgene can be maintained for 3-4 weeks, providing sufficient time for further functional studies. Overall, the current methodology will greatly facilitate the functional genomics analyses of candidate genes in root-rhizosphere interaction in this recalcitrant but economically important legume crop.
鹰嘴豆（Cicer arietinum L.）是一种重要的豆类作物，是膳食蛋白质的主要来源之一。开发一种高效且可重复的转化方法对于加速该作物的功能基因组学研究至关重要。在此，我们展示了一种用于鹰嘴豆根转化的优化且详细的方法。
接种后两周，在选择培养基上获得了转化阳性根。绿色荧光蛋白的表达进一步证实了转化的成功。我们证明我们的方法能够在早期发育阶段高效地充分转化鹰嘴豆根。此外，发现根转化与基因型无关，并且在七个测试的鹰嘴豆基因型中的两个（Annigiri和JG - 62）中，我们方案的效率最高。接下来，我们通过表达参与原花青素生物合成的AtMYB12基因对鹰嘴豆毛状根进行功能分析。AtMYB12的过表达提高了毛状根中原花青素的水平，导致真菌病原体Fusarium oxysporum的定殖减少。此外，转基因根的诱导不影响涉及真菌病原体感染根的功能研究。
表达感兴趣基因的转基因根将有助于使用反向遗传学研究进行下游功能表征。执行本研究中描述的根转化方案需要1天，并且表达转基因的根可以维持3 - 4周，为进一步的功能研究提供足够的时间。总体而言，当前方法将极大地促进这种顽固但经济上重要的豆类作物根际相互作用中候选基因的功能基因组学分析。
