Abstract
Background Chickpea (Cicer arietinum 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 Agrobacterium rhizogenes-mediated root transformation of chickpea.ResultsTransformation positive roots were obtained on selection medium after two weeks of A. rhizogenes 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 Arabidopsis TRANSPARENT TESTA 2 (AtTT2) gene involved in proanthocyanidins biosynthesis. Overexpression of AtTT2 enhanced the level of proanthocyanidins in hairy roots that led to the decreased colonization of fungal pathogen, Fusarium oxysporum. Furthermore, the induction of transgenic roots does not affect functional studies involving infection of roots by fungal pathogen.ConclusionsTransgenic 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.
Highlights
Chickpea (Cicer arietinum L.), an important legume crop is one of the major source of dietary protein
Induction of hairy roots in chickpea using Agrobacterium rhizogenes Agrobacterium rhizogenes-mediated root transformation in chickpea was performed following the protocol developed for Phaseolus with few modifications [40]
The binary vectors were transferred into competent A. rhizogenes strain K599 by electroporation as described earlier [48]
Summary
Chickpea (Cicer arietinum 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. Determining the function of genes/proteins identified through various such large scale OMICS studies is a major challenge, especially in this recalcitrant crop for conventional transformation methods. Reproducible and efficient method of plant transformation protocol is crucial for functional studies and crop improvement program. The transformation efficiency achieved in these studies was very low, genotype dependent and the approach is laborious as well as time consuming. To circumvent these shortcomings, a rapid, efficient and genotype-independent transformation method is a pre-requisite for functional genomics studies in this important grain legume
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