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Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies

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Title Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies
 
Creator Aggarwal, Pooja Rani
Nag, Papri
Choudhary, Pooja
Chakraborty, Niranjan
Chakraborty, Subhra
 
Subject Legumes
Cicer arietinum
Agrobacterium rhizogenes
strain K599
Transformation efficiency
Functional genomics
Green fluorescent protein (GFP) expression
TRANSPARENT TESTA 2
Proanthocyanidins
Fungal infection
 
Description Accepted date: 2 June 2018
Background: Chickpea (Cicer arietinum L.), an important legume crop is one of the major source of dietary protein.
Developing an efcient 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.
Results: Transformation positive roots were obtained on selection medium after two weeks of A. rhizogenes inoculation.
Expression of green fuorescent protein further confrmed the success of transformation. We demonstrate that
our method adequately transforms chickpea roots at early developmental stage with high efciency. In addition, root
transformation was found to be genotype-independent and the efcacy 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 afect functional studies
involving infection of roots by fungal pathogen.
Conclusions: 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 sufcient 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.
This work was supported by grants from the Department of Biotechnology (DBT) (BT/HRD/35/01/05/2013 and BT/AGR/CG-PhaseII/01/2014), Government of India and the National Institute of Plant Genome Research, New Delhi, India to S. C. P.R.A. is the recipient of pre-doctoral fellowship from the Council of Scientific and Industrial research (CSIR), Govt. of India. P.C. is the recipient of pre-doctoral fellowship from the University Grant Commission (UGC), Govt. of India.
 
Date 2018-07-12T09:36:14Z
2018-07-12T09:36:14Z
2018
 
Type Article
 
Identifier Plant Methods, 14: 55
1746-4811
http://223.31.159.10:8080/jspui/handle/123456789/875
https://plantmethods.biomedcentral.com/articles/10.1186/s13007-018-0315-6
https://doi.org/10.1186/s13007-018-0315-6
 
Language en_US
 
Format application/pdf
 
Publisher BioMed Central Ltd