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

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.

Results: Transformation 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.

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 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.

Keywords: Agrobacterium rhizogenes, strain K599; Cicer arietinum; Functional genomics; Fungal infection; Green fluorescent protein (GFP) expression; Legumes; Proanthocyanidins; TRANSPARENT TESTA 2; Transformation efficiency.

Fig. 1

Schematic representation of the timeline required for Agrobacterium rhizogenes-mediated root transformation of Cicer arietinum

Fig. 2

Effects of co-cultivation conditions on transformation efficiency of C. arietinum cultivar Annigeri. a Effects of duration of co-cultivation, b temperature during co-cultivation on transformation frequency were determined. Each value represents the mean of three independent experiments with standard deviation (SD). Approximately 50 seedlings were examined for each individual experiment. Values with different letters are significantly different at P < 0.05 (Fisher’s LSD test)

Fig. 3

Illustrations depicting the main steps for the root transformation of chickpea cultivar Annigeri. a Plants grown for 5 days in MS medium, b germinated chickpea seedling, c, d sectioning the root near hypocotyl region, e immersing the cut end into A. rhizogenes suspension, f explant transferred in co-cultivation medium, g explant transferred to selection medium after co-cultivation, h, i transformed roots grown in the selection medium after 3 and 7 days respectively. After 7 days, plantlets were transferred to pots, j plants grown in pot after 15 days, k completely grown hairy root system in chickpea. Scale bar represents 10 mm

Fig. 4

Green fluorescent protein (GFP) expression in transgenic chickpea (Annigeri) roots. GFP-derived fluorescence, bright field and merged image detected by laser scanning confocal microscope in a wild-type untransformed root (a, b) and transformed hairy root (c, d). Scale bar represents 100 µm

Fig. 5

Characterization of transformed roots in chickpea (Annigeri). a No GFP expression in root tip, b DAPI staining exhibiting intact nuclei, c merged image with bright field in untransformed wild-type chickpea roots. d Visualization of GFP expression in root tip, e DAPI staining exhibiting intact nuclei, f merged image with bright field in transformed chickpea roots. Scale bar represents 100 µm

Fig. 6

PA accumulation in transgenic roots of chickpea cultivar JG-62 expressing AtTT2:GFP. a Wild-type, b mock transformed and, c–e AtTT2-transformed roots after staining with DMACA reagent, f PCR amplification of TT2 (I) and virD (II) genes in A. rhizogens K599-p35S-TT2, untransformed wild-type (WT) and transgenic roots (T) and lane M is a 1-kb ladder (Invitrogen). Scale bars: for d 0.5 mm and for e 50 µm

Fig. 7

PAs accumulation leads to decreased fungal biomass in transformed chickpea (JG-62) roots. a Levels of proanthocyanidins (PAs) in untransformed wild-type (WT) and transgenic roots (T), b relative fungal biomass estimation in untransformed wild-type (WT) and transgenic roots (T). Each value represents the mean of three independent experiments with standard error (SE). Approximately 3-5 replicates were examined for each individual experiment. Statistically significant differences are indicated by an asterisk at P < 0.05 (Fisher’s LSD test). After 5 DPI, untransformed roots showing c, f no GFP fluorescence, d, g high fungal colonization, e, h merged image of root elongation and tip zones, respectively. AtTT2:GFP transformed roots showing i, l GFP fluorescence, j, m less fungal colonization, k, n merged image of root elongation and tip zones, respectively. Scale bars: 100 µm