Transgenic chickpea (Cicer arietinum L.) harbouring AtDREB1a are physiologically better adapted to water deficit

Abstract

Background: Chickpea (Cicer arietinum L.) is the second most widely grown pulse and drought (limiting water) is one of the major constraints leading to about 40-50% yield losses annually. Dehydration responsive element binding proteins (DREBs) are important plant transcription factors that regulate the expression of many stress-inducible genes and play a critical role in improving the abiotic stress tolerance. Transgenic chickpea lines harbouring transcription factor, Dehydration Responsive Element-Binding protein 1A from Arabidopsis thaliana (AtDREB1a gene) driven by stress inducible promoter rd29a were developed, with the intent of enhancing drought tolerance in chickpea. Performance of the progenies of one transgenic event and control were assessed based on key physiological traits imparting drought tolerance such as plant water relation characteristics, chlorophyll retention, photosynthesis, membrane stability and water use efficiency under water stressed conditions.

Results: Four transgenic chickpea lines harbouring stress inducible AtDREB1a were generated with transformation efficiency of 0.1%. The integration, transmission and regulated expression were confirmed by Polymerase Chain Reaction (PCR), Southern Blot hybridization and Reverse Transcriptase polymerase chain reaction (RT-PCR), respectively. Transgenic chickpea lines exhibited higher relative water content, longer chlorophyll retention capacity and higher osmotic adjustment under severe drought stress (stress level 4), as compared to control. The enhanced drought tolerance in transgenic chickpea lines were also manifested by undeterred photosynthesis involving enhanced quantum yield of PSII, electron transport rate at saturated irradiance levels and maintaining higher relative water content in leaves under relatively severe soil water deficit. Further, lower values of carbon isotope discrimination in some transgenic chickpea lines indicated higher water use efficiency. Transgenic chickpea lines exhibiting better OA resulted in higher seed yield, with progressive increase in water stress, as compared to control.

Conclusions: Based on precise phenotyping, involving non-invasive chlorophyll fluorescence imaging, carbon isotope discrimination, osmotic adjustment, higher chlorophyll retention and membrane stability index, it can be concluded that AtDREB1a transgenic chickpea lines were better adapted to water deficit by modifying important physiological traits. The selected transgenic chickpea event would be a valuable resource that can be used in pre-breeding or directly in varietal development programs for enhanced drought tolerance under parched conditions.

Keywords: AtDREB1a; Carbon isotope discrimination; Chlorophyll fluorescence; ETR; Genetic engineering; Osmotic adjustment; Phenotyping; Transcription factor; Yield.

Fig. 1

Molecular analysis of transgenic chickpea lines a: PCR analyses of four transgenic chickpea events (T₀); b: PCR analyses of transgenic chickpea progenies (T₁) derived from E₅; c: PCR analyses of transgenic chickpea progenies (T₁) derived from E₁₇; d: PCR analyses of transgenic chickpea progenies (T₁) derived from E₁₉; e: PCR analyses of transgenic chickpea progenies (T₁) derived from E_22; [L1–100 bp DNA ladder and L2–1 kb DNA ladder]; f: Southern blot analysis (L: DIG-labelled DNA ladder; I-IV: Four independent transgenic chickpea lines E₅, E₁₇, E₁₉ and E₂₂ (T1 stage); N: Non-transformed chickpea (DCP 92–3); P: Positive control (Binary plasmid). g: RT-PCR analysis (L1: 1Kb plus DNA ladder; P: Positive control; N: Negative control; I-IV: Transgenic chickpea lines (T₁ stage); V–X: Transgenic chickpea lines (T₂ stage); NTC: No Template Control; C: RNA as Template; L2: 100 bp DNA ladder) [Mean SM 11.8% and mean LWP −0.82 MPa]

Fig. 2

Association of OA with RWC observed in four transgenic events under Stress Level 4 (Soil moisture to 4.5%)

Fig. 3

Leaf RWC of transgenic lines and control at four different stress levels (Stress level 1, 2, 3 & 4). Error bar represents the deviation from the mean values of RWC (lsd bar shows significance level at 1%)

Fig. 4

OA of transgenic chickpea lines with progressive decrease in soil moisture from field capacity to Stress Level 4

Fig. 5

SPAD (chlorophyll content) values of leaves of transgenic chickpea lines and control at four different stress levels (Stress level 1, 2, 3 & 4). Error bar represents the deviation from the mean value of SPAD readings (lsd bar shows significance level at 1%)

Fig. 6

Images of photosynthetic quantum yield (Fv/Fm) of PSII under WW and WS in a transgenic chickpea line 17.6 and control. a, b & c- Quantum yield (Fv/Fm) images of control; d, e & f- Quantum yield (Fv/Fm) images of transgenic line 17.6. Corresponding values of SM, RWC and LWP were tabulated

Fig. 7

Photosynthetic ETR in response to increasing irradiance levels (PAR) in selected transgenic chickpea lines and control (lsd bar shows significance level at 1%)

Fig. 8

Histogram depicting CID values of transgenic chickpea lines and control under WW and WS conditions (lsd bar shows significance level at 1%)

Fig. 9

Histogram depicting the yield of the transgenic chickpea lines and control, under WW and WS conditions (lsd bar shows significance level at 1%)