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Physiological Studies on Moisture Stress Tolerance in Chickpea (Cicer Arietinum L.) Genotypes

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Physiological Studies on Moisture Stress Tolerance in Chickpea (Cicer Arietinum L.) Genotypes International Journal of Scientific Research in Agricultural Sciences, 1(2), pp. 23-31, 2014 Available online at http://www.ijsrpub.com/ijsras ISSN: 2345-6795; ©2014 IJSRPUB http://dx.doi.org/10.12983/ijsras-2014-p0023-0031 Full Length Research Paper Physiological Studies on Moisture Stress Tolerance in Chickpea (Cicer Arietinum L.) Genotypes Aqeel Hasan Rizvi1*, Vipin Kumar Dwivedi2, Raj Kumar Sairam3, Shyam Singh Yadav4, Chellapilla Bharadwaj4, Ashutosh Sarker1, Afroz Alam5 1International Center for Agricultural Research in the Dry Areas, South Asia & China Regional Program, NASC Complex, New Delhi* 2Department of Genetics and Plant Breeding, J.V. College, Baraut, Baghpat, U.P, India 3Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi, India 4Division of Genetics, Indian Agricultural Research Institute, New Delhi, India 5Department of Bioscience and Biotechnology, Banasthali Vidyapith, Rajasthan 304022, India *Corresponding Author: [email protected] Received 27 March 2014; Accepted 12 May 2014 Abstract. Drought is a major constraint that limits seed yield in chickpea (Cicer arietinum L.). It is important to establish the relative importance many of these drought-related traits for prioritizing their consideration in breeding for drought tolerance improvements. The objective of this study was to categorize the drought tolerant and susceptible chickpea genotypes on the basis of physiological parameters. An experiment was conducted with five chickpea genotypes in field under irrigated and rain fed conditions. Observations were recorded on relative water content (RWC), and the contents of chlorophyll (Chl), carotenoid (Car), proline (Pro) and protein in the five chickpea genotypes. RWC and contents of Chl, Car and protein decreased under moisture stress, whereas Pro content increased with the increase in moisture stress. Pusa-1108, Pusa-362, Pusa-1103 were able to maintain relatively higher RWC, Chl, Car and protein content and greater Pro accumulation, while Flip 90-166 and SBD- 377 showed comparatively greater decline in the RWC, Chl, Car and Protein and less accumulation of Pro under moisture stress. The data suggest that chickpea genotypes can be categorized into drought tolerant and susceptible types based on the variations in RWC, Chl, Car, Pro and protein content. Keywords: Carotenoid, chickpea, chlorophyll, moisture stress, proline, protein, relative water content 1. INTRODUCTION Relative water content (RWC) is one of the important parameter to measure water status of the Chickpea (Cicer arietinum L.) is world’s second most tissue (Barrs and Weatherley, 1962). Gradual decrease cultivated grain legume grown over 10.2 million in RWC with increase in stress and greater reduction hectare. Chickpea is grown as a winter crop in the afterwards under severe stress has been reported in Indian subcontinent, which accounts for nearly 85% chickpea (Deshmukh et al., 2000). High RWC under of the chickpea area sown worldwide. It is also an moisture stress denotes ability of plants to tolerate important crop in West Asia and Mediterranean moisture stress (Uprety and Sirohi, 1987, Ritchi et al., region. In India chickpea is generally grown using 1990).Under moisture stress conditions tolerant types stored soil moisture after rainy season. Moisture stress show less reduction in RWC as compared with is the most prevalent environmental factor limiting susceptible ones (Sairam et al., 1997). Baisak et al. plant growth, survival and productivity in chickpea (1994) reported the decline in chlorophyll content (Bohnert and Jenson, 1995). Moisture deficit affects with water stress. Reduction in chlorophyll content seed germination and seedling establishment in the upon exposure to oxidative stress and a comparatively field, however, genotypes vary in their capacity to higher chlorophyll content in tolerant wheat and tolerate moisture stress. Chickpea is believed to be maize genotypes than susceptible ones has also been tolerant to drought condition, but there is little reported (Kraus et al., 1995). Sairam (1994) reported published evidence to support this contention (Saxena, that under moisture stress chlorophyll content and 1984). chlorophyll stability index were higher in tolerant wheat genotypes in comparison to the susceptible 23 Rizvi et al. Physiological Studies on Moisture Stress Tolerance in Chickpea (Cicer Arietinum L.) Genotypes genotypes. Rahangdale et al. (1995) observed and Pusa-1108, Flip 90-166 (kabuli)grown under genotypic differences in chickpea for chlorophyll irrigated (control) and rain fed (moisture stressed) content under water deficit conditions. However, environments for two seasons following the tolerant genotypes maintained relatively higher recommended package of practices. Pre-sowing chlorophyll content. irrigation was provided to ensure proper germination. Carotenoid provides protection against oxidative In control plots irrigation was provided as and when damage (Schmitz and Noga, 1997). Jiang et al. (1991) required to avoid any stress to the crop, while under reported that carotenoids decreased markedly in rice rain fed plantings no irrigation was given during entire plants on induction of water stress. They further crop season. The various parameters were studied at reported that under osmotic stress the activities of two growth stages of the crop viz. flowering and pod endogenous protective enzyme systems and the formation stage. Experiment was laid down in split contents of ascorbic acid and carotenoids were plot design with 3 replications and the pooled data of negatively correlated with membrane lipid two years was analysed by factorial randomised block peroxidation. Osmolytes are involved in signalling design. /regulating plant response to multiple stresses, including reduced growth, which may be part of 2.2. Laboratory determinations plant’s adaptation against stress. Among all osmolytes, proline is probably most widely Relative water content (RWC) was determined in flag distributed, and its accumulation seems to be involved leaf as per the method of Barrs and Weatherley in the process of adaptation to osmotic stress (Yoshiba (1962). Hundred mg leaf samples was taken and kept et al., 1997). It has been shown to play an important in distilled water in a petridish for four hours to make role in ameliorating drought, salinity and heavy metal the leaf tissue turgid. The turgid weights of the leaf stresses (Andrade et al., 1995). The present study was, material were taken after carefully soaking the tissues therefore, conducted to study the variability in RWC, between two filter papers, subsequently the leaf Chl, Car, Pro and protein content in tolerant and material was kept in a butter paper bag and dried in susceptible chickpea genotypes. oven at 65 0C for 24 h and their dry weights were recorded. The RWC was calculated by using the 2. MATERIALS AND METHODS formula given below: 2.1. Sample preparation An experiment was conducted with five chickpea genotypes viz. Pusa-362, Pusa-1103, SBD-377 (desi) Fresh weight - Dry weight RWC (%) = x 100 Turgid weight - Dry weight Pigment extraction was done by the method of (model: Specord 200). The amount of chlorophyll ‘a’, Hiscox and Israelstam (1979). Fifty mg of leaf tissue chlorophyll ‘b’ and total chlorophyll were calculated from fully emerged leaf was incubated in 10 ml of as per the formula given by Arnon et al. (1949) and dimethyl sulphoxide (DMSO) for 4 h at 60 0C in an the amount of carotenoid were calculated using the incubator. The absorbances (A) values were recorded formula given by Lichtenthaler and Wellburn (1983). at 645, 663 and 470 nm in a digital spectrophotometer Chlorophyll ‘a’ = [12.7 (A663) - 2.69(A645)] x V/(W x 1000) Chlorophyll ‘b’ = [22.9 (A645) - 4.68(A663)] x V/(W x 1000) Total Chlorophyll = [22.2 (A645) + 8.02(A663)] x V/(W x 1000) where, A663 and A645 are the absorbance values at 663 and 645 nm respectively; W = weight of the sample in mg; V = volume of the solvent used (ml) Carotenoid= [1000 x A470 – (3.27 x Chl a + 104 x Chl b)] where, A470is the absorbance value at 470 nm; Chl ‘a’ = Chlorophyll ‘a’; Chl ‘b’ = Chlorophyll ‘b’ Proline content was estimated in leaf tissues as per sulpho-salicylic acid in chilled mortar and pestle. The the protocol given by Bates et al. (1973). Fresh leaf homogenate was centrifuged at 10,000 rpm for 10 sample was taken and homogenized with 10 ml min. 0.2 ml of supernatant was taken in test tube to 24 International Journal of Scientific Research in Agricultural Sciences, 1(2), pp. 23-31, 2014 which 2 ml of acid ninhydrin and 2 ml of glacial was homogenized in 10 ml extraction buffer (0.1 M acetic acid were added. The resultant mixture was phosphate buffer, pH 7.5, containing 0.5 mM EDTA) boiled at 1000C in a water bath. The reaction was with the help of pre chilled mortar and pestle. The brie stopped by keeping the test tubes in an ice bath. Then, was passed through a 4 layers of cheese cloth, and the 4 ml of toluene was added to each tube and mixed filtrate was centrifuged for 20 minutes at 15000 g vigorously on a vortex for 10-15 sec in order to (4oC) and supernatant was used0.1 ml supernatant was facilitate quick diffusion/movement of chromophores mixed with 3.8 ml DDW, 1.0 ml reagent ‘D’ then from the aqueous phase to non-aqueous phase. The shaken immediately and kept for 20 minutes. Added toluene layer (upper) was separated from the mixture 0.1 ml 1:1 diluted (with DDW) Folin-ceo-catleu and absorbance was read at 520 nm on a reagent and shaken immediately vigorously and leave spectrophotometer using toluene as blank. for 20 minutes. Finally read OD at 500 nm. Soluble protein was estimated as per the method given by Lowry et al. (1951). Fresh leaf material (1 g) 3. RESULTS AND DISCUSSIONS in RWC under moisture stress in all the cultivars. Under moisture stress condition the highest RWC at 3.1. Relative water content flowering stage was observed in Pusa-362 (86.33%) and at pod formation stage in Pusa-1103 (81.07%), Chickpea crop responds to water deficit in the form of while the lowest RWC was observed in SBD-377 changes in various physiological and biochemical (63.81%) and Flip 90-166 (52.64) at flowering and processes.
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