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World Journal of Science and Technology 2011, 1(1): 11-18 ISSN: 2231 2587

Ravi Ranjan Kumar, Krishna Karjol and G. R. Naik* * Department of Biotechnology, Gulbarga University, Gulbarga 585106 India
Corresponding authors e-mail:

The experiments were performed to evaluate seed germination, early seedling growth and other seedling parameters in stimulated drought to evaluate the ability to estimate drought tolerance in 10 local cultivated Pigeonpea cultivars among which six red cultivars (ICP 8863, ICPL 87119, GC-11-39, TAT 9903, G-RED and BSMR 736), three white cultivars (GRG 295, WRP-1 and TS 3) and one black cultivar (PG-12) were tested. Drought stress was imposed through polyethylene glycol 6000 of osmotic potential -0.15, -0.30, -0.45, -0.89 MPa. Percentage germination, early seedling growth such as radicle and hypocotyl length, seedling fresh and dry weight and other seedling vigor parameters were investigated in the present investigation. The results indicated significant differences among cultivars under drought stress level. In all cultivars, a significant decrease was observed with increase in stress level. The result clearly indicated that white and black cultivars were more tolerant than red cultivars. Sensitive cultivars showed no germination at -0.89 MPa osmotic potential, while germination was inhibited ~92% in moderately tolerant cultivars but tolerant cultivars showed about 20% germination. The germination was not seen beyond 0.89MPa. The increase in drought stress reduced the germination rate, seedling fresh and dry biomass, seedling vigour index and germination index in all cultivars. Mean germination time increased with increase in drought stress. Drought stress greatly affected the hypocotyl length rather than growth of radicle which indicates that the length of hypocotyl is more sensitive to drought stress. It was concluded that the inhibition in germination at equivalent water potentials of PEG was mainly due to an osmotic effect. Keywords: Drought stress, Pigeonpea, Polyethylene glycol, Seedling growth Abbreviations: GI- Germination Index, GR- Germination Rate, MGT- Mean Germination Time, PEGPolyethylene glycol, SVI- Seedling Vigor Index.

Abiotic stresses like drought, salinity, heat, temperature, cold and water logging have adverse effect on crop productivity and causes severe agriculture losses predominantly in arid and semi arid tropic region like India (Abdul et al; 2006, Rodriguez et al; 2005, Zhang et al; 2006). Water availability is one of the most important environmental factors influencing seed germination as it triggers the germination process and remains involved, directly or indirectly. Inadequate moisture availability and poor soil fertility are also the factors that affect failure of rainfed crops with mild to severe drought stress (Ramesh and Devasenapathy; 2007). Water stress

limits plant survival and early seedling growth by delaying its beginning or decreasing the final germinability (Gamze et al; 2005, Kaydan and Yagmur; 2008, Silva et al; 2000). Seed germination and early seedling growth are considered as most critical phases for seed establishment, determining successful crop production (Uniyal et al; 1998). Pigeonpea (Cajanus cajan [L.] Millspaugh) is one of the major grain legume crops of the tropics and subtropics. It is the most important pulse crop which is cultivated in the gross cropped area (3.58 million ha) under pulses providing 20% of the national pulse production (2.51 m tones). This accounts for 90% of the worlds Pigeonpea production (Johansen, 1990). Gulbarga region accounts for 70% of total Pigeonpea production of Karnataka state, is a major

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drought affected area. In recent year, significant loss has been seen in Pigeonpea production in this region. As seed germination and early seedling growth responses are critical phases to establish stress tolerance cultivars, there is need to study these responses to improve present Pigeonpea yield. Polyethylene glycol is commonly used to stimulate osmotic stress effects in petridishes to control water potential in seed germination studies. The non toxic PEG solution is used because of high molecular weight and cant pass through plant cell walls (Emmerich et al; 1990, Kaydan et al; 2007). The aim of the present study was to identify how pigeonpea varieties grown in Gulbarga region respond to different level of water stress induced by polyethylene glycol at seedling level. The studies were conducted to evaluate the effect of such a stress on germination and early seedling growth.

7th day, final germination percentage, radicle length, hypocotyl length, seedling fresh weight, dry weight and various seed vigour parameters were calculated as described by Uniyal et al; 1998 and Singh et al; 2007. Mean Germination Time (MGT) = (Dn)/ n Where n is the number of seeds germinated on day D (D representing the number of days since the sowing of seed)
Germination Index (GI) = Total percent germination Times (hr) taken for 50% germination

Germination rate (GR) = (100/n) (N3/3+N5/5) n= total no. of seeds, N3= Number of seeds germinated on 3rd day, N5= Number of seeds germinated on 5th day Seedling Vigour Index (SVI) = Percentage germination x root length Statistical Analysis Data recorded for different morphological characteristics were analyzed by one way analysis of variance (ANOVA) by using GraphPad Instat (Version 3.05 GraphPad software, Inc 2000) and mean values were compared by using Turkeys test at P 0.05 or P 0.01.

Materials and Methods

Ten locally cultivated pigeonpea accessions were examined for the evaluation of osmotic stress during germination and early seedling growth. The seeds obtained from Agriculture Research Station, Gulbarga were differing in their seed size, colour and maturity period. Among 10 pigeonpea cultivars; six red varieties (ICP-8863, ICPL-87119, TAT-9903, BSMR-736, G-RED and GC-11-39), three white varieties (GRG-295, WRP-1 and TS-3) and one black variety (PG-12) were selected for present investigation. Seeds were initially treated with 0.1% HgCl2 for surface sterilization and residual chlorine was eliminated by thoroughly washing of seeds with distilled water. PEG 6000 was used to induce the drought stress and diverse osmotic potentials of -0.15 MPa, 0.30 MPa, -0.45 MPa and -0.89 MPa were arranged as described by Michel and Kauffman; 1973. Three replicates of 50 seeds of each cultivar were germinated in petriplates on Whatman no. 1 filter paper poured with 10ml of respective test solution. Distilled water was served as control. Germination tests were carried in controlled environmental conditions at 241oC with 12 hr photoperiod. Observation on seed germination was recorded daily and 2 ml of respective treatment solution depending on requirement was applied. A seed was considered to be germinated when the emerging radicle elongated to 1 mm. Germination percentage was recorded after every 6hr for 7 days. At the end of the

The germination responses of all Pigeonpea accession at different level of osmotic potential is shown in Fig.1 and various attributes resulting to germination and early seedling growth are presented in Table 1, 2, 3 & 4. Various parameters were recorded under drought stress condition such as germination percentage, GR, GI, MGT, root and shoot length, fresh and dry weight of seedlings. Based on these parameters the drought tolerant and susceptible cultivars were identified among tested Pigeonpea accessions. PEG induced drought stress (P0.05 or P0.01) significantly affected the final germination in all cultivars of Pigeonpea. Maximum seed germination was experiential under control condition and germination in untreated seeds was rapid. Approximately 80% seeds were germinated within one day of sowing. It was seen that a decrease in

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osmotic potential of the medium resulted in gradual decrease in percentage germination. No germination was observed beyond -0.89 MPa, indicating that species resistance limit to water stress is between 0.30 to 0.70 MPa. With regards to the final percentage germination, only GRG-295, WRP-1, TS3 and PG-12 cultivars showed drought tolerant character, ICPL 87119 and G-Red moderately tolerant while ICP 8863, GC-11-39, BSMR 736 and TAT-9903 were sensitive to PEG induced stress. A significantly decrease in the percentage germination were observed in all the cultivars at -0.45 and 0.89MPa osmotic potential. Sensitive cultivars showed no germination at -0.89 MPa osmotic potential, while germination was inhibited ~92% in moderately tolerant cultivars but tolerant cultivars showed about 20% germination (Fig. 1).

GI were significantly higher than that of sensitive cultivars at severe drought stress of -0.45 and -0.89 MPa osmotic potential (Table 2). Seedling fresh weight and dry weight was significantly decreased in all the cultivars by increasing osmotic potential. ANOVA of seedling fresh and dry weight indicated highly significant differences in tolerant and sensitive cultivars. Fresh weight and dry weight of seedlings was higher in tolerant cultivars as compare to moderately tolerant and susceptible cultivars at all the concentrations (Table 4).

Water is one of the most essential environmental factor influencing seed germination and early seedling growth as it triggers the germination process and remains involved, directly or indirectly, in all subsequent stages of plant metabolism. It decisively participates in enzymatic reactions, metabolite solvency and transportation, and itself acts as an agent of hydrolytic digestion of proteins, carbohydrates and lipids from reserve seed tissues. Water and salt stresses are correlated, as excess of soluble salts reduces soil water potential and thus, hinders water absorption by seeds and plants in general. Several methods have been employed to create water stress in plants. It has been reasonably well established that polyethylene glycol solutions are more suitable to control water potential in seed germination studies (Emmerich and Hardegree; 1990, Grzesiak et al; 1996). Our study suggested that GRG-295, TS-3, WRP-1 and PG-12 cultivars had significant higher germination percentage at all the stress level. These cultivars were identified as drought tolerant and able to rapidly germinate under both normal and stress conditions. The cultivars like ICP-8863, GC-11-39, TAT-9903 and BSMR-736 showed good germination response under control condition but germinated poorly on decreasing the osmotic potential of the medium. These cultivars showed sensitivity to osmotic stress. ICPL-87119 and G-Red showed a moderate response to by increasing osmotic potential. Both these cultivars germinated moderately at higher osmotic potential. The locally cultivated white and black cultivars were much more tolerant as compare to the red cultivars. A decrease in the percentage of seed germination stimulated by PEG was reported by Gamze et al; 2005, Ghazi et al;

In all the cultivars, the time (in days) for 50% germination was delayed in respond to water stress. There were differential responses to drought stress within cultivars. At -0.15 MPa & -0.30 MPa osmotic potential, all cultivars reached 50% germination before 3rd day of experiment while at -0.45 MPa & 0.89 MPa, 50% germination seen by 5th day. At moderate stress (-0.15 & -0.30 MPa), there was no any significant differences seen in MGT in all cultivars. The significant increase in MGT was observed in sensitive cultivars at -0.45 MPa osmotic potential. The germination rate (GR) significantly declined as decrease in osmotic potential of PEG6000 (Table 1). Increase in PEG concentration causes decline in SVI and GI in all the cultivars but SVI and

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2007, Kaydan and Yagmur; 2008, Marin et al; 2004 and Silva et al; 2001. Reduction in the seeds water content due to low media water potential reduce the activity of hydrolytic enzymes such as proteases, amylase and lipases responsible for hydrolyzing cotyledon reserves required for providing energy in

the early stages of seeds growth by respiration. Thus, it is assumed that the cell expansion under osmotic stress is inhibited mainly by metabolic factors rather than by insufficient pressure potential (Gholani et al; 2010).

Table 1 Effect of different osmotic potential on mean germination time and germination rate in Pigeon pea cultivars

Mean Germination Time Osmotic Potential (MPa) Cultivars ICP8863 ICPL87119 GC-11-39 G-RED TAT-9903 BSMR-736 GRG-295 TS-3 WRP-1 PG-12 0.00 1.20a 1.20a 1.32a 1.20a 1.08a 1.44a 1.48a 1.14a 1.22a 1.14a -0.15 2.63b 1.93b 2.35b 1.82b 2.19b 2.07b 1.88b 1.94b 1.38b 1.52b -0.30 2.97c 2.90c 2.63b 3.38c 2.57bc 2.62bc 3.10c 2.34bc 1.98bc 1.87bc -0.45 5.40d 2.54d 3.56d 2.33bc 3.13cd 3.13cd 2.26cd 2.36cd 2.35b 2.33bc -0.89 0.00f 4.88de 0.00f 3.70de 0.00f 0.00f 4.19de 3.40e 3.50e 3.45e 0.00 53.33a 53.33a 53.33a 53.33a 53.33a 53.33a 53.33a 53.33a 53.33a 53.33a

Germination Rate

-0.15 41.20b 44.53b 34.92b 52.00b 49.87b 46.93b 51.20b 52.77a 48.00b 50.33b

-0.30 30.13c 21.87c 16.00c 12.00c 14.27c 13.87c 33.33c 40.40bc 40.67bc 38.53bc

-0.45 1.06d 18.40cd 6.27d 6.40d 7.20d 4.93d 19.60cd 12.93cd 23.20cd 19.20cd

-0.89 0.00f 2.26e 0.00f 1.46e 0.00f 0.00f 16.80cde 13.33cd 14.60cd 12.53cd

Mean separation by Tukey T test. Means within each varieties followed by the different letter along with rows are significantly different at P 0.01.

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Table 2 Effect of different osmotic potential on seedling vigor index and germination index in Pigeon pea varieties
Seedling Vigor Index Osmotic Potential (MPa) Cultivars ICP8863 ICPL87119 GC-11-39 G-RED TAT-9903 BSMR-736 GRG-295 TS-3 WRP-1 PG-12 0.00 353a 382a 563a 490a 687a 820a 612a 716a 599a 544a -0.15 246.72b 249.40b 211.48b 420.42ab 422.40ab 283.60b 323.52b 569.38ab 311.40b 343.68ab -0.30 182.78c 115.46c 71.70c 105.92c 49.84c 44.46c 303.20bc 489.54bc 385.60bc 264.48bc -0.45 6.60d 52.52cd 8.78d 7.20d 19.20d 13.68d 121.22cd 87.36cd 151.34cd 82.44cd -0.89 0.00f 5.49e 0.00f 3.36e 0.00f 0.00f 42.71de 28.37de 27.00de 21.31de 0.00 4.17a 4.17a 4.17a 4.17 a 5.56a 4.17 a 5.56a 4.17 a 5.56a 5.56a -0.15 2.67b 2.39b 1.89b 2.72b 2.67b 1.57b 3.20b 3.27b 3.75b 4.00b -0.30 1.54c 0.96c 0.83c 0.59c 0.67c 0.48c 1.48c 1.95bc 2.67bc 2.53bc -0.45 0.08d 0.54d 0.20d 0.21d 0.27d 0.29d 1.06cd 0.67c 1.53cd 0.86cd -0.89 0.00f 0.04e 0.00f 0.02e 0.00f 0.00f 0.31de 0.13e 0.14e 0.11e Germination Index

Mean separation by Turkey T test. Means within each varieties followed by the different letter along with rows are significantly different at P 0.01.

Table 3 Effect of different osmotic potential on radical and hypocotyl length in Pigeon pea varieties
Radical length (in cm) Osmotic Potential (MPa) Cultivars ICP8863 ICPL87119 GC-11-39 G-RED TAT-9903 BSMR-736 GRG-295 TS-3 WRP-1 PG-12 0.00 3.63a 3.88a 5.64a 5.02a 6.89a 7.88a 6.44a 7.31a 5.71a 5.61a -0.15 2.56b 2.82b 3.24b 4.3ab 4.16ab 3.26b 5.46ab 3.63a 3.57b 3.58b -0.30 2.55c 2.57c 2.34c 3.44c 1.93c 1.73c 6.18abc 3.87c 4.82c 3.55c -0.45 0.78d 1.94d 2.07cd 1.28d 3.06cd 1.34d 3.22cd 3.23cd 3.22cd 2.42cd -0.89 0.00f 0.75e 0.00f 0.72e 0.00f 0.00f 1.49de 1.52de 1.31de 1.23de 0.00 7.33a 7.04a 7.72a 7.83a 8.23a 7.71a 6.13a 6.49a 9.19a 7.31a -0.15 3.96b 2.98b 2.69b 1.84b 2.98b 1.32b 3.72b 3.84b 4.86b 4.81b -0.30 0.51c 0.38c 0.01c 0.21c 0.18c 0.48c 1.98bc 2.09bc 1.71bc 1.41bc -0.45 0.00d 0.00d 0.00d 0.00d 0.00d 0.00d 0.00d 0.00d 0.00d 0.00d -0.89 0.00d 0.00d 0.00d 0.00d 0.00d 0.00d 0.00d 0.00d 0.00d 0.00d Hypocotyl length (in cm)

Mean separation by Turkey T test. Means within each varieties followed by the different letter along with rows are significantly different at P 0.01.

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Table 4 Effect of different osmotic potential on seedling fresh weight and dry weight in Pigeon pea varieties
Seedling fresh weight (gm plant-1) Osmotic Potential (MPa) Cultivars ICP8863 ICPL8711 9 GC-11-39 G-RED TAT-9903 BSMR-736 GRG-295 TS-3 WRP-1 PG-12 0.00 0.329a 0.331a 0.329a 0.345a 0.391a 0.395a 0.438a 0.445a 0.346a 0.326a -0.15 0.246b 0.252b 0.237b 0.251b 0.256b 0.249b 0.365ab 0.308b 0.282b 0.281b -0.30 0.216c 0.242c 0.222c 0.210c 0.214c 0.202c 0.344bc 0.232c 0.232c 0.215c -0.45 0.191d 0.197d 0.201d 0.201d 0.198d 0.196d 0.296cd 0.199d 0.199d 0.177d -0.89 0.00g 0.095f 0.00g 0.098f 0.00g 0.00g 0.288cd 0.179de 0.159e 0.159e 0.00 0.072a 0.076a 0.075a 0.083a 0.096a 0.092a 0.101a 0.115a 0.082a 0.075a -0.15 0.061b 0.062b 0.059b 0.062b 0.062b 0.061b 0.086ab 0.081ab 0.073ab 0.061b -0.30 0.047c 0.052c 0.050c 0.047c 0.047c 0.043c 0.076a 0.069c 0.061c 0.048c -0.45 0.041d 0.041d 0.042d 0.042d 0.042d 0.040d 0.067d 0.042d 0.042d 0.041d -0.89 0.00e 0.00e 0.00e 0.00e 0.00e 0.00e 0.057de 0.039de 0.036de 0.035de Seedling dry weight (gm plant-1)

Mean separation by Turkey T test. Means within each varieties followed by the different letter along with rows are significantly different at P 0.01.

One way ANOVA showed a significant effect of PEG induced drought stress on early seedling growth (P0.05). Drought stress inhibited hypocotyl length more than radicle growth (Table 3). Radicle and hypocotyl length was significantly decreased as compare to control under all osmotic potentials. Hypocotyl growth was not observed at -0.45 MPa and -0.89 MPa in all cultivars which showed that hypocotyl growth significantly affected under drought stress condition. At -0.89 MPa osmotic potential, the radicle as well as hypocotyl growth was completely inhibited in susceptible cultivars (ICP 8863, GC-1139, BSMR-736 and TAT-9903) while radicle growth was recorded in tolerant (GRG-295, WRP-1, TS-3, PG-12) and moderately tolerant (ICPL 87119 and GRed) cultivars. More number of secondary roots was observed when radicle growth was less (data not shown). Root length is also a vital trait against drought stress in plant cultivars. The plant embryo grows at germination and develops radicles that become the primary roots and penetrate down into

the soil. After radicle emergence, hypocotyl emerges and lifts the growing tip above the ground. These hypocotyls are the primary organ of extension and develop into the stem. Under drought stress condition, the root develops faster than the hypocotyls to acclimatize the drought stress. Therefore, the growth of radicle and hypocotyls should reflect the adaptability of plant to drought stress (Zhu et al; 2006). Thus, root plays a role in plant survival during of drought and also drought tolerant can be characterized by extensive root growth and reduction of shoot growth. Root growth inhibition involves spatially variable reductions in growth rate, outward portion pumping, wall extensibility and gene expression (Neumann; 2008). Similar results were observed by Fransiani and Ghobadi; 2009, Kaydan and Yagmur; 2008, and Kulkarni and Deshpande; 2007. Our results suggest that white and black variety had more resistance than red cultivar. The ratio of radicle to hypocotyl in white and black cultivars was higher than that of red cultivars under 20% PEG

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stress treatment suggest the high resistance of that seeds. It is also observed that tolerant cultivars like GRG-295, WRP-1, TS-3 and PG-12 showed increased seedling fresh and dry biomass at all level of stress than moderately tolerant and sensitive cultivars. The increased biomass in resistance cultivars can be correlated with their radicle and hypocotyl length. A significant decrease in seedling fresh and dry weight was observed in all the cultivars with increasing the PEG concentration. This phenomenon is also reported in Pea by Gamze et al; 2005 and in Ougeinia by Uniyal et al; 1998. GR, MGT, GI and SVI were also significantly affected under drought stress condition. The rate of germination seems to be more sensitive to the water stress than final germination percentage as even moderate stress (-0.30 MPa and -0.45 MPa) caused a significant increase in mean germination time and decrease in germination index, germination rate and seedling vigour index. Our reports are strongly supported by the finding of Farsiani et al; 2009, Singh et al; 2007 and Uniyal et al; 1998. Thus, these significant higher MGT, SVI, GR and GI of tolerant cultivars as contrast to sensitive cultivars signify that quality of tolerant cultivars seeds are better than that of sensitive cultivars. The germination responses show that tolerant cultivar seeds have high viability and maintained high germination capacity under moderate drought stress (Zhu et al; 2006). Since, the response of plants to drought is very complex and diverse, it is unrealistic to consider that there is single gene for drought tolerance and hence the need of an understanding of physiological response of plants under water limited conditions is necessary (Mundree et al; 2002). The knowledge of biochemical and molecular basis of plant responses to stress is required to effectively generate improved stress tolerance legumes (Diaz et al; 2005). The results of present study can be concluded that water stress has no adverse effect on seed germination but it affects early seedling growth. It also indicates that there are much genetic variations among all cultivated Pigeonpea cultivars under drought stress condition. White cultivars like GRG295, TS-3, WRP-1 and black cultivar PG-12 was more tolerant to water stress as compare to other red cultivars. Germination failure due to PEG resulted from osmotic barrier caused by PEG. It can also be concluded that seed size and quality contributes vital role to cope with drought stress. However, a detail

work on genetic basis of drought tolerance in Pigeonpea (work in progress) is needed that can be utilizing genetic potential to improve present cultivars.

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