Screening of Blackgram Genotypes for Drought Tolerance using PEG (Polyethylene Glycol) Induced Drought Stress at Seedling Stage

Global climate change, in the form of increasing temperature and decreasing soil moisture should results in the projected decrease in the yield of food crops over the next 50 years. The lack of moisture in soil directly affects seed germination and seedling growth of a plant. Germinating the seeds in solutions of different water potentials is a convenient method to study the responses of seeds at germination against low water potential. Polyethylene glycol (PEG) compounds have been used to simulate osmotic stress effects in vitro to maintain uniform water potentials. PEG 6000 found to be convenient solute to create osmotic stress without causing any toxicity to the plant cells (Verslues et al., 2006; Van den Berg and Zeng 2006 and Datta et al., 2011). Molecules of PEG 6000 are too small to influence the osmotic potential but large enough not to be absorbed by plant. Moreover PEG withdraws water from the cell without entering into apoplast which mimics dry soil (Hatem Zgallai et al., 2005 and Radhouane 2007). It is envisaged from the above findings that PEG solution can be used in the laboratory for screening drought tolerance varieties. Therefore, present experiment was designed to screen the germplasm of twenty nine blackgram genotypes against moisture stress at germination and seedling stages under in vitro conditions. Blackgram is one of the most highly prized pulse crop, cultivated in almost all parts of India. It has inevitably marked itself as the most popular pulse and can be most appropriately referred to as the “king of pulses” due to its mouthwatering taste and numerous other nutritional qualities. Blackgram is a perfect combination of all nutrients which include 20-25% of proteins, 40-47% of starch along with ash, carbohydrates and essential vitamins. It is boiled and eaten directly or used after splitting into dhal. It is extensively used in various culinary preparations and recommended for diabetes. The green pods are eaten as a vegetable which is highly nutritious. The hulls or the outer covering of green and straw are used as a cattle feed (Baroowa and Gogoi 2015). Blackgram is grown mostly in marginal and rainfed areas and the major constraint for higher productivity is inadequacy of moisture. Hence, developing a drought tolerant genotypes is necessary for cultivation in these areas. The aim of this study was to assess the effect of polyethylene glycol on germination ability of some blackgram genotypes and to identifying the superior genotypes for drought tolerance.

The experimental material used in the present investigation consisted of 28 blackgram genotypes were evaluated at the Department of Genetics and Plant Breeding, Faculty of Agriculture, Annamalai University, Annamalainagar. Healthy and seeds of uniform size were surface sterilized with 0.1% Mercuric chloride (HgCl₂) for 2-3 min and then washed thoroughly with distilled water. Sterilized (10) seeds were sown in petridishes containing moistened germination paper with various water potential viz., 0.0 (control), 10%, 20% and 30% of PEG 6000 in 100 ml of distilled water following the method of Hadas (1976) and three replication were maintained for each treatment. The emergence of 2 mm radicle was set as the criteria for germination (Kaur et al., 2017 and Yadav et al., 2013). Numbers of seeds germinated were counted from day-3 to day-6 after sowing to determine the germination percentage. Seedling growth parameters such as plumule length and radicle length were recorded on 6th day after sowing in all the seedlings. Fresh weight of seedling, Dry weight of seedling, Seedling vigour index and drought tolerance index (DTI) were measured using the following formula:
           
PEG 6000 concentration of 30% that induce 50% seedling mortality was the stress level for screening the blackgram genotypes.
 
Screening of the blackgram genotypes for drought stress tolerance at seedling stage
 
A set of 28 blackgram genotypes (Table 1) were used for this study to identify the tolerant genotypes. Surface sterilized blackgram seeds were sown in petridishes containing germination paper moistened with water 0.0 and 30% of PEG 6000 solution. Three replications with 10 seeds in each were maintained. Plumule length and radicle length of seedlings were recorded on 6^th day after sowing in all the seedlings in each replication.
 

 
Statistical analysis
 
The experimental design was factorial experiment under completely randomized design (FCRD) with five replications  for standardization of drought stress and three replications for screening the set of 28 green gram genotypes. Statistical analysis was performed using Agres software.

Based on the drought stress was imposed on the blackgram genotypes at 30% and distilled water was used for control. The same seedling growth characteristics were recorded on 6th day after sowing.
       
A significant delay in the initiation and completion of germination was also observed in PEG 6000 treated varieties. The decreased germination in response to water stress is not uniform in all cultivars. A significant decrease in the germination percentage was observed at 30% concentration the germination percentage was higher (93.33%) in VBN 8 followed by (90.00%) ADT 4 (Table 2). At control the genotypes CO 6, ADT 6, Nirmal 7, S-07, IPU-95, GOLDSTAR NUL7 VISWAS, IPG 104, VBN (Bg) 3, LBG 17, AUB-8, AUB-3, AUB-21, VBN 7 recorded higher (100%) germination percentage. The rate of germination was varying with the genotypes at 30% concentration. Decline in germination percentage due to lower water potential have been reported earlier in Babu and Rosaiah (2017) and Rana et al., (2017).
 

       
The radicle length and plumule length in different genotypes were recorded in all the germinated seedlings of blackgram genotypes. Maximum radicle length was observed in Nirmal 7 (2.20 cm) and VBN 8 (2.16 cm). The  mean radicle length of all genotypes measured 1.89 and 2.92 cm in control and 0.91 cm in 30% level respectively (Table 2).
       
Plumule length was higher in NRIB-002 (1.10 cm), T9 (0.96 cm), NUL 7 VISWAS (0.96 cm), AUB-39 (0.90 cm) and IPU 94-1 (0.83 cm) at 30% level (Table 3). Therefore, the lower water potential during germination inhibits the seed germination or suppress the growth and development of seedlings (Khodarampour 2011; Bibi Kaur et al., 2017 and Sivakumar et al., 2017).
 

       
Decreasing water potential was caused a remarkable reduction in fresh and dry weight of seedlings. Significant reduction in seedling growth in terms of length and weight of seedlings among the genotypes might be attributed to their differential response in term of tolerance level to moisture stress. The fresh weight of seedling was higher in NUL 7 VISWAS (1.72 g) followed by MDU 1 (1.60 g) at 30% level respectively (Table 4).
 

               
Under control condition the dry weight of seedling varied between 0.08 g (S-07) to 0.38 (AUB-23). Lowering of water potential resulted in significant reduction in dry weight of seedling of all the genotypes. The genotypes MDU 1 (0.18 g), NUL 7 VISWAS (0.17 g), Nirmal 7 (0.16 g) and VBN (Bg) 4 (0.15 g) showed higher dry weight of seedling (Table 4). Similar kind of reduction in fresh and dry weight of seedling was reported by Gupta et al., (1993) and Fatemeh et al., (2011).

The seedling vigour index and drought tolerance index was also calculated for different genotypes (Table 5). The drought tolerance index was higher in Nirmal-7 (0.42), VBN 8 (0.35) and IPU 94-1 (0.34) genotypes which indicates higher level of tolerance to drought. The seedling vigour index is one of the indicative measurements for the speed of germination and development of seedlings at lower water potential. The genotypes has recorded high drought tolerance index has faster development. The present results are in accordance with the reports of Sabesan and Saravanan (2016) and Patheeban et al., (2017). The seedling vigour index was higher in VBN 8 (273.33).
 

The blackgram genotypes were screened for their responses to drought on the basis of root length, dry weight and drought tolerance index. The blackgram genotypes Nirmal 7, NRIB 002, MDU 1, VBN 8 and NUL 7 VISWAS were showed better tolerance (>50% germination percentage) to drought stress during germination stage. The selected genotypes have been observed to possess tolerance traits to withstand under drought stress and can be used for further investigation of field condition.

Authors are acknowledging the help rendered by Dr. K.R. Saravanan, Asst. Professor, Dept. Genetics and plant Breeding, Faculty of Agriculture, Annamalai University, Annamalainagar.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

C. Shobanadevi performed the lab experiments, measurements, data analysis and drafted the manuscript, K. Vadivel and R. Elangaimannan supervised the work, worked on the manuscript and aided in interpreting the results, R. Elangaimannan, K. Vadivel and C. Shobanadevi were involved in planning. All authors provided critical feedback on research, analysis and manuscript.