Physiological Studies on Six Wheat (Triticum Aestivum l.) Genotypes for Drought Stress Tolerance at Seedling Stage

Screening of drought tolerant varieties of wheat are important for ameliorating productivity of water scarce areas. Six wheat genotypes including two local checks were evaluated for drought tolerance against different osmotic stress levels induced by Polyethylene Glycol (PEG)6000. Experiment was set in completely randomized design (CRD) with three replicates and four treatments [0, -0.5 MPa, -0.75 MPa and -1.0 MPa PEG concentrations]. Wheat genotypes Khirman and Chakwal-86 were employed as check varieties. Four test genotypes evaluated were SD-621, SD-222, NIA Saarang and 22-03. Data were recorded on various seedling parameters such as germination percentage, germination index, shoot length, root length, fresh and dry weight of shoot and root, chlorophyll contents (a and b) and ionic contents (K+ and Ca2+, and K+ /Ca2+ ratio). No seedling survived at -1.0 MPa osmotic stresses. Best results were illustrated by wheat genotypes SD-222 and SD-621 with 98.89% and 96.7% germination at -0.5MPa and -0.75MPa osmotic stress, respectively. NIA Saarang and genotype 22-03 also exhibited better results at moisture stress. Reduction in chlorophyll and ionic contents was occurred with rise in water deficit in all genotypes. These findings appreciate the future use of these genotypes for obtaining high yield in water sparse areas.


Introduction
Drought is a worldwide problem and present climatic changes have even made it is worse in many parts of the world. Exposing plants to water stress adversely affect plant growth and productivity. Osmotic stress is produced when inadequate moisture is present for plant's growth and development [1]. Drought is one of the major environmental issues affecting the wheat production. Pakistan is facing extreme drought conditions from last three decades especially in Sindh and Balochistan.
Wheat is an important cereal food and feeds one third population of the world fulfilling 50% of their protein requirements [2]. It is the staple food of Pakistan. Seed germination is the vital stage in plant's life cycle and it is regulated by genetic and environmental factors as well as seed treatment methods. The time and degree of seedling establishment are also highly important factors in determining the yield [3].
In case of wheat, the main threatening issue is the deficit water at seedling stage, mid season stress and terminal stress.
Various factors affect the yield of a crop like seed germination, seedling vigor, mean emergence time, growth rate and desiccation tolerance [4]. Germination and seedling establishment are rigorously declined by drought [5]. Thus, development of drought tolerant wheat cultivars is encouraged to gain high yield under moisture stress. Growth involves cell division, cell elongation and differentiation, and is regulated by genetic, ecological, physiological and morphological phenomenons. Cell growth is the most drought-sensitive physiological processes due to the turgor pressure reduction in cell [6].
Osmotic adjustment is the process of maintaining water relations in osmotic stress. Under water deficit, accumulation of solutes occurs which results in lowering of the osmotic potential of the cell, which pulls water molecules in the cell and helps in maintaining turgor. PEG is used for artificial induction of osmotic stress. [3,7]. Poly ethylene glycol (PEG) molecules are non-ionic, inert and have impermeable chains that induce water stress with no physiological damage and maintain the same water potential throughout the trial [8]. As PEG molecules do

Materials and Methods
The experiment was conducted in plastic bowls to assess the effects of different levels of drought stress at seedling stage in four wheat varieties at Plant Physiology laboratory, Nuclear Institute of Agriculture, Tandojam, Pakistan. The layout of the experiment was in completely randomized design (CRD) with four treatments (0, -0.5, -0.75, -1.0MPa PEG solution) and three replicates. Day and night lengths were 14/10 h, with 25°C and 20°C temperatures, respectively. Wheat genotypes, Khirman (C 1 ) and Chakwal-86 (C 2 ) were used as check varieties. Four test varieties included SD-62, SD-222, NIA-Saarang and 22-03. Healthy seeds were first sort out, sterilized in 3% solution of sodium hypochlorite for about 10 minutes and thoroughly washed with distilled water. 30 seeds of each variety were placed in each bowl containing PEG solution and Hoagland's solution. Bowls were covered and placed in dark for 48 hours for germination. After germination had started, bowls were transferred in light in growth chamber (Vindon, England) and germination was noted up to 96 hours. Germination percentage and germination index was computed. Two weeks old seedlings were subjected to the following measurements.

Growth analysis
Seedling growth was assessed by taking into account following characters, like root and shoots length (cm) of 10 randomly selected seedlings while 20 randomly selected seedlings were subjected to fresh and dry weight (g) of root and shoot.

Biochemical analysis
Chlorophyll a and b were determined following the method of Lichtenthaler [9] based on fresh weight by using the following formulae.

Determination of ionic constituents
Potassium and calcium concentrations, in roots and shoots, were analyzed by Ansari and Flowers's [10] methodology. According to it, 0.1g dried plant material was digested with 0.1M acetic acid (CH 3 COOH) in water bath for 1 hour at 95°C, the extract was filtered and suitable dilution was made. K + and Ca 2+ contents were determined by flame photometer (Jenway, Model PFP7).

Statistical analysis
All parameters were taken thrice and data was statistically analyzed using analysis of variance techniques to check the significant differences among wheat genotypes using least significance difference (LSD) test at 0.05 probability levels [11].

Results
Early wheat seedling's growth and physiology was adversely affected by the increase in level of drought stress. Significant reduction in shoot and root length, fresh and dry weight at higher concentrations of PEG was observed as compare to control. Deceleration in percentage germination and germination index (GI) was also occurred with alleviated levels of water stress. The variations among genotypes were also momentous for all studied traits. All varieties expressed better results in control treatment especially SD-222 and SD-621 along with local checks.

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Genotype 22-03 executed maximum shoot and root fresh weight with minimum % reduction over control, i.e. 0.079g (28.83% reduction over control) and 0.036g (16.28% reduction) respectively at -0.5MPa PEG stress which had decreased up to 0.073g (34.23% reduction) and 0.025g (41.86% reduction) under -0.75MPa PEG stress. SD-222 also showed good results in case of seedling fresh and dry weight (Figure 3, 4).

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Chlorophyll contents of all wheat genotypes were affected by drought. Results of total chlorophyll contents expressed the presence of more chl. b as compare to chl. at in all treatments (Table 1). Highest chlorophyll contents were observed in SD-621 and NIA Saarang which were reduced slightly in comparison with chlorophyll contents of other genotypes with alleviated levels of PEG concentrations (Table 2). Low chlorophyll contents were present in wheat genotype 22-03. NIA Saarang had highest shoot K + /Ca 2+ ratio (0.61) equalent to check variety Khirman (0.62) under normal conditions followed by SD-621(0.57) and SD-222 (0.49). Under drought, shoot K + /Ca 2+ ratio boost up in genotype 22-03 from 0.47 (control) up to 0.72 under -0.75 PEG stress. Similar ascending trend was observed in genotypes SD-621 and SD-222. Root K + /Ca 2+ ratio was not significantly affected by osmotic stress. Maximum K + /Ca 2+ ratio was marked in roots of genotype SD-222 (1.15) at -0.5MPa water deficit conditions (Table 2).

Discussion
The present study showed that osmotic stress induced by PEG-6000 had inhibitory effects on plant seedling's growth and physiology. All wheat varieties showed diversity in their ability to tolerate chemical dehydration induced by PEG during the seedling's growth and development. The plant growth related parameters such as root and shoot length, seedlings fresh weight etc. are visualized as major characteristics for screening of drought resistant wheat varieties [12]. Limited water conditions inhibited plant growth resulting in decline in biomass. The decrease in the shoot and root length and biomass had been observed in all genotypes at water deficit conditions. It may be the result of diminish relative turgidity and dehydration of protoplasm which is correlated with turgor loss and reduced cell expansion and impediment of cell division. All the under tested varieties fall in the category of drought sensitive genotypes as their % reduction is greater than 30%. Khakwani et al. [7] also documented the deduction in shoot and root length and fresh seedling weight while studying early growth responses in wheat varieties by inducing osmotic stress using 15% and 25% PEG-6000 solutions. Mean followed by same letters are non-significantly different at a level of P < 0.05.

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Drought stress produces changes in photosynthetic constituents [13]. Reduction of chlorophyll was the result of disappearance of thylakoid structures and chloroplast disintegration [14]. SD-621 and NIA-Saarang exhibited better chlorophyll contents under moisture stress which showed their tolerance against drought. These results are supported by Khayatnezhad et al. [15] according to which chlorophyll contents of tolerant cultivars increased under drought stress.
The under tested wheat genotypes also varied in response to solute accumulation and mineral uptake in water stress. Wheat genotype 22-03 better maintained its turgor by accumulation of higher inorganic ions as compare to other genotypes under scarce water conditions. All other genotypes also had better K + and Ca 2+ contents in their root and shoot under stress condition.

Conclusion
All the tested wheat genotypes exhibited tolerance against osmotic stress under laboratory conditions. Hence, it is recommended that these genotypes which performed better in water stress condition could increase production of arid lands.