Abstract: Soil salinity is one of the most serious worldwide threats to agriculture. In Pakistan, salinity has affected a vast potentially agricultural area, the extent of which is increasing day by day. Having been identified as an important national issue, continued efforts are mandatory to find ways and means to tackle this problem to get more yields from salinity-affected soils. Although various approaches like conventional breeding, genetic engineering and molecular biology techniques, and amelioration of salt-affected soils have been employed in the past, the success has not been up to mark so far, despite the fact that they took a lot of time and cost heavily. On the other hand, agronomic practices are usually success-oriented in a short time, without incurring large sums of extra money. CO2 has multiple effects on the plant growth and its environment. It is anticipated that by the end of this century, ambient CO2 level will be double (>700 ppm) the present level (350 ppm). Studies have shown that increased CO2 level has a favourable impact on photosynthesis and plant productivity in enhancing 30-50% or even greater. Salinity, on the other hand, has an adverse effect on photosynthesis. There are very few reports on the salinity-high CO2 interaction, but whatsoever data is available indicates that elevated CO2 levels have amicable effect on the salinity-grown plants. However, it needs to be established on firm grounds. We propose this project to study the salinity-high CO2 interaction using wheat as a model plant, keeping in view our current and upcoming agro-climatic conditions. In addition to resource generation in terms of facilities and manpower training, we will provide a model system for crop water requirement, stress tolerance and future breeding strategies for the future wheat crop production

Problem statement: In our country, the problems of soil salinity and high aerial CO2 levels prevail. Soil salinity is increasing mainly due to substandard irrigation water and partly due to seepage or flow of industries effluent into the adjacent fields that change the physico-chemical properties (pH and electrical conductivity) of thousands of acres. Ambient CO2 level is increasing due to excessive use of automobiles and ill-planned mushroom growth of industry in the urban, sub-urban and rural areas. As most of the major crops are glycophytes, they show substantial growth reduction under low to moderate salt levels

Objectives:

• To determine any shift in the salt tolerance threshold/limit of wheat varieties under CO2 enriched environment
• To study the patterns of dry matter (assimilate) partitioning to above- and below-ground parts under salinity combined with CO2 enrichment.
• To determine the whole plant net rate of photosynthesis, transpiration and water use efficiency under saline conditions combined with aerial CO2 enrichment
• To study the changes in water and osmotic relations characteristics of wheat leaves under the interaction of soil salinity and ambient CO2 elevation.
• To determine the changes in net photosynthesis of flag leaf and implication for grain filling and grain yield and yield components under salinity and elevated CO2 levels.
• To study source-sink relationship of vegetative and reproductive parts under high CO2 and salinity.
• To make the recommendations available for wheat breeders, agronomists and plant physiologists for future production of wheat varieties to grow under salinity and elevated CO2 conditions

Methodology:

For second year of the project activities, certified seed of five wheat varieties viz., BK-2002, AS-2002, Sehar, Shafaq and Inq-91 was obtained from Federal Seed Certification and Registration Department (FSC&RD), Faisalabad. For CO2 enrichment, stainless steel and plexiglass fitted open-top canopies were constructed, with the aid of funds from some other source, as the provided funds under the project were meager and therefore not spent.
To determine any change in the growth, yield and physiological attributes of the varieties, pot experiments were started in the Botanical Garden of the Department of Botany, University of Agriculture, Faisalabad. The pots were filled with loam soil, having ECe of 2.75. At each stage, desired levels of salinity (6, 8, 10, 12, 14 and 16 dS/m) were developed gradually at the rate of 2 dS/m NaCl per day in order to avoid the osmotic shock. After the development of salinity levels, one half of the plants were shifted to the canopies at 350 (control) and the other half at 700 ppm CO2 enriched environment. The CO2 was supplied using CO2 cylinders, which was mixed with air to accomplish the required concentration. The CO2 level inside the canopies was measured with the infra-red gas analyzer (IRGA) and maintained the required level. The plants were grown inside both the canopies for 15 days at respective growth stages and then harvested. The design of the experiment was completely randomized with three replications per treatment. At each harvest, determinations were made for the growth, yield, gas exchange, water relations, and ionic relations parameters at various growth stages.

Growth and yield determinations: Before harvest leaf area per plant was determined as leaf length × leaf width × 0.68 (correction factor). Shoot length was determined of intact plants, while root length was taken after carefully removing the roots from the soil. The fresh shoot and root were transferred to paper bags and dried in an oven at 70oC for two weeks and determined for their dry weight. Flag leaf area was determined 15 days after the application of salinity at the onset of grain filling. For the determination of yield and yield components at maturity, the gains were extracted from the spike and counted. One hundred grain weight and grain yield per plant were determined. Above-ground dry mass was determined to determine the harvest index (HI) as: HI = (grain yield) × 100/straw yield
Gas exchange characteristics: Third leaf from the top of each plant was measured for the gas exchange attributes including net rate of photosynthesis (Pn), transpiration rate (E), water use efficiency (as Pn/E ratio) and stomatal conductance using infra-red CO2 analyzer (IRGA; LCA-4, Analytical Development Company, Hoddesdon, England). Measurement were taken between 10:00 and 11:00 am with the following leaf chamber adjustments: leaf surface area 11.35 cm2, ambient CO2 concentration 357 ppm, temperature of leaf chamber varied from 28.5 to 30oC, leaf chamber gas flow rate (v) 392.8 ml/min, molar flow of  air per unit leaf  area  440 µmol m2/s, ambient pressure (P) 99.6 kPa , water vapor pressure  in to chamber ranged from 20.5 to 23.1 mbar, photosynthetically active radiations (Q leaf) on leaf surface ranged from 975-1250 µmol m2/s.
To determine the phtotsynthetic attributes of flag leaf, the glass chambers were used. The flag leaf (at grain filling stage) was placed inside the chamber and photosynthetic determinations were made under CO2 enriched atmosphere from the salinity treated and non-treated plants. The prescribed levels of CO2 were passed through the chamabers, which were clamped in position to enclose the flag leaf. The set of leaf chamber and IRGA conditions for these determinations remained the same except ambient tempearture which was 30.5 – 32.5oC.
Leaf water relations: Leaf water potential (ψw) was measured with Scholander pressure chamber (Scholander Bomb, Germany). Penultimate leaf was used to determine the leaf osmotic potential (ψs). For this purpose, the leaf was excised and immediately on dry ice and stored in a freezer (at -30oC) for seven days, thawed and cell sap extracted. The sap so extracted was centrifuged at 13000 rpm for 5 min and determined for ψs with osmometer (Wescor-5500). Turgor potential (ψp) was calculated as the difference of ψw and ψs.
Osmolytes determination: Free proline was determined from the frozen fresh leaves according to the method of Bates et al. (1973). A 0.5 g of fresh leaf tissue was homogenized with 10 mL of 3% aqueous sulphosalicylic acid and filtered homogenate through Whatman No. 2 filter paper. Two mL each of acid ninhydrin and glacial acetic acid were added to 2 mL of filtrate. The mixture was vortexed, heated at 100oC in a water bath for 1 h and reaction terminated in an ice bath. The reaction mixture was extracted with 4 mL of toluene by vigorously vortexing for 15-20 sec. The chromophore containing free proline was aspirated and added to a test tube, warmed to room temperature and measured the absorbance at 520 nm. Same procedure was followed for blanks using 2 mL of 3% aqueous sulphosalicylic acid. A standard curve was constructed by running the proline standards (10 to 50 µg 2 mL-1) along the unknown samples. The amount of free proline in the leaves was calculated from the standard curve.
The GB was determined using the method of Grieve and Grattan (1983). Fresh bud extracts were prepared by vigorously shaking in 2N H2SO4 and cooled. These extracts were mixed with equal volume of periodide (prepared by dissolving excess of iodine in potassium iodide solution, vortexed and kept at 4oC for 16 h). The mixture was centrifuged at 10,000×g at 4oC for 15 min and supernatant discarded. The periodide crystals left in the bottom of the test tube were dissolved in 10 mL of 1, 2-dichloroethane, vortexed and left at room temperature for 15-20 min. The absorbance of the colored solution was taken at 365 nm using spectrophotometer.
For the determination of glucose equivalent soluble sugars, chopped fresh material (0.1 g) was extracted in 5 ml of 0.2 M phosphate buffer (pH 7) overnight. A 0.1 ml of aliquot was taken in a test tube and 3 ml of freshly prepared anthrone reagent was added. Mixture was heated at 95oC for 15 min. Reaction was terminated by placing the test tubes in an ice bath and absorbance was read at 625 nm on spectrophotometer (Hitachi U-2001, Japan). A standard series of glucose (20, 40, 60, 80 and 100 μg/ml) was prepared to calculate the amount of soluble sugars in samples.
Total Na+, K+ and Cl-: For these determinations, dried powdered (0.5 g) material was put in test tubes (15 ml) and 8 ml of the distilled water was added. The samples were extracted by boiling in distilled water for one hour in a water bath, filtered and volume made up to 50 mL. The analysis of Na+ and K+ was carried out using flame photometer (Sherwood Model 410, Cambridge). The actual quantity of Na+ and K+ in the samples was determined by constructing standard curves separately for both these ions. The K+/Na+ ratio was derived by dividing the content K+ by Na+. The Cl- concentration was determined from simple water extraction with Chloride Analyzer (Model 926, Sherwood Scientific Ltd., Cambridge). The Cl- concentration was directly measured in mg/kg dry weight.
Data analysis: Data obtained at the final harvest were analyzed statistically using computer software COSTAT for various growth parameters at seedling and tillering stage and flag leaf area and yield parameters at grain filling and maturity stages, respectively. The analysis of variance (ANOVA) was performed to find significant differences in various factors and possible interactions of these factors, if any.

Results and Discussion

 The results of this project revealed that, despite varietal difference, although wheat growth was hampered by the increased salinity levels, the increased CO2 levels were helpful in counteracting the detrimental effects of salinity. The rising CO2 level proved more beneficial to the sensitive varieties rather than the tolerant varieties, as evident from a greater increase in the net rate of photosynthesis third leaf from the top at seedling stage and flag leaf at grain filling stage. Increased CO2 levels was also helpful in reducing the tissue Na+ and Cl- contents and increasing the K+ contents, improving leaf water potential as well as increased synthesis of cytosolutes (free proline, glycinebetaine and soluble sugars) at all stages of growth of wheat varieties. The increased net rate of photosynthesis is the result of increased Rubisco affinity towards CO2 (Wahid and Rasul, 2005), which is an important manifestation of salinity tolerance
It is evident that rising levels of atmospheric carbon dioxide (CO2) can affect air temperature and precipitation patterns, thereby, causing global change in ways yet to be determined. Because of potential alteration in future climatic conditions, soil–water content may be affected in regions of the globe where the foremost grain source, wheat (Triticum aestivum L.), is grown (Food and Agricultural Organization of the United Nation (FAOSTAT, 2007). In general, these studies have shown that increases in CO2 (e.g. a doubling from 350 to 700 ppm) increase photosynthetic rates for C4 species and decrease water use per unit area of vegetation for C3 species (Morison, 1987; Drake et al., 1996; Kimball et al., 2002).  Findings of this project clearly show that to cater with the upcoming climatic changes, breeding of varieties may be carried out which are although sensitive to salinity but responsive to increased CO2 levels to certain limits.

Conclusion

There was a substantial improvement in the gas exchange properties particularly Pn and WUE and reduction in the tissue ionic concentrations for Na+ and Cl- but an improvement in K+ and K+/Na+ ratio at seedling, tillering and grain filling stages. Applied salinity hampered the leaf water relations, while elevated CO2 levels helped the plants to maintain high turgor by virtue of the accumulation of osmolytes free proline > glycinebetaine and soluble sugars at all growth stages. Improvements produced in the flag leaf for the physiological attributes were of greater significance due to its significance in the grain filling at maturity. The net photosynthesis of flag leaf appeared to play a greater role in the improved grain yield and harvest index under CO2 enriched environment. Based on the physiological attributes, although salt tolerance of varieties improved under elevated CO2 levels, the basic trend of changes was similar under both the CO2 levels. The growth enhancements due to elevated CO2 levels support the assertion that it acts as foliar fertilizer and supports the plant growth. An upcoming increase in the atmospheric CO2 is likely to improve the net photosynthesis of the salinity stressed wheat

Recommendation

In view of the anticipated increase in CO2 levels in our environment, as well as increased salinization of the soils, it is recommended that breeding efforts should be made to produce such wheat varieties, which can efficiently assimilate CO2 under moderately saline conditions.