Evaluation of Chickpea Cultivation Approaches in Terms of Environmental Resilience and Future Protein Security

The commercial growth in chickpea production for exportation purposes is not keeping pace with increasing demand for protein and protein derived products. In this concern, a pot experiment was conducted under field conditions during winter 2013-2014 at Botany department, AMU, Aligarh, India. Treatment consists of (1) FW (2) FP (3) FS (4) FPS (5) SGA (6) SGA+FP (7) SGA+FS (8) SGA+FPS (9) FGA (10) FGAP (11) FGAS (12) FGAPS (13) SGA+FGA (14) SGA+FGAP9 (15) SGA+FGAS (16) SGA+FGAPS. Before sowing, the seeds of chickpea are soaked for 8 h in 10-6M GA3. After 60 and 70 days of sowing, the plants were sprayed with 10MGA3 along with 2 kg P and /or S/ha in equal splits. Performance of the crop was assessed especially in terms of nodule number per plant, nitrate reductase activity (NR), nitrogenase (N-ase) two most significant N-fixing enzymes, leghaemoglobin content (Lb), pod weight per plant, seed yield per plant, and seed protein content. Nitrogen (N), phosphorus (P) and potassium (K) content in leaves were influenced almost non-significantly due to applied P and S level. Treatment (16) SGA+FGAPS proved best, it enhanced NR by 22.37% and 22.46%; Lb by 206.113 and 215.38% respectively at 90 and 100 DAS. Seed yield per plant and seed protein content enhanced by 86 and 21% by the same treatment at harvest without compromising the N-fixing activity.

Chickpea is a crop that is environment friendly and sustains soil productivity.The benefits of the crop thus extend beyond the increase to the farmers and the farming systems.Chickpea has been well recognized as a valuable source of proteins particularly in the developing countries where majority of the populations depends on the low proceeds food for meeting the dietary requirements [1][2][3] .Its magnitude of significance is more among Indians due to their reliance on vegetarians diet besides limited buying capacity of more than 200-250 millions (27%) people living below the poverty line (BPL).Like other pulses, supplementation of chickpea with cereals based diets is considered to be one of the possible solutions to the problems associated with protein energy malnutrition (PEM).The daily per capita availability of the chickpea is a source of approximately 2.3 % (56 Kcal) energy and 4.7 (27g) proteins to Indian population besides being a major source of calcium and iron 4 .
The area occupied by the crop is 16% of the total pulse area but in some countries, e.g., India and Pakistan, it is the most important pulse crop and the area occupied could well be around 50% of the total pulse area 5 .The productivity of chickpea is much low as compared to cereals crops due to varied reasons.Two of these are described here (i) excessive cold conditions adversely affect productivity, (ii) farmers are ignorant of precise doses of fertilizers recommended by the Agriculture Department for a particular cultivar and region, the techniques of cultivation of high yielding cultivars [6][7] .
Among the factors determining the desired yield of chickpea, use of quality seeds and yield stability of cultivars over varied environments are most critically important.The study of cultivar environment interaction provides useful information to identify stable cultivars over a range of environments.The growth and development of any individual plant species proceed at a speed and extent predetermined by the genetic constitution.The eventual expression of the pattern, however, is modified by many interlocking environmental complexities which individual plant inhabitants.It is already have been stated that vigorous seed germinates rapidly and uniformly after planting and the emerged seedlings have the ability to grow vigorously under the wide range of environmental conditions.However, with the release of new crop cultivars, it is imperative to work out the precise package of farm practices for each agroclimatic conditions so as to exploit their genetic potential fully as cultivars of the same species, differ in their inputs including nutrients 8 .Most of the Indian soils supplementing chickpea are low in available P and S. As mentioned earlier, there is limitation on increasing the acreage for cultivation, it is, therefore, highly logical to innovate ways that can improve the productivity as well as environment sustainability.In this regard, an approach could be to make plants utilize fully the available resources leading to maximum harvesting of solar energy and subsequently enhancing the active sites but without interfere with better N2-fixation efficiency.To attain such goal, the use of GA 3 may play various important roles [9][10][11] .Moreover, enhancing P and S availability to chickpea along with gibberellic acid (GA 3 ) holds promise in the present scenario of escalating prices of phosphatic and sulphatic fertilizers and a general deficiency of P in Indian soils [12][13] .After the determination of the promising responses of chickpea to P, S or GA 3 or combined application in terms of maximum yield, protein value and economic profitability of production and environmental resilience (better N-fixation) in a particular environment as well as a very little investigation is in the existing literature, the present study was undertaken.

MATERiAlS And METhodS
The soil sample was analysed in the Soil Testing Laboratory, Government Agriculture Farm, Quarsi, Aligarh for various physico-chemical properties.The physico-chemical analysis of the mixture of soil and FYM used for filling of the pots is as; texture, sandy loam; pH, 8.04; E.C (dSm-1), 0.65; N-P-K (Kg/ha), 210-34.32-209.20 respectively and calcium carbonate (%), 0.10.Aligarh district has the same soil composition and the appearances as those found generally in the plains of Northern India.The average temperatures for December and January are about 15ÚC and 13ÚC respectively.
Besides this, the average rainfall is 847.3 mm.More than 85% of the total rainfall occurs during June to September.The relative humidity of the winter season ranges between 56% to 77% with an average 66.5% that of the summer between 37% and 49% with an average of 43% and that of the monsoon seasons, between 63% and 73% with an average of 68%.Subsequently, seeds were inoculated with the recommended strain of Rhizobium and then were sown.The amount of GA 3 was dissolved in 10 Ml ethyl alcohol and the final volume was made 100 Ml using DDW.Further dilutions of the stock solutions (10 -6 M GA 3 ) were made with DDW.A uniform recommended basal dose of 40 kg N + 30 kg P 2 O 5 / ha (17.9 mg N+13.4 mg P/kg soil) was applied to all pots with the half dose of N and full dose of P giving at the time of sowing and the remaining half dose of N after 30 DAS with the help of Pora.
The remaining amount of N dose was compensated with urea.Finally, four plants per pot were maintained.A water-sprayed control was also included in the scheme of treatments.The experiment was performed according to a simple randomized design.

Sampling techniques
One plant from each replicate was uprooted randomly at the various sampling stages to assess the performance of the crop on the basis of growth and biochemical parameters at 90 and 100 DAS while yield associated attributes at harvest.

Growth characteristics
Length of shoot on per plant basis was determined with the help of a metre scale.LAI is determined by the following formula suggested by Watson 14 :

Ground area
At the flowering stage, sample plants were collected from 1-2 cm linearly for the collection of nodules.The roots of the collected samples were washed carefully and all the nodules were separated manually for counting their number and later oven dried at 70ÚC for 72 hours to record the dry weight of the nodules.

biochemical characteristics
The NR and N-ase activities in fresh leaves were estimated by the methods of Jaworski 15 and acetylene reduction assays method of Turner and Gibson 16 respectively.The Lb content in fresh nodules was estimated following the method of Sadasivam and Manickam 17 .The Lb content was calculated by the following formula: Where, D is the initial dilution.A556 and A539 are the absorbances at 556 and 539 nm, respectively.The calculation is based upon the equation E = 23.4x 10 3 mol -1 cm -1 .N and P were estimated according to the methods of Lindner 18 and Fiske and Subbarow 19 respectively whereas K was estimated flame photometrically.

yield characteristics
Number of pods per plant was determined at physiological maturity from two remaining plants.The pods were manually removed from all the harvested plants and number of pods per plant determined.Pod weight per plant was estimated with the help of electronic balance.The total seeds of two plants were threshed, cleaned and allowed to dry in the sun for some time and their weight was obtained with the help of an electronic balance, with  20 .

data collection and analysis
All data were analyzed statistically adopting the analysis of variance technique, according to Gomez and Gomez 21 .In applying the F test, the error due to replicates was also determined.When 'F' value was found to be significant at 5% level of probability, critical difference (CD) was calculated.Mean differences among the treatments were adjusted by using the Ducan multiple range test (DMRT) at 5% level of significance also.

RESulTS
Among treatments, combination of presowing seed treatment with GA and foliar spray of GA, P and S (S GA + F GAPS ) gave the maximum value for shoot length per plant at both sampling stages (90 and 100 DAS).Its effect was, however, equal to that of S GA + F GAS at each sampling stage.Treatment S GA + F GAPS gave 86.71 and 78.95% higher value at 90 and 100 DAS respectively than the water-sprayed control, i.e.F W (Table 1).Treatment S GA + F GAPS gave the maximum value of LAI at both sampling stages.Its effect was followed by that of S GA + F GAP and S GA + F GAS at each stage of sampling and also by that of S GA + F GA at 90 DAS.Treatment S GA + F GAPS gave 218.42 and 186.21% higher value at 90 and 100 DAS respectively than F W (Table 1).
Treatment S GA + F GAPS gave the maximum value of nodule number per plant at each sampling stage.Its effect was followed by that of S GA +F GAS and S GA +F GAP at 90 DAS and was, however, equal to that of S GA + F GAS at 100 DAS.Treatment S GA + F GAPS gave 136.84 and 145.00% higher value at 90 and 100 DAS respectively than F W (Fig. 5a).Treatment S GA +F GAPS gave the maximum value of nodule fresh weight per plant at each sampling stage.Its effect was, however, equal to that of S GA + F GAS at both sampling stages.Treatment S GA + F GAPS gave 161.90 and 136.00% higher value at 90 and 100 DAS respectively than F W (Table 1).Treatment S GA + F GAPS gave the maximum value of nodule dry weight per plant at each sampling stage.Its effect was, however, equal to that of S GA + F GAS at 90 DAS and was followed by that of the same treatment at 100 DAS.Treatment S GA + F GAPS gave 233.34 and 250.00% higher value at 90 and 100 DAS respectively than F W (Table 1).
Treatment S GA + F GAPS gave the maximum value of NRA at each sampling stage.Its effect was, however, equalled by that of S GA + F PS , F GAPS , S GA + F GAS at both stages and was also by that of S GA + F S at 100 DAS.Treatment S GA + F GAPS gave 22.37 and 22.46 % higher value at 90 and 100 DAS respectively than F W (Fig. 5b).Treatments S GA + F GAPS and S GA + F GAS were at par in their effect and gave maximum value of N-ase at each stage of sampling.Treatments, F GAPS , S GA + F GAP and F GAS being at par occupied second position at each stage and treatments F GAP , S GA + F PS and F GAS also at 90 DAS.Treatment S GA + F GAPS gave 206.13 and 215.38% higher value at 90 and 100 DAS respectively than F W (Fig. 5c).The effect of treatments on N content was not found significant at both stages of sampling (Table 2).Treatment S GA + F GAPS gave the maximum value of P content at each sampling stage.Its effect was, however, equal to that of F GAPS at both sampling stages and also to that of S GA + F GAS at 90 DAS.
Treatment S GA + F GAPS gave 37.50 and 41.46% higher value at 90 and 100 DAS respectively than F W (Table 2).The effect of treatments on K content was not found significant at both stages of sampling (Table 2).Treatment S GA + F GAPS gave the maximum value of nodule Lb content at each sampling stage.Its effect was, however, equal to that of F GAPS at both sampling stages and also to that of S GA + F GAS at 90 DAS.Treatment S GA + F GAPS gave 17.50 and 11.46% higher value at 90 and 100 DAS respectively than F W (Table 2) .
Treatment S GA + F GAPS gave the maximum value for pod number per plant.Its effect was, however, equal to that of S GA + F GAS .Treatment S GA + F GAPS gave 121.72% higher value than F W (Table 3).Treatment S GA + F GAPS exhibited the maximum value for seed weight per plant.Its effect was, followed by that of S GA + F GAS .Treatment S GA + F GAPS gave 200% higher value than F W (Table 3).Treatment S GA + F GAPS gave the maximum value of seed number per pod.Its effect was, however, equal to that of S GA + F GAS , S GA + F GAP and S GA + F GA .Treatment S GA + F GAPS gave 86.14% higher value than F W (Table 3).Treatment S GA + F GAPS gave the maximum value of seed yield per plant.Its effect was, however, equal to that of F GAPS , F GA , S GA + F GA , S GA + F GAS , F GAS , S GA + F GAP , S GA + F S , S GA + F P , F GAP , S GA and F PS .Treatment S GA + F GAPS gave 20.59 % higher value than F W (Fig. 6a).Treatment S GA + F GAPS gave the maximum value of seed protein content.Its effect was, however, equal to that of S GA + F GAS and S GA + F GAP .Treatment S GA + F GAPS gave 16.11% higher value than F W (Fig. 6b).

diSCuSSion
The improvement of a crop depends to a larger extent on the genetic variability as it provides us the raw materials for selection of better genotypes.Little natural variability is found in chickpea for conspicuous morphological and physiological characters.Overwhelmingly, GA 3 serve manifold growth related functions in plants by enhancing replication, transcription and different enzymatic  which are crucial for plant growth and also provide effective targets for herbicide development also.This enhancement could be the results of increased uptake of nutrients, enhanced photosynthesis and improved translocation of photosynthates and other metabolites to the reproductive parts.This sustained increase in the N-P-K contents of the treated plants which is expected to culminate the maximization of the seed yield and seed protein content 25 .Needless to say, GA 3 might be involved in formation of seeds in pods and their optimum nourishments have resulted in less number of aborted seeds and thus maximized the survival of fertile seeds/pods.
The growth improving effect of pre-sowing seed treatment for 8 h and foliar treatment at 60 and 70 DAS with 10 -6 M GA 3 over their respective water treated control on plant height and LAI of chickpea grown with a dose of N and P could be explained on the basis of its roles and the fact that the supply of GA 3 by pre-sowing seed treatment or by foliar application would more than compensate the inadequate level of GA 3 to growing crops.Also, GA 3 foliar spray increased plant height and leaf length.An increase in growth parameters like shoot length in plants sprayed with GA 3 in accordance with the known fact that exogenous application of PGRs evoke the intrinsic genetic potential of the plant causing increase in elongation of internodes as a consequence of cell division and cell wall extensibility [26][27][28] .The improving effect of the spray of a small quantity of P and /or S alone or in combination with the soaking and/or foliar spray treatment of GA 3 over the respective control on the growth parameters of chickpea is a noteworthy observation.The promoting effect of P and S on the growth parameters can be traced to their various roles.Thus, being important essential nutrients, P and S are directly or indirectly involved in growth of chickpea like other crops through the production of metabolic compounds.These metabolites, in turn, encourage the formation and enlargement of new cells in treated plants, hence the increased height of plants, extra leaf formation and larger LAI.It may be added that these results on the improving effect of foliar application of P and S broadly corroborate the earlier findings of Naqvi 29 and Khan 30 .Improvement in shoot length and LAI would have contributed in improving the ability of treated plants for nodule and biomass production.
The augmenting effect of leaf-applied GA 3 over the water-sprayed control as also its superior effect on N-ase and NR activities and leaf NPK content is worth mentioning.The increase in NR and N-ase activities can be attributes to the hormoneinduced increase in transcription and/or translation of the gene that codes for NR and N-ase as well as in NPK content to its role in enhancing the permeability of membranes and absorption of nutrients 31 .These results are also in accordance with the data of earlier workers including Premabatidevi 32 , on NR activity; and Hassanpouraghdam 33 on N-ase and NPK content.Enhancement in leaf-nutrients, particularly N due to GA 3 application could be attributed to the compositional or chemical change in plants leading to alterations in N concentration 33 .
The enhancing effect of pre-sowing seed treatment for 8 h and foliar treatment at 60 and 70 DAS with 10 -6 M GA over their respective water treated control NR and N-ase activities and nutrient contents of chickpea grown with the recommended This may also be attributed, as for growth characters, to its roles on one hand and compensation of the 'hidden hunger' for GA by its pre-sowing seed treatment or foliar application on the other.The augmenting effect of foliar spray of P and /or S alone or in combination with the soaking and /or foliar spray treatment of GA over the respective control on NR and N-ase activities and N, P and K content of chickpea grown with a basal dose of N and P is not far to seek.P and S being component of the various metabolites involve in the production of organic compounds which, in turn, encourage the formation and proper supply of proteins to be involved in the formation of enzymes.Hence higher activities of NR and N-ase in treated plants.The higher values for N, P and K content in leaves of P and /or S sprayed plants may be due to the stimulatory effect of leafapplied P and S on absorption of nutrients from the soil 34 .Increased NR and N-ase activities might be responsible for increasing biosynthesis of protein of treated plants.
Moreover, improvement in N, P and K content would also have enhanced content of protein on one hand and Lb content on the other.Furthermore, GA 3 contributes towards enhancing the capacity of the treated plants for biomass production as reflected in shoot and root dry weight of the plants.This enhancement could be the results of increased uptake of nutrients, enhanced photosynthesis and improved translocation of photosynthates and other metabolites to the reproductive parts.This sustained increase in the above mentioned parameters of the treated plants which is expected to culminate the maximization of seed yield and seed protein content.
The increase in the number of pods per plant and seed number per pod weight resulting from the foliar application of GA 3 in comparison with the water-sprayed control as well as its superior effect applied at 10 -6 M on these parameters of chickpea receiving the recommended basal dose of N and P is worth mentioning.The increase in these yield attributes may be traced to its various role leading to observed higher values for growth characters and, physiological and biochemical parameters of treated plants [35][36][37][38] .
The augmenting effect of pre-sowing seed treatment with 10 -6 M GA for 8 h over watersoaking treatment on pods per plant and seeds per pod and of spray treatment at 60 and 70 DAS with the same concentration of GA 3 in comparison with the water-sprayed control on these parameters is understandable.The augmenting effect of foliar treatment with 10 -6 M GA for 60-70 DAS over water-spray treatment on seeds per pod is understandable.In addition, GA 3 might have increased the translocation of assimilates to the reproductive organs which resulted in the maximum number of pods per plant up to a certain levels of GA 3 application 39 .Instinctly like other growth regulators, GA 3 might be involved in formation of seeds in pods and their optimum nourishments have resulted in less number of aborted seeds and thus maximized the survival of fertile seeds/pods in chickpea.Lastly, Makarem and Mokhtar 40 reported that GA 3 could lead to an increase in fruit set of deciduous trees and played a major role in enlarging fruit size.
Similarly, El-Seginy and Khalil 41 reported also that GA 3 application increased fruit set, fruit weight, and as a result increased the yield.
The improvement in pods per plant, seeds per pod, pod and seed weight per plant of chickpea grown with the recommended basal dose of N and P, due to foliar application of a small quantity of P and /or S alone or in combination with the soaking and /or spray of GA 3 over the respective control is not far to seek.Also, P and S influence differentiation in plants 42 .The improvement in growth, physiological and biochemical parameters resulted from the spray of these nutrients together with enhancement in differentiation may lead to the improvement in pods per plant, seeds per pod 43 on foliar application of P and S. The increased yield attributing parameters of treated plants, particularly pods per plant is likely to have contributed to the improved seed yield.The observed increase in seed protein content due to foliar application and pre-sowing seed treatment of GA 3 is not surprising.An improvement in protein synthesis may result from the application of GA 3 and P and S 32,43 , hence higher values for seed protein content on P and S although on basal application 40- 41 .

systems 22 - 24 .
An increase in growth parameters like shoot and root lengths, fresh and dry weights in plants treated with GA 3 in accordance with the known fact that exogenous application of PGRs evoke the intrinsic genetic potential of the plant causing increase in elongation of internodes as a consequence of cell division and cell wall extensibility.Enhancement in leaf-nutrients, particularly N due to GA 3 application could be attributed to the compositional or chemical change in plants leading to alterations in N concentration.Acquisition and assimilation of N is a fundamental process that is essential for the growth and development of plants.N is available to plants mainly in the form of nitrate (NO 3 ).The NO 3 taken up by the plants is first reduced to NH 3 by the enzyme namely, nitrate reduction (NR) and nitrite reductase (NiR).N assimilating enzymes (NR and N-ase)

Fig. 1 :Fig. 2 :
Fig.1: (a) Showing the relative distribution of chickpea in three major kinds of environments; (b) Showing the probable sites for origin of chickpea on world map

Fig. 3 :Fig. 4 :
Fig. 3: Annual relationship between the relative temperature ratios with each month of the year

Fig. 5 :
Fig.5: Effect of spray of P and / or S in the presence or absence of soaking and / or spray treatment of GA 3 on (a) nitrate reductase activity (b) nodule number per plant (c) n-ase activity of cultivar dCP 92-3 of chickpea

Fig. 6 :
Fig. 6: (a & b): Effect of spray of P and / or S in the presence or absence of soaking and / or spray treatment of GA 3 on seed yield and protein content of cultivar dCP 92-3 of chickpea