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International Journal of Bio-Technology and Research (IJBTR) ISSN 2249-6858 Vol.2, Issue 3 Dec 2012 13-20 TJPRC Pvt.

. Ltd.,




Crop Physiology Ph.D student at the Islamic Azad University, Science and Research Unit, Khuzestan, Iran

Faculty of Agriculture and Natural Resources Research Center of Khuzestan, Iran Faculty of Islamic Azad University, Science and Research Unit, Khuzestan, Iran


Faculty of Agricultural Biotechnology Research Center, Karaj, Iran

The aim of this study is to understand the evolution of Cytokinin affect on biological nitrogen fixation in chickpea. Azad, Hashem and Local mass cultivars and four levels of cytokinin including: control (non- consumption ),5, 10 and 15 ppm were studied. . The results showed that there is a significant difference between cultivars regarding shoot dry weight, nodule dry weight, grain yield, nitrogen percent and fixed nitrogen amount by nodule, shoot, grain and total nitrogen-fixed by plants. Local mass and Azad had the highest (141/1 kg/ha) and the lowest (121/9) amount of nitrogen fixation respectively. 10 ppm treatment (143 /1kg/ha) and control (121/9 kg/ha) had the highest and lowest amount of nitrogen fixation respectively. Of the total nitrogen fixed by the plants, 51/9, 40/4 and 5/7 percent, were accumulated in shoot, grain and nodes respectively. Generally, the highest amount of nitrogen fixed was obtained by local mass in 10 ppm.

KEYWORDS: Chickpea, Biological Nitrogen Fixation, Cytokinin, Cultivar, Nodule, Shoot INTRODUCTION
Nitrogen is the most important nutritional element in chemical compound of the plant (Parsa and Bagheri, 2008), metabolism and protein synthesis (Khatak et al., 2006). Although, the amount of nitrogen in plant, is estimated about 1 to 2 and maximum 4 to 6 percent, however, nitrogen is the limiting factor for crop production and there is a lack of nitrogen in all the world agricultural lands (Parsa and Bagheri, 2008). An important feature of legumes is their ability to fix atmospheric nitrogen through symbiosis with Rhizobium bacteria in nodules. The legumes can do biological nitrogen fixation of nitrogen effectively, and therefore they grow with less applied nitrogen (Hardarson and Danso, 1993). The importance of biological nitrogen fixation is due to the lost nitrogen by crop residual, leaching, volatilization, runoff, erosion and denitrification is recovered (Evers and Parsons, 2011). Chickpea doesnt need to nitrogenous fertilizers in case of providing environmental conditions with atmospheric nitrogen fixation; and it is possible to provide some parts of the required nitrogen for next crop in optimal rotations (Fatima et al, 2008). Hardarson and Danso (1993) stated that if nitrogen is the main limiting factor for growth, there is a highly positive correlation between dry matter yield of legumes and fixed nitrogen amount. Fatima et al (2008) reported that there is a high correlation between nitrogen accumulation and biomass and seed growth in legumes. Fixed nitrogen in chickpea is estimated from 0 to 176 kg /ha in a agricultural season that this amount is 0 to 82 percent of total plant nitrogen (Parsa and Bagheri, 2008). Fatima et al (2008) reported that 81% of nitrogen fixation in chickpea is 138 kg/ha. In another study, Islam et al (2011) estimated the fixed nitrogen by fertilizer treatments of phosphate and sulfur from 52 to 55 percent and amount of 30 kg/ha. Evers et al (2011) in a study, estimated the maximum nitrogen fixation at 296 kg/ha on clover cultivars. in this study, the portion of root nodules was 12 kg/ha.


Kianoosh Hamidian , Ahmad Naderi , Sharam Lak, Mojtaba Alavi Fazel & Islam Majidi

In general, cytokinins promote cell growth and division (Arteca; 2000 and Fransworth; 2004) and their decreasing in root reduce nitrogen fixation by limiting nodule protein synthesis , loss of leghaemoglobin, limiting nodule respiration, changes in nodule metabolism or affect on nitrogenase enzyme activity(Goicoechea et al.,1996). Thompson et al (1987) reported that cytokinins increase cell wall flexibility and protect cell membranes by inhibiting from forming free radicals and fast breakdown of them. Cytokinins also delay leaf senescence by reducing free radicals and increasing the plant tissues capacity as active sinks for phloem transporting of sugars and nutrients (Jordi et al. 2000). Cytokinins play an important role in forming of plant mutualisms with bacteria and mycorhizae through symbiont. These hormones increase the responsible nodulation genes activity by stimulating symbionts and facilitate the nutrient transport between the plant and its partner (Ng et al. 1982). The increasingly use of nitrogenous fertilizers is one of the most important aspects in design of crop rotation due to their prices and environmental issues in which, legume plants are considered with the aim of improving soil conditions and biological nitrogen fixation. On the other hand, increasing of cytokinin usefulness in plant in symbiosis between legumes and fixing bacteria of atmospheric nitrogen as biological nitrogen was confirmed. Nitrogen fixation and bacteria activity are influenced by environmental conditions. This study was performed with the aim of evaluating cultivars response to this hormone application and their interaction in environmental conditions, due to the increasingly cultivation of chickpea in Lorestan and lack of research about genetic diversity of chickpea cultivars and cytokinin use in this conditions.


This study was performed as a factorial experiment in randomized complete block design in four replications at Grit Agricultural Services Center (48 70' long. E; 33 34' lat. N, and 1794 m above sea level) located in 35 kilometers Eastern-North of Khorramabad. Treatment combination was studied with three cultivars: Azad, Hashem and Grit local mass and four levels of cytokinines (6-Benzyl aminopurin) including: control (lack of consumption), 5, 10 and 15 ppm. Each plot consisted of five rows with 6 m length that space between rows and plants space on rows were considered 25 and 15 cm respectively. Planting was done according to the planting date and atmospheric conditions in April 2011. Before planting, soil sample prepared from the 0-30 cm depth, and nitrogen amount in soil were measured(0.1%). In this study, neither macro and nor micro fertilizers were applied. Hormone was sprayed in the vegetative growth stage (45 leaf stage). In 50% flowering stage, two lateral rows and a half meters from the beginning of each plot were removed as the border effects and from the three middle rows ,three plants with 30 cm3 soil around it was harvested, after washing the soil surrounding the roots, number of nodules on the roots and surrounding soil were collected; then nodules were dried for 72 hours in the air and were measured with a digital scale with this stage, a sample consisting of six plants was prepared from each plot. in harvest stage, 20 seeds selected randomly from each plot and in the laboratory, using Kjeldahl method, N percentages in nodules, shoots and seeds were measured and using the following formulas fixed nitrogen in each part of plant , total nitrogen fixed in plant and biological nitrogen fixation percent were calculated: Amount of nitrogen fixed (kg/ ha)= nitrogen Percent shoot, nodule or grain dry weight (kg/ ha) Total nitrogen fixed(kg/ha) = shoot nitrogen + node nitrogen + seed nitrogen

Percentage of nitrogen fixation =

Effect of Cytokinin on Biological Nitrogen Fixation in Chickpea


Where A: total nitrogen fixed by treated plant and B: total nitrogen fixed by control plant. Data were analyzed using MSTAT-C software and graphs were drawn using EXCEL2010. Means were compared using Duncans Multiple Range Test.


The results showed that the difference of cultivars, the effect of hormone levels and hormone cultivar interaction regarding shoot dry weight, nodule dry weight, grain yield, nitrogen percent and fixed nitrogen by nodules, shoot, grain and total nitrogen fixed by the plant were significant (Tables 1 and 2). Nodule, Grain and Shoot Dry Weight Cytokinin use increased significantly shoot, grain and node dry matter. using hormone, both shoot dry matter and grain showed a similar reactions, their difference with control was significant but there wasnt a significant difference between various concentration of hormone (Table 1). Increased shoot dry weight could be attributed to cytokinin regulation effect on photosynthetic capacity , delay in senescence (Fatima et al, 2005) and plant height increase (Robbie, 1996).Also increased grain weight could be attributed to assimilates translocation increase from vegetative to reproductive part due to cytokinin stimulating effect (Fatima et al, 2005). Kim et al (1993) suggested that a significant increase in soybean seed yield due to cytokinin use is related to increase in Amino acid and minerals. On the other hand, Peterson et al (1990) stated that increase in soybean seed yield is related to the significantl reduction of pod abscission and increase in total number of pods and seeds. The effect of cytokinin on nodule dry weight was very significant. The maximum dry weight of nodule was obtained in 10 ppm treatment. As hormone concentration increased, an intense reduction in dry weight of nodules was observed (Table 1). Dayal et al (1991) reported that kinetin caused to increase nitrogenase enzyme activity in chickpea root nodules as a growth-inducing hormone and this increased the haemoglobin amount and finally lead to increase in nodule weight. Torrey (1962) showed that lateral root development of chickpea in cutting pieces were stimulated by low concentration of kinetin and in higher concentrations, elongation and lateral branches stopped. Cultivar differences were significant in shoot, nodule and grain dry weight (Table 1). Local mass had the maximum shoot and nodule dry matter and Hashem produced the highest seed dry weight while Azad had the lowest amount in all three cases.different studies showed that the genotype such as environment and growth period is an important factor that can effect on chickpea growth (Khattak et al.,2000). James et al (2007) found that biomass, shoot and pod dry weight were influenced significantly by the cultivar. Mansour et al (2009), in another study reported that there was a meaningful difference between cultivars regarding grain yield and biomass. ICCV-2 with 1840 kg/ha grain yield and 2159 kg/ha biomass had a sensible superiority than BG-267. Biabani and carpenter (2011) stated in a study of 30 chickpea that commercial cultivars had maximum root and shoot biomass and lowest number of nodes and node weight. Interaction effects study showed that local mass in 15 ppm concentration and Hashem in 0 ppm concentration produced the highest and lowest shoot dry matter per plant with 5.488 g and 3.362 g respectively. Increasing hormone concentration, grain dry weight showed a positive reaction in cultivars. Azad in 15 ppm with 3.888 g had the highest grain yield per plant. But had no significant difference with Hashem treatments in 10 and 15 ppm concentrations and local mass in 15 ppm concentration (Table 2). Fatima et al (2005) showed that kinetin was very effective in increasing of root biomass, shoot, grain yield and nitrogen fixation.


Kianoosh Hamidian , Ahmad Naderi , Sharam Lak, Mojtaba Alavi Fazel & Islam Majidi

Nitrogen Percent of Shoot, Grain and Nodules Effect of cytokinin on nitrogen Percent of shoot, grain and nodules was significant (Table 1). Shoot in concentration of 10 ppm with 3.77 percent had the highest percentage of nitrogen fixation while nodules at the concentration of 5 ppm fixed 6.63% of nitrogen. In grain, control treatment with 4.17% had the highest percentage of nitrogen fixation and were placed with 15 ppm in one group. The highest percentage of nitrogen fixation in chickpea cultivars have been accumulated in the nodes (Figure 1). The nodules are the site of nitrogen fixation in plants and from there nitrogen is exported to other plant organs.
7 6 Fixation percent 5 4 3 2 1 0 azad hashem cultivar Figure1: percentage of nitrogen fixation in plant parts of chickpea cultivars local mass nodule grain shoot

Different between cultivars were significant (Table 1). Local mass and Azad cultivars had the highest nitrogen fixation percent and their differences was significant with Hashem cultivar. Local mass and Azad are cultivars with adaptation to cold and moderate conditions while Hashem cultivar shows the highest efficiency in semi-hot areas. Interaction effects showed that all cultivars had the highest nitrogen fixation percent of shoot in 10 ppm concentration , while, the best efficiency of nitrogen fixation in nodes observed in concentration of 5 ppm. The trend of nitrogen fixation in grain was different. Local mass in control treatment and Azad in concentration of 10 ppm had the highest percentage of fixation whereas Hashem in different concentrations of hormone had the lowest percentage of fixation. Total Nitrogen Fixed Nitrogen accumulated in the plant was outcome of nitrogen fixed in shoot, grain and nodes. shoot (53.9%) and grain (40.42%) had a major portion in nitrogen fixation by plant while the share of nodes in total nitrogen fixation was 5.71% (Figure 2). more dry weight of shoot and grain are the main reason for their role in nitrogen fixation. (Table 1).
60 50 40 30 20 10 0
azad hashem local mass 0 5 10 15

shoot nodule grain

nitrogen accumulation

tre atme nt Figure 2:Pe rce ntage of nitroge n accumulation in applie d tre atme nt

Study of correlation coefficients showed that total nitrogen fixation with shoot dry weight (R = 0.94) and grain dry weight (R = 0.5) had a strong positive correlation while this correlation was weakly with nodule dry weight (R = 0.08).

Effect of Cytokinin on Biological Nitrogen Fixation in Chickpea


Fatima et al (2008) were expressed in the grain legumes, nitrogen accumulation is high correlated with the amount of biomass and grain growth. Biabani et al (2011) was estimated positive correlation between nitrogen fixed with shoot dry weight and total plant biomass but did not significantly correlated with nodule number and nodule dry weight. Fatima et al (2011) were expressed that increase amount of nitrogen fixed is due to increasing in plant growth and increase absorption of nitrogen, particularly absorbed nitrogen from atmosphere. They concluded that grain yield (R = 0.9) had a very strong positive correlation with nitrogen fixed. Local mass had highest rate of nitrogen fixation with 141.1 kg/ha and was placed with Azad in a same groups while Hashem with 126/7 kg/ ha had lowest of nitrogen fixation. Among varieties, local mass had highest amount of nitrogen fixation in shoot, nodule and grain, while Hashem had the lowest (Table 1). Nitrogen fixation by legumes, influenced by genotype, Rhizobium efficiency, soil and climate conditions and methodology used in evaluation of fixation (Montanez,2000). Thavarajah et al (2004) reported that in comparison of different genotypes of chickpea cultivars, CDC-NIKA and Myles cultivars had higher nitrogen fixation and this difference was significant. Application of cytokinin hormone had a significant effect on total nitrogen fixation by plant. the trend of nitrogen fixation in different concentrations of hormone are shown in Figure 3. with increasing hormone concentration, the total amount of nitrogen fixation by plant increased and decreased in concentrations of 15 ppm. in concentration of 10 ppm, nitrogen fixation rate was 17.5% over control. It seems as Hardarson et al (1993) expressed cytokinin has been increased fixation through the induction of photosynthesis and growth, delay in senescence and accelerate the transfer of nutrients and organic matter .
amount of nitrogen fixed(kg/ha) 160 140 120 100 80 60 40 20 0 0 5 10 15 cytokinin concentration Figur3: Total nitrogen fixed in plant parts at cytokinin treatments shoot nodule grain

Survey of interaction effects showed that although the local mass in the 15 ppm treatment had the greatest amount of nitrogen fixation but its difference with other cultivars and concentration of 10 ppm was not significant and were placed in a statistical group (Table 2). Seems, the local mass due to long-term adaptation with environmental conditions and proper bacterial strains, have shown positive reactions to the use of hormones.

In this study, hormone increases the nitrogen fixation. It seems, cytokinin increased plant dry matter and thus increase the amount of nitrogen fixation through the stimulation of growth and delay in senescence of leaves. The local mass mostly used of these conditions due to long-term adaptation with environmental conditions and proper bacterial strain.


Kianoosh Hamidian , Ahmad Naderi , Sharam Lak, Mojtaba Alavi Fazel & Islam Majidi

1. Arteca, R. N. 2000. Plant Growth Sustances: Principles and aplications. Translated by: Fathi,G., B. Esmaaielpour. Mashhad,Iran. Jahad-e-daneshgahi Press.288. 2. Biabani, A,. L. Carpenter-Boggs. 2011. Nitrogen fixation Potential global chickpea mini-core collection. Biol Fertil Soils. 47:679-685. 3. Dayal, J. and S. Bharti. 1991. Effects of Kinetin, cycocel and colchicine on nitrogenous activity and ATP production in Chickpea (Cicer arietinum L.). In: New trends in Plant Physiology.Proceeding, national symposium on growth and differentiation in plant (Eds.): K.K. Dhir, I.S.2012 ZARRIN FATIMA ET AL., Dua & K.S. Chark). New Del India,Today and Tomorrows Printers and Publishers 235-238 ISBN 81: pp. 7019-375-3(India); 1-55528-217-2 (USA). 4. Evers, G. W and M. J. Parsons. 2011. Estimated N2-Fixation of cool- season annual clovers by the difference method. Crop science. 51:2276-2283. 5. Fatima,Z., A.Bano, R. Sial and M. Aslam. 2005. Response of chickpea to plant growth regulators on nitrogen fixation and yeild. Pak. J. Bot., 40(5): 2005-2013. 6. Fatima,Z., M. Aslam and A.Bano. 2008. Chickpea nitrogen fixation increases production of subsequent wheat in rain red system. Pak. J. Bot., 40(1): 369-376. 7. 8. Fransworth, E. 2004. Hormones and shifting ecology throughout plant development.Ecology, 85(1): 5-15. Goicoechea, N., M. C. Antolin, M. Strand and M. Sanchez-Diaz. 1996. Root cytokinins, acid phosphatase and nodule activity in dtought- stressed mycorrhizal or nitrogen-fixing alfalfa plants. Journal of Botany., Vol. 47, NO. 298:683-686. 9. Hardarson, G and S. K. A. Danso. 1993 . Methods for measuring biological nitrogen fixation in grain legumes. Plant and soil. 152: 19-23. 10. Islam, M., M. Saleem, A. Safdar, R. Khlid, F. Hassan, A. Mahmood and A. Subhani. 2011. Growth, Nitrogen Fixation and Nutrient Uptake by Chickpea(Cicer arietinum) in Response to Phosphorus and Sulfur Application under Rainfed Conditions in Pakistan. J. Agric. Biol., Vol.13, No.5:725-730. Experimental

11. James, E., T. C. Mackown and S. C. Rao. 2007. Dry weight and nitrogen content of chickpea and winter wheat grown in pots for three rotations. Jornal of Plant Nutrition 30: 1541-1553. 12. Jordi, W., A. Schapendonk, E. Davelaar, G. M. Stoopen, C.S. Pot, R. De visser, J. A. Van Rhijn, S. Gan, and R. M. Amasing. 2000. Increased cytokinin levels in Transgenic PSAG12-IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis, and N partitioning. Plant, Cell and Environment 23:279289. 13. Khattak, S. G., D. F. Khan, S. H. Shah, M. S. Madani and T. Khan. 2006. Role of rhizobial inoculation in the production of chickpea crop. Soil and Environ. 25(2): 143-145. 14. Kim, D.H., Y.K. Kim, C.H.K. Heo, Y.S. Lee and D.S. Chung. 1993. Effects of plant growth regulators on the growth yield and chemical components of soybean seed. RDA Journal of Agricultural Science, upLand and Industrial Crops 35(1): 89-98. 15. Majnoun Hosseini, N. 2008 . Grain legume Production. 4th ed. Tehran .Iran. Jahad-e- daneshgahi Press. 281.

Effect of Cytokinin on Biological Nitrogen Fixation in Chickpea


16. Mansur, C. P., Y. B. Palled, S. L. Halikatti, P. M. Salimath and M. B. Chetti. 2009. Uptake of nitrogen phosphorus and protein content in Kabuli chickpea genotypes as influenced by plant densities and phosphorus levels. Karnataka j. Agric. Sci. 22(1): 5-7. 17. Montanez,A. 2000. Overview and case studies on biological nitrogen fixation: perspectives and limitations. Science in Agriculture (2000). Pages: 1-11. 18. Ng, P. P., A. L. Cole, P. A. Jameson, and J. A. Mcwha. 1982. Cytokinin production by ectomycorrhizal fungi. New Phytologist. 91:5762. 19. Parsa, M., A, Bagheri. 2008. Pulses. Mashhad. Iran. Jahad-e-daneshgahi Press. 522. 20. Peterson, C.M., J.C. Williams and A.X. Kuang. 1990. Increased pod set of determinate cultivars of soybean, Glycine max L., with 6- benzylaminopurine. Botanical Gazette.151(3): 322-330. 21. Rabie, K.A.E. 1996. Studies on the interaction between gibberellins and Benzyladenine inregulating growth yield and phytohoromone content in wheat plants. Field CropAbstract. 49: 12 22. Thavarajah, D and R. A. Ball. 2004. Metabolic products of nitrogen. Saskatchewan, Plant sciences 51 Campus Drive, Poster Board Number:1040 Table 1: Mean Comparison of Measured Traits in Cultivar and Hormone Treatments Dry Weight(g/plant) Treatment Shoot Cultivar(A) azad hashem Local mass cytokinin 0 ppm 5 ppm 10 ppm 15 ppm 4.383 b 5.006a 5.131a 4.257b 4.961a 50118a 5.024a 0.252b 0.261b 0.303a 0.252b 0.264b 0.322a 0.25 b 3.245c 3.79 a 3.434b 3.346b 3.577a 3.516a 3.521a 3.733a 3.507b 3.658a 3.509c 3.598bc 3.773a 3.651 b 6.463 a 6.362b 6.507a 6.35b 6.636a 6.353b 6.437b 4.191a 3.931b 4.229a 4.169a 4.1b 4.129ab 4.07b 65.77b 70.22b 75.2a 59.78c 71.22b 77.24a 73.34ab 6.5b 6.621b 7.912a 6.423b 7.009b 8.182a 6.43b 54.41b 59.6a 58.01a 55.7b 58.53a 57.92ab 57.21ab 126.7b 136.4a 141.1a 121.9c 136.8b 143.3a 137b Nodule Grain Shoot Nodule Grain Shoot Nodule Grain Nitrogen Percent Fixed Nitrogen (kg/ha) Total Fixed Nitrogen (kg/ha)

Means in each column followed by similar letter(s) are not significantly different using Duncans Multiple Range Test.


Kianoosh Hamidian , Ahmad Naderi , Sharam Lak, Mojtaba Alavi Fazel & Islam Majidi

Table 2: Mean Comparison of Measured Traits in Intraction Effects of Cultivar Hormone

Dry Weight(G/Plant) Shoot
Cytokinin cultivar a1 b 1 a1b2 a1b3 a1b4 a2b1 a2b2 a2b3 a2 b 4 a3 b 1 a3 b 2 a3 b 3 a3 b 4 3.362f 4.34e 5.235abc 4.592cde 4.825bcde 5.267ab 4.94abcde 4.992abcd 4.582de 5.275ab 5.18abcd 5.488a 0.185e 0.223de 0.365a 0.236ce 0.264bcd 0.261bcd 0.291bc 0.227cde 0.306ab 0.309ab 0.31ab 0.287bcd 3.182fg 3.443cde 3.22efg 3.135g 3.795 ab 3.883a 3.717ab 3.765ab 3.06g 3.405def 3.61bcd 3.662abc 3.533bc 3.765a 3.827a 3.808a 3.483c 3.35c 3.725 a 3.47c 3.513bc 3.678ab 3.765a 3.675ab 6.488abc 6.655a 6.382bcd 6.328cde 6.17e 6.68a 6.193de 6.405bc 6.392bcd 6.573ab 6.485abc 6.578ab 4.075e 4.19cd 4.325ab 4.175cde 4.075e 3.888f 3.832f 3.93f 4.358a 4.222bc 4.23bc 4.105de 47.59e 65.4d 80.07ab 70.01bcd 67.32d 70.62abcd 73.6abcd 69.32cd 64.43d 77.63abc 78.04abc 80.69a 6.807e 5.925de 9.302a 5.965de 6.512cd 6.972bcd 7.215bc 5.783de 7.947 b 8.13b 8.028b 7.543bc 51.87e 57.7abc 55.7bcde 52.35de 61.88a 60.37ab 57abcd 59.17ab 53.34cde 57.5abc 61.07a 60.11ab 104.3f 129de 145.1abc 128.3de 135.7bcde 138abcd 137.8abcd 134.3cde 125.7e 143.3abc 147.1ab 148.3a


Nitrogen Percent Shoot Nodule Grain

Fixed Nitrogen (Kg/Ha) Shoot Nodule Grain



Total Fixed Nitrogen (kg/ha)

Means in each column followed by similar letter(s) are not significantly different using Duncans Multiple Range Test.