*Corresponding author: romiarora@yahoo.com
COMBINED EFFECT OF CYTOKININ, PACLOBUTRAZOL AND ASCORBIC ACID ON
NITROGEN METABOLISM AND YIELD OF WHEAT (
TRITICUM AESTIVUM
L.) UNDER
WATER DEFICIT STRESS CONDITION
S.K. DWIVEDI, V.P. SINGH, G.P. SINGH AND AJAY ARORA*
Division of Plant Physiology Indian Agricultural Research Institute, New Delhi-110 012 Received on 15th Jan., 2012, Revised and accepted on 15th August, 2012
SUMMARY
Drought is the most significant factor restricting wheat production in the majority of agricultural fields not only in India but also in the world. A pot culture study was conducted to study the effect of exogenous application of cytokinin (TDZ), paclobutrazol (PBZ) and ascorbic acid (AsA) in combination on two contrasting wheat cultivars viz., HD 2733 and HD 2987 differing on the basis of their tolerance to drought. The results revealed that water stress condition leads to decrease in the activity of two main enzymes of nitrogen assimilation pathway i.e. nitrate reductase (NR) and glutamine synthetase (GS). Cytokinin, paclobutrazol and ascorbic acid application in combination enhanced the activity of both the enzymes across the cultivar and water regimes. Increase in level of total nitrogen was also found in treated plants under two water regimes. Protease activity was also found to be up regulated under water stress condition, which led to the degradation of protein, while treated plants showed significant reduction in the level of protease activity under stress condition. Yield related traits also showed significant reduction under water stress condition, while combined application of cytokinin, paclobutrazol and ascorbic acid enhanced the yield and its components.
Key words: Ascorbic acid, cytokinin, glutamine synthetase, nitrate reductase, nitrogen metabolism, paclobutrazol, protease
Abbreviations: TDZ- thidiazuron; PBZ- paclobutrazol; AsA- ascorbic acid; NR- nitrate reductase; GS-glutamine synthetase; PGRs- plant growth regulators
INTRODUCTION
Water stress is a global problem, which is a severe threat for sustainable agriculture (Shao et al. 2005). In
developing countries 37% of the area is semiarid in which available moisture is the primary constrain to wheat production. Development of adequate agricultural strategies to counteract this effect and minimize drought-related yield loss could be oriented towards emphasizing the urgent need to develop adaptive agricultural strategies for a changing environment. These ranges from changes
in traditional management and agronomic practices to the use of marker-assisted selection for the improvement of drought-related traits and the development of transgenic crops with enhanced tolerance of drought and improved water use efficiency that could minimize drought-related losses and ensure food production for a growing population.
Senescence can be described as a nutrient remobilization process. In wheat (Triticum aestivum),
remobilization of N stored in roots and shoots before anthesis (Hirel et al. 2005) determine the grain protein
concentration and the baking quality of flour. Among stresses, limited N availability is one of the main factors that accelerate the senescence in crop leaves (Crafts-Brandner et al. 1998). Photosynthetic activity is closely
associated with leaf nitrogen (Llorens et al. 2003). The
two key enzymes involved in assimilating intracellular ammonium into organic compounds are nitrate reductase (NR; EC 1.6.6.1) and glutamine synthetase (GS; EC 6.3.1.2), which also participate in photosynthesis and carbohydrate metabolism (Sibout and Guerrier 1998). Although high N level enhances photosynthesis and delays senescence (Sinclair et al. 2000), generally,
drought results in a decrease in leaf N content (Llorens
et al. 2003), NR and GS activities. This is important
because GS can be associated with amino acid conversion (Lam et al. 1996), and amino acid
composition might be altered due to drought, which could promote stress-resistance.
Various plant hormones/PGRs like ascorbic acid, paclobutrazol, cytokinin etc. play a crucial role as a protective agent under stress and protect the crop plants from severe injury. Ascorbic acid plays a diverse role in plants. Increasing evidence suggests that ascorbate peroxidase provides resistance to various environmental stresses in plants (Kwon et al. 2002). It is essential for
plant growth participates in stress resistance, and seems to control flowering time and the commencement of senescence (Davey et al. 2000). Cytokinins are a group
of plant growth regulators which plays a role in many aspects of plant growth and development, such as cell division, photosynthesis, senescence, chloroplast development and assimilate partitioning. The way by which physiological effects are evoked at the molecular level and, especially, how cytokinins regulate assimilate partitioning remains to be elucidated (Roitsch and Ehness 2000). Besides, only few reports have focused on the cytokinin-induced expression/activity of enzymes such as nitrate reductase (NR) and glutamine synthetase (Brenner et al. 2005). Paclobutrazol (PBZ) is one of
triazole compounds, widely used as growth retardant, induces mild stress tolerance in seedlings and adult plants. More specifically, PBZ has been reported to protect plants against drought stress (Fletcher et al.
2000). In ornamental crops, PBZ is used for reducing the size of plants, improving compactness, and increasing other functional aspects, such as the ability to resist both abiotic and biotic stresses. Several studies have demonstrated the efficiency of this compound to reduce the negative effects of water stress.
Reports (Davidson and Chevalier 1987) have shown that the growth and yield of cereals are reduced by soil moisture deficits, and the extent to which yields are affected in cereal crops is dependent on the stage of development at which the deficits occur. In wheat, growth is affected by water stress which can reduce the final number of tillers per plant by reduced tiller production and (or) increased tiller mortality (Fischer I973).The number of kernels per spike of wheat has been reported to be most severely reduced by water stress during the 15 days prior to anthesis, while water stress during grain filling reduces kernel weight, yield decreases from stress at this stage are usually less severe than from stress just prior to anthesis (Musick and Dusek 1980). Spray of vitamin C increased grain yield of wheat by influencing many physiological processes such as stimulates respiration, activates cell division and many enzymes activities as reported by Zewail (2007). Ascorbic acid foliar application increased stem and leaf dry weight and leaf fresh weight. In addition, an increase was observed in grain weight when plants were treated by 150 mg/L ascorbic acid at two growth phases. Paclobutrazol, a potential plant growth regulator also perform multiple task to mitigate the drought upto some extent and enhance the grain yield. It was shown that, without PBZ treatment, grain yield and WUE of water-stressed plants were significantly lower than normal grown plants. Irrespective of water stress treatment, grain yield and WUE of PBZ-treated plants were significantly higher than non- PBZ-treated plants (Fletcher et al. 2000).
N economy is necessary for improving N use efficiency under water limited environment and reducing excessive input of fertilizers with maintaining an acceptable yield.
MATERIALS AND METHODS
Plant material and growth conditions: Two wheat
varieties were selected HD 2987 (relatively drought tolerant) and HD 2733 (relatively drought sensitive), as suggested by breeders and procured from Division of Genetics, Indian Agricultural Research Institute, New Delhi. Sowing was done in 30 cm earthen pots with clay loam soil and farmyard manure in 3:1 ratio during winter season and supplied with 60, 60 and 60 kg ha-1of N, P, K, respectively, in the form of urea, single super phosphate and muriate of potash at the time of sowing. Remaining 60 kg N ha-1 was given after 25 days of sowing. Plants were subjected to water stress after 55 days after sowing for imposing water deficit stress during reproductive growth stage. Cytokinin, paclobutrazol and ascorbic acid treatment in combination was provided by spraying Thidiazuron (TDZ, 0.01 ÎĽL/L), paclobutrazol (PBZ, 30 ÎĽL/L) and ascorbic acid (AsA, 100 ÎĽL/L) at 40 DAS and 60 DAS. The plants were sampled and observations were taken for growth, physiological, biochemical parameters and molecular study at pre-anthesis (67 DAS); pre-anthesis (77 DAS) and post-pre-anthesis stages (87 DAS) of the wheat plants as described above. Three replications with five pots per replication were taken for each variety. Fully expanded flag leaves were used for recording biochemical observations.
Nitrate reductase activity (NR; EC 1.6.6.1): Nitrate
reductase activity was assayed following the method of klepper et al. (1969) as modified by Nair and Abrol
(1973). The NR activity was calculated as the amount of nitrite formed from the standard curve of nitrite (1OD = 152.46 nmoles nitrite). The NR activity was expressed as ÎĽmoles NO2- formed g-1 dw h-1.
Glutamine synthetase (GS; EC 6.3.1.2): The activity
was assayed following the method of Mohanty and Fletcher (1980). The absorbance was recorded at 540 nm. The GS activity was calculated from the standard curve of Îł-glutamyl hydroxymate as the amount of ferric
Îł-glutamyl hydroxymate formed (1OD = 3.72 ÎĽmoles).
The GS activity was expressed ÎĽmoleses Îł-glutamyl hydroxymate formed g-1 dw h-1
Total protease activity: The activity was assayed
following the method of Basha and Beere (1975). The protease activity expressed as ÎĽM of amino acid produced per gram fresh weight or per hour per gram dry weight per hour.
Determination of total nitrogen: Total N was
estimated by using Kjeldhal method (1883). N per cent in the tissues and the N content per plant was calculated using the formula.
Growth and yield parameters: Plant biomass was
calculated by the plants harvested at various growth stages and separated into stems and leaves then dried in an oven at 80ÂşC for four hours and then at 60ÂşC till constant dry weight was recorded. Dry weights were recorded and expressed as g plant-1. Leaf area of flag leaf, main shoot leaves and total plant leaf area was measured using a standard leaf area meter (Model LiCOR 3100) and was expressed as cm2 plant-1. Following yield components were recorded at the time of final harvest: number of tillers; number of grains per ear, grain yield per plant, 1000-grain weight (g).The harvest index was calculated as the ratio of the economic yield to biological yield and was expressed as percentage.
Harvest index = (Economic yield / Biological Yield) X 100
Statistical analyses:The data was analysed statistically
using 3 factorial CRD (Biochemical analysis and growth parameters) and 2 factorial CRD (Yield attributes) design and CD at 5% and ANOVA was calculated. The analysis was done using OPSTAT programme available online on CCS Agricultural University, HISAR web site.
RESULTS AND DISCUSSION
Nitrate reductase activity: Nitrate reductase activity
wheat plants affected by PGR's treatment in combination. Combined treatment of PGRs enhanced the NR activity by 7% and 26% in control and water deficit plant of cultivar HD 2987 at pre-anthesis, while, in HD 2733 it was 16% and 36%, respectively. Sensitive cultivar (HD 2733) was found more responsive to PGRs treatment in combination as compared to tolerant cultivar (HD 2987) under water stress condition. Drought can also have adverse effects on nitrate reductase (NR) activity (Burman et al. 2004) which is involved in
carbohydrate metabolism. Since NR is the first enzyme of the nitrate assimilation pathway, the increased NR activity observed in the TDZ-treated plants indicates that TDZ also modifies the N assimilatory capacity of plants. Our finding strongly supports that NR is also induced by cytokinins. In F.arundinacea the activity of nitrate
reductase in all treatment was higher than that of CK and showed a trend of first increasing and then decreasing with the increase of paclobutrazol concentration. Study showed that ascorbate applications
enhanced the correlative effects of NR activity and leaf protein content and this was particularly so under high temperature stress conditions. The enhancement effect of ascorbate could be through the release of NADPH2 to the enzyme. The enzyme is NADPH2-dependent (Bako 2006).
Glutamine synthetase activity: Glutamine synthetase
(GS) activity (ÎĽmoles Îł-glutamyl hydroxymate g-1dw h-1) was estimated to assess the nitrogen status of plants affected by cytokinin, paclobutrazol and ascorbic acid treatment in combination in two cultivars differing in their tolerance to water deficit stress at reproductive stages of wheat (Fig. 1). Cultivar differences were highly significant under water deficit stress as compared to the control wheat plants affected by PGR's treatment in combination. Control wheat plants of cultivar HD 2987 showed enhancement of GS activity only by 5% treated by PGR's treatment in combination as compared to 22% in water deficit plants treated with PGRs treatment at
Fig. 1. Combined effect of cytokinin {Thidiazuron; TDZ (0.01 ÎĽL/L)}, paclobutrazol (PBZ; 30 ÎĽL/L) and ascorbic acid (AsA; 100 ÎĽL/L) on nitrate reductase activity (ÎĽmoles NO2- formed g-1dw h-1) ( A & B) and glutamine synthetase activity (ÎĽmoles
γγγγγ-glutamyl hydroxymate g-1dw h-1) (C & D) at three different growth stages in two contrasting cultivars affected by water
pre-anthesis stage. While, in HD 2733 it was 13% and 42%, respectively. Glutamine synthetase activity is closely associated with protein hydrolysis (Tsai et al. 2003) and
the ability of adapting to drought. In the present study, water deficit stress reduced the activities of two enzymes (NR and GS) involved in N assimilation.
Total nitrogen content: Total nitrogen content (%) was
measured to study effect of two water regimes at three different developmental stages of wheat (Fig. 2). Significant cultivar differences were also observed under water deficit stress. Control wheat plants of cultivar HD 2987 showed enhancement of total nitrogen by 5% treated by PGR's as compared to 31% in water deficit plants treated with PGRs at pre-anthesis stages. While, in HD 2733 it was 26% and 38%, respectively.The total N-translocation amount decreases under water stress, because early leaf senescence enhanced by drought can lead to a decrease in leaf N levels (Seligman and Sinclair 1995). The present results demonstrate that water deficit stress can decrease grain yield and leaf N concentration
through adversely affecting N metabolism with early flag leaf senescence in both the tolerant and sensitive cultivar of wheat.
Total protease activity: Total protease activity (ÎĽmoles
g-1dw h-1) was also measured at three different developmental stages of wheat (Fig. 2). Cultivar differences were observed under water deficit stress. Control wheat plants of cultivar HD 2987 showed reduction in total protease activity of around 3% treated by PGR's as compared to 18% in water deficit plants treated with PGR's at pre-anthesis stages. While, in HD 2733 it was 5% and 14%, respectively. In the present study, water deficit stress increased the activity of proteases. Proteases were negatively and significantly correlated with photosynthetic rates. Therefore, it is suggested that the N metabolism regulation could play an important role in the photosynthetic adaptation of wheat plants to water stress. Reduced levels of protease activity in leaves of the drought resistant cultivar (HD 2987) suggested that suppressed expression of specific
Fig. 2. Combined effect of cytokinin {Thidiazuron; TDZ (0.01 ÎĽL/L)}, paclobutrazol (PBZ; 30 ÎĽL/L) and ascorbic acid (AsA; 100 ÎĽL/L) on total leaf nitrogen (%) (A & B) and total protease activity (ÎĽmoles g-1dw h-1) (C & D) at three different growth
protease activity, accompanied only by slight changes in proteolytic activity, could be regarded as a prerequisite or at least as a marker for drought tolerance. This proteolytic activity was further declined by the treatment of cytokinins (TDZ), paclobutrazol and ascorbic acid in combination, across the cultivars and water regimes.
Growth and Yield parameters: Total biomass (g
plant-1) was measured at three different developmental stages of wheat i.e., pre-anthesis, anthesis and post-anthesis (Fig. 3). Cultivar differences on total biomass were observed more under water deficit stress as compared to the control wheat plants affected by PGR's. Control wheat plants of cultivar HD 2987 showed enhancement of total biomass by 6% treated by cytokinin, paclobutrazol and ascorbic acid treatment in combination as compared to 18% in water deficit plants treated with same PGRs at pre-anthesis stage. While, in HD 2733 it was 9% and 18%, respectively. The sensitive cultivar, HD 2733 was more responsive to PGR's as compared to tolerant cultivar, HD 2987 under
water stress condition. Leaf area (cm2 plant-1) was also measured at three different developmental stages of wheat (Fig. 3). Cultivar differences on leaf area were observed more under water deficit stress as compared to the control wheat plants affected by cytokinin, paclobutrazol and ascorbic acid treatment in combination. Control wheat plants of cultivar HD 2987 showed enhancement of leaf area of around 20% treated by PGR's as compared to 74.8% in water deficit plants with PGR's at pre-anthesis stage. While, in HD 2733 it was 9 % and 122 %, respectively.
Grain yield per plant (g) were also recorded at the time of harvest in wheat plants (Table 1). There was 4% increased in grain yield per plant in treated control plants while it was 7% increased in water deficit condition with PGR's in tolerant HD 2987 cultivar of wheat. Similarly, in HD 2733 it was 14% and 4%, respectively. Grain numbers per panicle were also recorded at the time of harvest in wheat plants (Table 1). There was 1% increased in grain number per panicle
Fig. 3. Combined effect of cytokinin {Thidiazuron; TDZ (0.01 ÎĽL/L)}, paclobutrazol (PBZ; 30 ÎĽL/L) and ascorbic acid (AsA; 100 ÎĽL/L) on total leaf area (cm2 plant-1) (A & B) and total biomass (gm plant-1) (C & D) at three different growth stages
in treated control plants while there was 24% increased in water deficit condition with PGR's treated plants in tolerant HD 2987 cultivar of wheat. While, in HD 2733 it was 21% and 29%, respectively.Thousand grain weight (g) was also recorded at the time of harvest in wheat plants (Table 1). There was 8% increased in 1000 grain weight in treated control plants while it was 3% increased in water deficit condition with PGR's in combination in tolerant HD 2987 cultivar of wheat. Similarly, in HD 2733 it was 7% and 6%, respectively. Harvest index (HI) was also recorded to assess the partitioning efficiency of wheat plants at the time of harvest (Table 1). There was 2% increase in HI in treated control plants while it was 5% increase in water deficit condition with PGR's in tolerant HD 2987 cultivar of wheat.
The present study results showed significant improvement in grain yield and harvest index of both the cultivars treated with cytokinins, paclobutrazol and ascorbic acid in combination in control as well as stressed plants. This reflected towards the enhancement of total biomass, spike number and leaf area of the treated plants. Similar kind of observation were observed (Shah and Paulsen 2011) in cytokinin treated black cumin plants where they observed appreciable improvement in the yield parameters. This is because of higher values for nitrate reductase, net photosynthetic rate, leaf protein content and dry mass, as compared to the control plants. Different concentration of paclobutrazol could increase the tiller number and root-shoot ratio of F.arundinacea
and treatment with higher concentration of paclobutrazol had extremely significant difference with cytokinin (CK). Chlorophyll content and root activity in all treatment were higher than that of CK and showed an increasing trend with the increase of paclobutrazol concentration.
In conclusion, the reduction in the activities of two important enzymes (NR and GS) involved in N assimilation and enhancement in the activities of proteases are involved in the decline of plant growth and grain yield caused by water deficit stress. However, changes in leaf N concentration and key enzyme activities for the two varieties were complex. This could have resulted from higher responses of the cytokinin , paclobutrzol and ascorbic acid treatment in combination
and different genotypes to drought. Table 1.
Combined effect of cytokinin {Thidaizuron; TDZ (0.01
ÎĽ
L/L)}, paclobutrazol (PBZ; 30
ÎĽ
L/L) and ascorbic acid (AsA; 100
ÎĽ
L/
L) on yield and yield attributes at harvest in two contrasting cultivars affected by water deficit stress in wheat.
--- ---Treatment Control (C) C + (TDZ+PBZ+AsA) Water stress (WS)
WS + (TDZ+PBZ+AsA)
HD2733 HD2987 Mean HD2733 HD2987 Mean HD2733 HD2987 Mean HD2733 HD2987 Mean ---
---Grain No. ear
-1 32.00 34.67 33.33 38.67 35.00 36.83 28.00 30.00 29.00 36.00 37.33 36.66
Grain yield Plant
-1 (
g) 10.67 12.00 11.33 9.33 12.50 10.91 8.33 09.17 8.75 8.67 9.83 9.25
1000 grain wt. (g)
46.29 53.38 49.83 49.64 57.97 53.80 40.27 46.97 43.62 42.97 48.25 45.61
Harvest Index (%)
---ACKNOWLEDGEMENT
Authors thankfully acknowledge the technical help provided by Dr. D.V.Singh. Financial assistance provided by CSIR is gratefully acknowledged.
REFERENCES
Bako, S.P. (2006). Effects of plant age, ascorbate and kinetin applications on nitrate reductase activity and leaf
protein content of maize (Zea mays L.) plants grown
under heat stress. Asian J. Plant Sci. 5: 363-367.
Basha, S.M.M. and Beever, L. (1975). The development of proteolytic activity and protein degradation during the
germination of Pisum sativum L. Planta. 124: 77-87.
Brenner, W.G., Romanov, G.A., Kollmer, I., Burkle, L. and Schmulling, T. (2005). Immediate early and delayed
cytokinin response genes of Arabidopsis thaliana
identified by genome wide expression profiling reveal novel cytokinin sensitive processes and suggest
cytokinin action through transcriptional cascades. Plant
Mol. Biol. 44: 314–33.
Burman, U., Garg, B.K. and Kathju, S. (2004). Interactive effects of thiourea and phosphorus on cluster bean under water
stress. Biol. Plant. 48: 61-65.
Crafts-Brandner, S.J., Holzer, R. and Feller, U. (1998). Influence of nitrogen deficiency on senescence and the amounts
of RNA and proteins in wheat leaves. Physiol. Plant.
102: 192-200.
Davey, M.W., Mantagu, M.V., Dirk, I., Maite, S., Angelos, K., Smirnoff, N., Binenzie, I.J.J., Strain, J.J., Favell, D. and Fletcher, J. (2000). Plant ascorbic: acid chemistry, function, metabolism, bioavailbility and effects of
processing. J. Sci. Food and Agri. 80: 825-850.
Davidson, D.J. and Chevalier, P.M. (1987). Influence of poly ethylene glycol-induced water deficits on tiller
production in spring wheat. Crop Sci. 27: 1185-1187.
Fischer, R.A. (1973). Developmental orphology of the inflorescene in hexaploid wheat cultivars with and
without Norin 10 in their ancestry. Can. J. Plant Sci.
53: 715.
Fletcher, R.A., Gilley, A., Sankhla, N. and Davis, T.V. (2000). Triazoles as plant growth regulators and stress
protectants. Horticultural Reviews. 24: 55-138.
Hirel, B., Andrieu, B., Valadier, M.H., Renard, S., Quillere, I.,
Chelle, M., Pommel, B., Fournier, C., Drouet, J.L. (2005).
Physiology of maize II: Identification of physiological
markers representative of the nitrogen status of maize
(Zea mays) leaves during grain filling. Physiol. Plant.
124: 178-188.
Kjeldhal, J.Z. (1883). A new method for determination of
nitrogen in organic bodies. Analytical Chemistry. 22:
366.
Klepper, L. And Hageman, R.H. (1969). The occurrence of
nitrate reductase in apple leaves. Plant Physiol. 44:
110-114.
Kwon, S.Y., Jeong, Y.J., Lee, H.S., Kim, J.S., Cho, K.Y. and Allen R.D. (2002). Enhanced tolerances of transgenic tobacco plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl
viologen mediated oxidative stress. Plant Cell Environ.
25: 873-882.
Lam, H.M., Coschigano, K.T., Oliveira, I.C., Melo-Oliveira, R. and Coruzzi, G.M. (1996). The molecular-genetics of
nitrogen assimilation into amino acids in higher plants.
Ann. Rev. Plant Physiol. Plant Mol. Biol. 47: 569-593.
Llorens, L., Penuelas, J. and Estiarte, M. (2003). Ecophysiological responses of two Mediterranean shrubs, Erica multiflora and Globularia alypum, to
experimentally drier and warmer conditions. Physiol.
Plant.119: 231-243.
Mohanty, B. and Fletcher, J.S. (1980). Ammonium influence on nitrogen assimilating enzymes and protein accumulation in suspension cultures of paul’s scarlet
rose. Physiologia Plantarum. 48: 453-459
Musick, J.K. and Dusek, D.A. (1980). Planting date and water deiicit eifects on development and yield of irrigated
winter wheat. Agron J. 72: 45-52.
Nair, T.V.R. and Abrol, Y.P. (1973). Nitrate reductase activity
in developing wheat ears. Experientia. 29: 1480-1481
Roitsch, T. and Ehness, R. (2000). Regulation of source/sink
relations by cytokinins. Plant Growth Reg. 32: 359-367.
Seligman, N.G. and Sinclair, T.R. (1995). Global environment change and simulated forage quality of wheat. II. Water
and nitrogen stress. Field Crops Res. 40: 29-37.
and high temperature on photosynthesis and
grain-filling of wheat. Plant Soil. 257: 219-226.
Sibout, R. and Guerrier, G. (1998). Solute incompatibility with
glutamine synthetase in water-stressed Populus nigra.
Environ. Exp. Bot. 40: 173-178.
Sinclair, T.R., Pinter, P.J., Kimball, B.A., Adamsen, F.J., Morte,
R.L., Thompson, T., Leavitt, S., Mattias, A.(2000). Leaf
nitrogen concentration of wheat subjected to elevated
[CO2] and either water or N deficits. Agr. Ecosyst.
Environ. 79: 53-60.
Tsai, Y.C., Hsu, Y.T. and Kao, C.H. (2003). Proline accumulation induced by phosphinothricin in rice
leaves. Biol. Plant. 46: 317-322.
Zewail, Y.M.R. (2007). Improvement of wheat productivity by
using some biofertilizers and antioxdants. M.Sc. Thesis,
