Indian J. Agric. Res., 43 (3) : 194-199, 2009 AGRICULTURAL RESEARCH COMMUNICATION CENTRE www.arccjournals.com / indianjournals.com EFFECT OF EXOGENOUS APPLICATION OF BRASSINOLIDE AND SALICYLIC ACID ON CERTAIN PHYSIOLOGICAL AND BIOCHEMICAL ASPECTS OF GREEN GRAM (VIGNA RADIATA L. WILCZEK) U. Maity and A.K. Bera Department of Plant Physiology Bidhan Chandra Krishi Viswavidyalaya Mohanpur 741 252, West Bengal, India ABSTRACT Brassinolide, a steroidal compound of plant origin and salicylic acid, a phenolic compound have been found to increase yield in green gram. But, the physiological and biochemical basis of yield improvement are yet to be ascertained. A field experiment was conducted to understand the influence of brassinolide (0.10, 0.25 and 0.50 ppm) and salicylic acid (500, 1000 and 1500 ppm) on certain physiological and biochemical aspects of green gram plant leading to higher yield in this crop. Altogether,8 treatments were given as foliar spray including two types of control namely water spray and no spray. Foliar application of these plant growth regulators, once at pre-flowering and second time at flowering stage of the crop significantly increased chlorophyll-a, b and total chlorophyll content, Hill activity in the leaves of green gram plant. Reducing, non-reducing sugar, starch and soluble protein content in the leaves were also increased over control and water spray due to these treatments. Brassinolide at 0.25 ppm and salicylic acid at 1000 ppm were found to be most effective indicating optimum doses respectively and brassinolide seems to be superior than salicylic acid to influence these physiological and biochemical yield parameters. Key words: Brassinolide, Salicylic acid, Physiological aspect, Biohemical aspects, Green gram INTRODUCTION Green gram is an important grain legume crop in India. The productivity of this crop is low when compared with other crops. Generally, the productivity of any crop depends on various physiological and biochemical events at different stages of crop growth. The invention of brassinosteroids and salicylic acid as plant growth regulators creates an outstanding achievement over traditional plant growth regulators like IAA, GA 3, kinetin, ethylene and ABA which have contributed a promise for yield improvement in various field crops. Brassinolide is an important steroidal component obtained from pollen grains of Brassica napus L. and salicylic acid is a phenolic compound obtained from bark of willow tree ( Salix sp.). Although, these compounds are now extensively used in Agriculture to increase crop productivity, yet the physiological and biochemical basis of yield improvement by application of these plant growth regulators are yet to be explored (Clouse, 1998; Kananbala and Singh, 2003). Moreover, the precise action depends on concentration, frequency and stages of the crop for application of the substance used. Keeping all these aspects in view, the present studies
Table 1: Influence of Brassinolide and Salicylic acid on chlorophyll-a, chlorophyll-b and total chlorophyll content in leaf at different growth stages of green gram cv. PDM 54 (Data expressed as mg g -1 fr. Wt.) Chl.a Chl.b Total Chl.a Chl.b Total Chl.a Chl.b Total Chl.a Chl.b Total Chl.a Chl.b Total Chl. Chl. Chl. Chl. Chl. : Control 0.905 0.272 1.177 1.090 0.420 1.500 1.200 0.365 1.565 0.826 0.362 1.188 0.775 0.190 0.965 : Control (Water spray) 0.970 0.226 1.247 1.225 0.395 1.620 1.230 0.443 1.673 0.970 0.226 1.247 0.728 0.320 1.048 : Brassinolide 0.10 1.221 0.500 1.722 1.519 0.530 2.050 1.047 0.434 1.481 1.018 0.363 1.381 : Brassinolide 0.25 1.373 0.502 1.875 1.670 0.590 2.260 1.171 0.527 1.698 1.150 0.370 1.525 : Brassinolide 0.50 1.031 0.480 1.512 1.367 0.589 1.956 1.026 0.409 1.436 0.822 0.281 1.105 : Salicylic acid 500 1.210 0.425 1.635 1.241 0.491 1.732 1.155 0.356 1.511 0.816 0.178 0.995 : Salicylic acid 1000 1.250 0.410 1.660 1.330 0.440 1.770 1.185 0.371 1.556 0.945 0.367 1.312 : Salicylic acid 1500 1.200 0.365 1.565 1.210 0.425 1.635 1.018 0.425 1.443 0.768 0.340 1.109 SEm (±) 0.013 0.0012 0.001 0.0025 0.0011 0.0020 0.0018 0.0023 0.0020 0.0015 0.0013 0.0006 0.0016 0.0022 0.0020 C.D. (P=0.05) 0.003 0.0002 0.00002 0.0078 0.0033 0.0062 0.0056 0.0070 0.0061 0.0047 0.0041 0.0020 0.0049 0.0069 0.0062 Vol. 43, No. 3, 2009 195 were undertaken to assess the influence of brassinolide (BR) and salicylic acid (SA) on certain physiological and biochemical aspects of green gram plant at different growth stages. MATERIAL AND METHODS The experiment was carried out in the Instructional farm of Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India during kharif seasons of 2005 and 2006 in a randomized block design with three replicates. Seeds of mungbean (Vigna radiata L. Wilczek cv. PDM 54) were sown in the well manured field where, a population of 35 plants per m 2 was maintained in a plot size of 12 m 2. Recommended doses of N, P 2 O 5, and K 2 O were applied before sowing. Brassinolide as Godrej double obtained from Godrej Agrovet Limited, Godrej Bhavan, Block GN, Sector V, Salt Lake City, Kolkata 700 091 and salicylic acid purchased from Emerck India Private Limited, Bombay were used in this experiment. Different concentrations of brassinolide (viz. 0.10, 0.25 and 0.50 ppm) and salicylic acid (viz. 500, 1000 and 1500 ppm) were applied twice till drenched, once at pre-flowering stage and second time at flowering stage. Spraying was done between 8 to 10 a.m. of bright sunny days using 0.1% (v/v) Tween 20 as detergent. Altogether, 8 treatments were given as foliar spray including two types of control namely water spray and no spray. Leaves from control and treated plants were procured at 10 days interval starting from 30 DAS to till harvest (70 DAS). Chlorophyll content, Hill activity and soluble protein content were measured in the fresh samples following the methods of Arnon, (1949); Naidu et. al. (1984) and Lowry et. al. (1951) respectively. Reducing, non-reducing sugar and starch content were measured from leaf samples dried in a hot air oven at 80 ± 20C for 48 hours following the methods described by Sadasivam and Manickam, (1991). The entire data were analysed statistically by using
196 INDIAN JOURNAL OF AGRICULTURAL RESEARCH Table 2: Effect of Brassinolide and Salicylic acid on Hill activity in the leaves of green gram cv PDM 54 at different stages of growth (Data expressed as μ M DCPIP reduced mg 1 chlorophyll h-1) : Control 2.22 2.46 2.74 2.51 2.05 : Control (Water spray) 2.38 2.85 3.06 2.66 2.48 : Brassinolide 0.10 3.12 3.64 3.05 2.87 : Brassinolide 0.25 3.54 4.16 3.42 3.14 : Brassinolide 0.50 3.28 3.60 3.16 2.86 : Salicylic acid 500 3.00 3.24 3.02 2.64 : Salicylic acid 1000 3.32 3.68 3.08 2.78 : Salicylic acid 1500 3.14 3.25 2.92 2.62 SEm (±) 0.0245 0.0584 0.0255 0.0212 0.0213 C.D. (P=0.05) 0.0221 0.1771 0.0774 0.0644 0.0648 analysis of variance as given by Panse and Sukhatme, (1989). RESULTS AND DISCUSSION Chlorophyll, the pigment responsible for photosynthesis was found to increase in all the growth stages of plant over control and water spray due to application of brassinolide and salicylic acid. Chlorophyll-a, b and total chlorophyll content were highest at 50 DAS, which gradually decreased afterwards as the plants entered into senescence phase and this decrease was more marked in control and water spray than the plants treated with either BR and SA (Table 1). Among the treatments, brassinolide at 0.25 ppm and salicylic acid at 1000 ppm recorded higher chlorophyll content over other treatments and BR was found to be superior than SA in influencing chlorophyll content in green gram leaves. Increase in chlorophyll content by BR treatment was reported in groundnut by Prakash et. al. (2003) and SA in soybean by Sharma and Kaur, (2003). The increase in chlorophyll content with leaf maturation (50 DAS) and their decline with the onset of senescence may be due to increased synthesis and degradation of chlorophyll respectively. It is also possible that the chlorophyll synthesis was enhanced by these bio-regulators because the chlorophyllase enzyme which is responsible for chlorophyll depletion might have been inhibited leading to higher accumulation of chlorophyll (Paricha et. al., 1977). Soleman, (1988) reported that seed yields have been directly related to leaf chlorophyll content and thus, the foliar application of BR (0.25 ppm) and SA (1000 ppm) at pre-flowering and flowering stage can bring about changes in assimilatory component and thereby improvement of yield in green gram has been recorded by application of these bio-regulators (Maity, 2007). Data presented in Table 2 revealed that BR at 0.25 ppm significantly increased Hill activity up to 50 DAS (4.16) and decreased thereafter as the plant enter into senescence phase. SA also influenced Hill activity similar to BR and out of these two bio-regulators tested, BR and SA at 0.25 ppm and 1000 ppm respectively proved to be superior over their other concentrations. Hill activity was low in control and water spray at every periodical stage compared to BR and SA (Table 2). The enhancement of Hill activity with leaf maturation (50 DAS) may be due to increased synthesis of chlorophyll along with modification of photosynthetic apparatus by BR and SA. However, decline in Hill activity afterwords i.e. 50 DAS might be attributed to degradation of chlorophyll coupled with senescence. Mukherjee and Rao, (1993)
Vol. 43, No. 3, 2009 197 Table 3: Effect of Brassinolide and Salicylic acid on the carbohydrate fractions of green gram leaf cv. PDM 54 at different stages of growth (Data expressed as mg/100 mg dry wt.) Reducing Non Starch Reducing Non Starch Reducing Non Starch Reducing Non Starch Reducing Non Starch reducing reducing reducing reducing reducing : Control 5.62 5.14 8.36 5.96 5.62 8.84 6.72 6.84 9.70 6.22 6.14 8.52 5.28 5.00 7.05 : Control 5.85 5.33 8.72 6.14 6.02 9.36 7.23 7.00 9.82 6.60 6.18 8.68 5.36 5.04 7.16 (Water spray) :Brassinolide 8.38 7.56 11.72 8.44 7.58 11.94 7.12 6.85 8.74 6.56 6.08 7.72 0.10 : Brassinolide 9.14 8.85 12.46 9.52 9.18 13.60 8.76 7.56 10.42 7.22 6.96 8.66 0.25 : Brassinolide 8.48 7.62 11.84 9.16 8.74 12.82 7.80 7.42 10.00 6.44 6.12 7.92 0.50 : Salicylic acid 7.28 7.02 10.66 8.12 7.33 11.34 7.70 6.22 9.46 6.22 5.60 7.36 500 : Salicylic acid 8.48 7.76 11.62 9.24 8.56 11.92 8.44 7.30 10.22 6.98 6.65 8.15 1000 : Salicylic acid 7.24 7.12 10.66 8.44 8.28 11.22 7.16 7.12 9.76 6.52 6.44 8.02 1500 SEm (±) 0.0286 0.0125 0.0245 0.0092 0.0158 0.0175 0.0179 0.0197 0.0225 0.0383 0.0192 0.0086 0.0158 0.0187 0.0201 C.D. (P=0.05) 0.0213 0.0059 0.0100 0.0280 0.0481 0.0533 0.0543 0.0598 0.0684 0.1163 0.0584 0.0263 0.481 0.0567 0.0612
198 INDIAN JOURNAL OF AGRICULTURAL RESEARCH Table 4; Effect of Brassinolide and Salicylic acid on soluble protein content in green gram leaf cv. PDM 54 at different stages of growth (Data expressed as mg g-1 fr. wt.) : Control 15.67 18.02 20.38 18.02 15.22 : Control (Water spray) 16.27 19.23 20.66 18.23 15.49 : Brassinolide 0.10 24.65 27.57 21.57 17.16 : Brassinolide 0.25 26.87 29.38 22.02 18.12 : Brassinolide 0.50 22.63 26.45 21.63 16.75 : Salicylic acid 500 22.15 25.10 20.74 16.27 : Salicylic acid 1000 24.40 26.75 21.88 17.18 : Salicylic acid 1500 21.62 23.42 20.58 16.52 SEm (±) 0.078 0.4430 0.5225 0.2169 0.3098 C.D. (P=0.05) 0.111 1.3438 1.5848 0.6580 0.9397 noticed significant increase in Hill activity from young to mature leaves of pigeon pea but decreased sharply thereafter as the leaves undergo senescence. Brassinolide and salicylic acid increased all the fractions of carbohydrate up to 50 DAS over control and water spray and decreased thereafter as the senescence stage advanced (70 DAS). Increase in reducing, non-reducing sugar and starch content in leaves at 50 DAS by various treatments ranged from 8.12 to 9.52, 7.33 to 9.18 and 11.22 to 13.60 over control (6.72, 6.84 and 9.70) and water spray (7.23, 7.00 and 9.82) respectively (Table 3). Comparatively, higher levels of available carbohydrate fractions were observed with BR 0.25 ppm and SA 1000 ppm which might be attributed to the optimum concentration of these bio-regulators respectively for this crop in particular. The decrease of carbohydrate fractions with rise in the concentration of BR and SA again indicated the inhibitory activity of these plant growth regulators at higher concentration. These results corroborated with the findings of Salem, (1989) in soybean with plant growth regulators. Higher chlorophyll content (Table 1) and Hill activity (Table 2) in response to foliar application of BR and SA might account for synthesis of more carbohydrates in treated plants. Decrease in carbohydrate fractions 50 DAS may result from transport of sugars from the senescent leaves to the developing pod as in the present study these organs viz. subtending leaves and pods were very close to each other and enhanced transport of 14C labeled sugar from subtending leaf to axillary developing pod by brassinosteroid treated leaves of Vicia faba has been reported (Petzold et. al., 1992). The rise in the rate of respiration in senescent leaves may also be held responsible for the decline in the amount of sugar(tetley and Thiman, 1974). Soluble protein is an important constituent of leaf protein, approximately 50 per cent of which is composed of RUBISCO, the key enzyme of CO 2 fixation in C 3 photosynthetic pathway. It was observed that both BR and SA increased soluble protein content in the leaves up to 50 DAS over control and water spray and decreased thereafter as the plant enter into senescence phase (Table 4). Highest protein content was recorded in plants at 50 DAS treated with BR 0.25 ppm (29.38) followed by BR 0.10 ppm (27.57) and SA 1000 ppm (26.75) in the descending order over water spray (20.66) and control (20.38). A similar report of increased soluble protein with BR treatment has been reported by Vardhini and Rao, (1998) in groundnut. It is assumed that increase in leaf protein content
by BR treatment might be due to increase in amino acid biosynthesis. However, stimulation of NRase activity can not be ruled out as enhancement of NRase activity in black gram by BR treatment has been reported by Mathur et. al. (1988). Phenolic compounds play paramount role in nitrogen metabolism of plants. Salicylic acid induces NRase activity by interacting with NRase specific inhibitor which has been reported in many plants (Srivastava, 1980; Jain and Srivastava, 1981). Alternatively, the effect of SA on NRase may be mediated via phytohormones. Many phenolic compounds are known to provide protection to auxin against oxidation (Schneider and Whitman, 1974). The increased levels of auxin may increase NRase, as it has been demonstrated that external supply of IAA increases NRase in leaves (Roth- Bejerano and Lips, 1970). Vol. 43, No. 3, 2009 199 The present study revealed that foliar application of BR and SA once at preflowering and second time at flowering stage influences different physiological and biochemical aspects of green gram plant. BR and SA might have increased assimilation rate as evidenced by increased chlorophyll content and Hill activity in the leaf. Sugar, starch and soluble protein content of leaf are important yield determinants of crop which have been found to increase by application of these bioregulators. It is assumed that increase in yield of crop by application of BR and SA might occur through enhancement of assimilatory rate in conjunction with increased production of sugar, starch and protein content in the leaf followed by accumulation of these biochemical components into major sink. However, out of these two plant growth regulators tested, the former one appears to be more promising. REFERENCES Arnon, D.I. (1949). Plant Physiol., 24 : 1 15. Clouse, S.D. (1998). Ann. Rev. Plant Physiol. Plant Mol. Biol., 49 : 427 451. Jain, A. and Srivastava, H.S. (1981). Physiol. Plantarum, 51 : 331 342. Kananbala, S. and Singh, T.N. (2003). Crop Res., 26(2) : 355 360. Lowry, O.H. et al. (1951). J. Biol. Chem., 193 : 265 275. Maity, U. (2007). Ph.D. Thesis, Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India. Mathur, S. et al. (1988). J. Indian Bot. Soc., 67 : 212. Mukherjee, D. and Rao, K.U.M. (1993). Indian J. Plant Physiol., 36(1) : 13 16. Naidu, R.A.M. et al. (1984). Physiol. Plant Path., 25 : 181 190. Panse, V.G. and Sukhatme, P.V. (1989). Statistical Methods for Agricultural Workers. ICAR, New Delhi. Paricha, P.C. et al. (1977). Sci. & Cult., 43 : 230-231. Petzold, U. et al. (1992). Acta Bot. Neerl., 41(4) : 469 479. Prakash, M. et al. (2003) Indian J. Plant Physiol., 8(3) : 313 315. Roth-Bejerano, N. and Lips, H. (1970). New Phytol., 69 : 165 169. Sadasivam, S. and Manickam, A. (1991). Biochemical Methods for Agricultural Sciences. Wiley Eastern Limited, New Delhi. Salem, S.M. (1989). Bull. Faculty Agric., Univ. Cairo, 40(1) : 213 223. Schneider, E.A. and Whitman, F. (1974). Ann. Rev. Plant Physiol., 25 : 487 513. Sharma, K. and Kaur, S. (2003). Indian Agriculturist, 47 : (1/2) : 79 84. Soleman, N.J. (1988). Intern. Biol. (Sci. Engg.)., 49 : 265 268. Srivastava, H.S. (1980). Phytochemistry, 13 : 725 733. Tetley, R.M. and Thiman, K.V. (1974). Plant Physiol., 54 : 294 303. Vardhini, B.V. and Rao, R.S.S. (1998). Phytochemistry, 48 (6) : 927 930.