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Utilizing the γ-Irradiated Sodium Alginate as a Plant Growth Promoter for Enhancing the Growth, Physiological Activities, and Alkaloids Production in Catharanthus roseus L.
Agricultural Sciences in China
August 2011
2011, 10(8): 1213-1221
Utilizing the γ-Irradiated Sodium Alginate as a Plant Growth Promoter for Enhancing the Growth, Physiological Activities, and Alkaloids Production in Catharanthus roseus L. Mohd Idrees1, Mohd Naeem1, Masidur Alam1, Tariq Aftab1, Nadeem Hashmi1, Mohd Masroor Akhtar Khan1, Moinuddin1 and Lalit Varshney2 1 2
Department of Botany, Aligarh Muslim University, Aligarh 202002, India Bhabha Atomic Research Centre, Mumbai 400085, India
Abstract Sodium alginate is a polysaccharide that is largely obtained from the brown algae (Sargassum sp.). It has been used as a wonderful growth promoting substance in its depolymerized form for various plants. The aim of this study was to find out the effects of various concentrations of γ-irradiated sodium alginate (ISA), viz., deionized water (control, T 0), 20 (T 1), 40 (T2), 60 (T3), 80 (T4), and 100 ppm (T5) on the agricultural performance of Catharanthus roseus L. (Rosea) in terms of growth attributes, photosynthesis, physiological activities, and alkaloid production. The present work revealed that ISA applied as leaf-sprays at concentrations from 20 to 100 ppm might improve growth, photosynthesis, physiological activities, and alkaloid production in C. roseus L. significantly. Of the various ISA concentrations, 80 ppm proved to be the best one compared to other concentrations applied. Key words: γ-irradiated sodium alginate, plant growth promoter, chlorophyll and carotenoids content, carbonic anhydrase and nitrate reductase activities, growth attributes, photosynthesis
INTRODUCTION Alginates occupy a prominent position among natural polysaccharides available as structural part of the brown algae Sargassum (Anthony et al. 2007) in large amounts. Sodium alginate (SA), a new multifunctional marine bioactive material derived from the brown algae, consist of homopolymeric poly-β-(1 4) D-mannuronic acid residues and poly-α-(1 4) L-gluronic acid residues. Depolymerised sodium alginate, known as oligo-alginate, is prepared by acid hydrolysis or enzymatic degradation of sodium alginate (Hien et al. 2000). Applying ionizing radiation to degrade natural bioactive agents
and then using them as growth promoting substances is a novel emerging technology to exploit the full genetic potential of crops in terms of growth, yield, and quality. Polysaccharides, such as sodium alginate, have been used as wonderful growth promoting substances in their depolymerized form regarding various plants (Nagasawa et al. 2000). The SA irradiated by gamma rays has growth promoting activities like other plant growth promoters and acts as bio-fertilizer (Mollah et al. 2009). Depolymerised sodium alginate showed various biological effects on plants including enhanced seed germination, shoot elongation, and root growth (Yonemoto et al. 1993; Natsume et al. 1994; Hu et al. 2004). It also acts as the endogenous elicitor to pro-
Received 2 August, 2010 Accepted 16 November, 2010 Correspondence Mohd Naeem, Ph D, Mobile: +91-9719341207, E-mail: [email protected]
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60112-0
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mote the production of certain enzymes and plant growth (Akimoto et al. 1999; Khan et al. 2011; Sarfaraz et al. 2011). Catharanthus roseus L., a member of Apocyanacea family, has gained commercial importance due to the anticancer properties of its alkaloids (Nwafor et al. 2001). It is identified by two common varieties, namely, Rosea and Alba, found in India. It has several alkaloids which are distributed in all the parts of the plant. The physiologically important and antineoplastic alkaloids, vinblastine and vincristine are mainly present in the leaves, while the antihypertensive alkaloids such as ajmalicine, serpentine, and reserpine are found in roots (Singh et al. 2000). The variety Rosea proved superior over Alba in overall growth performance and alkaloids production in our previous study (Idrees et al. 2010). Taking into consideration, the importance of irradiated sodium alginate (ISA) as a plant growth promoter and the medicinal value of C. roseus L., the present study was undertaken to find out the effect of foliar spray of depolymerised sodium alginate on the agricultural performance of C. roseus L. in terms of growth, photosynthesis, physiological activities, and alkaloids production.
MATERIALS AND METHODS Preparation of aqueous solution of sodium alginate Sodium alginate was purchased from Sigma-Aldrich, USA. Sodium alginate was kept in water for swelling over night at room temperature to obtain 4% aquous solution. Then the solution was stired for several hours untill completely dissolved. The samples of sodium alginate were irradiated by γ rays using Co-60 as a source at 520 kilo Gray (kGy) in Gamma Radiation Chamber, Bhabha Atomic Research Centre (BARC), Mumbai, Maharashtra, India. The solid sodium alginate, thus prepared, was sealed in a glass tube with atmospheric air. A 20 ppm concentration was prepared by dissolving 2 mg of the ISA in 100 mL DDW. Different concentrations of the ISA, viz., 20, 40, 60, 80, and 100 ppm were finally prepared using double distilled water.
Mohd Idrees et al.
Plant materials and growth conditions Healthy seeds of periwinkle obtained from Indian Agricultural Research Institute, New Delhi, India, were surface sterilized by incubating in 0.02% HgCl2 solution for 5 min with frequent shaking. Later, the seeds were washed with de-ionized water. The seeds were sown in earthen pots (25 cm diameter and 25 cm height) and filled with 5 kg of homogenous mixture of the field soil.
Experimental design, growth and yield analyses The experiment was conducted on C. roseus L. according to simple randomized block design in the natural conditions of net house at the Botany Department, A.M.U., Aligarh U.P., India (27°52´N latitude, 78°51´E longitude, and 187.45 m altitude). Foliar spray of the γ-irradiated sodium alginate was applied to the plants at the stage of 2-3 true leaves. Total five sprays of the ISA were carried out using a hand sprayer. The treatments applied were: control (deionised water) (T0), 20 (T1), 40 (T2), 60 (T3), 80 (T4), and 100 ppm ISA (T5). Growth and biochemical attributes of the crop were determined at 90 d after sowing (DAS). Analysis of the crop was performed in terms of growth and other physiological attributes and total alkaloids content. At 90 DAS, four plants from each treatment pot were harvested and their roots were washed carefully with tap water to remove all adhering foreign particles. Water adhering to the roots was removed with a blotting paper and the fresh weights of plants were recorded thereafter. The plants were dried at 80°C for 24 h using a hot air oven and the dry weights of the plants were recorded subsequently. Each treatment was replicated four times and each replicate had three plants. Thus, each treatment consisted of 12 pots, and each pot having a single healthy plant. Plants were grown under naturally illuminated environmental conditions. Before transplanting, uniform basal doses of nitrogen, phosphorus, and potassium were given at the rate of 15, 25, and 25 kg ha -1, respectively, in the form of urea, potassium dihydrogen orthophosphate and muriate of potash. The plants were sufficiently watered as needed. The height of the plants was measured with the help
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Utilizing the γ-Irradiated Sodium Alginate as a Plant Growth Promoter for Enhancing the Growth, Physiological Activities
of a meter scale. Leaf-area index (LAI) was determined using the following formula described by Watson (1947): LAI=Leaf-area per plant/Area occupied per plant
Net photosynthetic rate, stomatal conductance, and water use efficiency Net photosynthetic rate, stomatal conductance, and water use efficiency were measured on sunny days at light saturation intensity between 1 100 and 1 200 h on youngest fully expanded periwinkle leaves using the infra red gas analyzer (IRGA), Li-Cor 6400 Portable Photosynthesis System (Lincoln, Nebraska, USA). The atmospheric conditions during the measurements were photosynthetically active radiation (PAR) (1 016±6 l) μmol m-2 s-1, relative humidity (60 ± 3)%, atmospheric temperature (38 ± 5)°C, and atmospheric CO2 concentration 360 μmol mol-1. The ratio of atmospheric CO2 to intercellular CO2 concentration was constant. The leaves of the periwinkle plants were enclosed in a gas exchange chamber for the photosynthetic measurements. These measurements were recorded three times for each treatment.
Total chlorophyll and carotenoids content Total chlorophyll and carotenoids content were estimated in the youngest fully developed leaves using the method of Lichtenthaler and Buschmann (2001). The fresh tissues from interveinal leaf area were ground using a mortar and pestle with 80% acetone. The optical density (OD) of the solution was read at 662 and 645 nm for chlorophyll a and b contents, respectively, and at 470 nm for carotenoids content, using a spectrophotometer (Shimadzu UV-1700, Tokoyo, Japan). The contents of photosynthetic pigments were expressed as mg g-1 FW.
Nitrate reductase (NR) activity Nitrate reductase (E.C. 1.7.1.1) activity was estimated by the intact tissue assay method developed by Jaworski (1971), which is based on the reduction of nitrate to nitrite according to the following biochemical reaction:
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The amount of nitrite formed was determined spectrophotometrically. Fresh chopped leaves, weighing 200 mg were transferred to a plastic vial. Each vial contained 2.5 mL phosphate buffer (pH 7.5), 0.5 mL potassium nitrate solution, and 2.5 mL of 5% isopropanol. After incubation for 2 h at 30°C, 0.4 mL of the contents were transferred from the vial to the test tube. To it, 0.3 mL each of 1% sulphanilamide and 0.02% N-(1-naphthyl) ethylenediamine dihydrochloride (NED-HCL) solution was added. After waiting for 20 min at room temperature for maximum colour development, the contents were diluted to a volume of 5 mL with distilled water. The OD of the contents was recorded at 540 nm using the spectrophotometer. NR activity was expressed as nano moles of nitrite produced per g fresh weight leaf tissue per hour (nmol NO2- g-1 FW h-1).
Carbonic anhydrase (CA) activity The carbonic anhydrase (E.C. 4.2.1.1) activity in the youngest fully developed leaves was determined using the method described by Dwivedi and Randhawa (1974). 200 mg of fresh leaf tissue was transferred to Petri plates, followed by incubation of the leaf tissue in 10 mL of 0.2 mol L-1 cystein hydrochloride solution for 20 min at 4°C. Thereafter, 4 mL of 0.2 mol L-1 sodium bicarbonate solution and 0.2 mL of 0.02% bromothymol blue was added to the homogenate. The reaction mixture was titrated against 0.05 N HCl using methyl red as indicator. The activity of CA was expressed as micromoles of CO2 produced per kg of fresh leaf tissue per second (μmol CO2 kg-1 FW s-1).
Total alkaloid content in leaves Total alkaloid content was estimated in the dry leafpowder as described by Afaq et al. (1994). 500 mg of fine powder of leaves was taken in a 100-mL round bottom reflux flask. To this, 50 mL of ethyl alcohol was added and the mixture was refluxed for 6 h. The mixture was then filtered, and followed by addition of 50 mL of diluted HCl. Later, the mixture was transferred to a separating funnel, to which 50 mL of diethyl ether was added. The contents were shaken for 15-20 min. The upper diethyl ether layer was discarded and the
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lower water layer was decanted in a beaker and was made slightly basic by adding ammonia solution. Subequently, the mixture was again transferred into a separating funnel adding 50 mL of diethyl ether. The contents were shaken for 15-20 min. The lower layer was discarded and the upper diethyl ether layer was decanted. To the decant, anhydrous sodium carbonate was added. The mixture was decanted and transferred to a pre-weighed dry porcelain dish and then it was evaporated till dryness. The weight of this porcelain dish was then taken again. Total alkaloids were expressed as percent alkaloid content in the dry leaves.
Statistical analysis The data were analysed statistically with SPSS-17 statistical software (SPSS Inc., Chicago, IL, USA) according to simple randomized block design. Mean values were statistically compared by Duncan’s multiple range test (DMRT) at P<0.05% level.
RESULTS AND DISCUSSION Data presented in Figs. 1-3 indicated that all the treatments had strong promotive effects on all growth parameters studied. Treatment T1 showed the poorest results for most of the parameters studied (Figs. 1-3). Of the five ISA concentrations, 80 ppm resulted in the
Mohd Idrees et al.
best values compared to the other ISA concentrations. This concentration (80 ppm ISA) exhibited 24.48, 24.57, 34.48, 21.94, and 16.05% higher values of plant height, leaf-area index, shoot fresh weight, root fresh weight, and root dry weight, respectively, in comparison to the control (Figs. 1-3). Treatment T5 did not further increase the values of the growth attributes studied; hitherto it significantly improved the above mentioned growth attributes compared to the control (Figs. 1-3). In this regard, previous studies have shown that a range of concentrations of radiation degraded sodium alginate depend upon the source and unit (kGy) of irradiation for a particular plant (Tomoda et al. 1994). The oligomers produced by depolymerisation of alginates have been reported to stimulate plant growth, promotion of germination, and shoot elongation in plants. Among different concentrations of radiation degraded oligomers of sodium alginate (irradiated at 520 kGy), 80 ppm concentration exhibited promotive effects on all the growth parameters studied (Figs. 1-3). In line with the present results, significant improvement in plant growth attributes by the application of radiation-derived oligosaccharides of alginate has earlier been reported. Hien et al. (2000) reported that γ-irradiated sodium alginate at concentrations of 20-100 ppm promoted the productivity of various crops. The results are in agreement with the findings of Hien et al. (2000), Thama et al. (2001), Kume et al. (2002), Luan et al. (2003, 2005, 2009), Khan et al. (2011), Jamsheer (2010),
Fig. 1 Effect of different concentrations of γ-irradiated sodium alginate on plant height (A) and leaf-area index (B) of C. roseus L. Bars showing the same letter are not significantly different at P=0.05 as determined by Duncan’s multiple range test. Error bars mean SE. The same as below.
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Utilizing the γ-Irradiated Sodium Alginate as a Plant Growth Promoter for Enhancing the Growth, Physiological Activities
Qureshi (2010), and Sarfaraz et al. (2011) in the case of various crops. The values of physiological and biochemical parameters were significantly increased in T4 treated plants (Figs. 4-6). The depolymerised sodium alginate significantly stimulated the photosynthetic parameters in the treated plants as depicted in Fig. 4. Among all the treatments applied, T4 exhibited the best results regarding photosynthetic parameters. The plants that received
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the treatment T4 registered 19.6, 20.1, and 20.0% higher values for net-photosynthetic rate, stomatal conductance, and water use efficiency, respectively, as compared to the control (Fig. 4). For the photosynthetic pigments viz., total chlorophyll and carotenoids content, application of ISA at 80 ppm always proved optimum (the best), giving 21.90 and 8.54% higher values for these pigments, respectively (Fig. 5). Presumably, ISA-treated plants might trap more sun-
Fig. 2 Effect of different concentrations of γ-irradiated sodium alginate on shoot fresh (A) and dry weights (B) of C. roseus L.
Fig. 3 Effect of different concentrations of γ-irradiated sodium alginate on root fresh (A) and dry weights (B) of C. roseus L.
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
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Fig. 4 Effect of different concentrations of γ-irradiated sodium alginate on net photosynthetic rate (A), stomatal conductance (B) and water use efficiency (C) of C. roseus L.
light to increase the rate of photosynthesis as compared to the control plants. The ISA-improved contents of photosynthetic pigments might presumably result in the increased photosynthetic rate in this study
Mohd Idrees et al.
and in our previous studies as well (Khan et al. 2011; Sarfaraz et al. 2011). Hien et al. (2000) also observed significant enhancement in net-photosynthesis and CO2 assimilation as a result of application of depolymerised SA. In the present study, ISA-sprayed leaves exhibited greater CA activity (18.23%) than the control (Fig. 6). Such a response of the plants to the applied ISA was expected because spray-treatment of ISA also increased the stomatal conductance (Fig. 4) that might have facilitated the diffusion of carbon dioxide into the stomata. In turn, the CO2 might have been acted upon by the CA. Finally, the CO2 could be reduced by ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) enzyme in the stroma region of chloroplast. In this connection, our results are in accordance with the findings of Tomoda et al. (1994), Natsume et al. (1994), Kume et al. (2002), and Luan et al. (2003), who reported that foliar spray of γ-irradiated sodium alginate enhanced the enzyme activities in plants. Luan et al. (2003) also suggested a key role of γ-irradiated sodium alginate in enhancing the biological activity of the plants. Nitrate reductase is the key enzyme in nitrogen metabolism and is responsible for initiation of nitrate assimilation and hence that of protein synthesis. Fig. 6 indicated that an increase in NR activity (16.47% higher than the control) by the application of γ-irradiated sodium alginate might have exerted a pivotal role in enhancing photosynthetic rate. Thus, the ultimate culmination of enhancement of NR activity might have increased the overall growth of ISA-treated plants in this study. Ultimately, these results might justify the increase in plant fresh and dry matter contents as a result of foliar application of γ-irradiated sodium alginate via improvement in the biochemical and physiological parameters studied in the ISA-treated plants (Figs. 2 and 3). The irradiated sodium alginate spray was also effective in increasing total alkaloid content compared to the water sprayed plants (control). Plants treated with spray of γ-irradiated sodium alginate at a concentration of 80 ppm (T4) registered an increase in the total alkaloid content by 14.81% compared to the control (Fig. 7). The increase in alkaloid content owing to foliar application of ISA might be ascribed to the expected increase in the leaf nitrogen content that might have promoted
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Utilizing the γ-Irradiated Sodium Alginate as a Plant Growth Promoter for Enhancing the Growth, Physiological Activities
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Fig. 5 Effect of different concentrations of γ-irradiated sodium alginate on total chlorophyll (A) and carotenoids contents (B) of C. roseus L.
Fig. 6 Effect of different concentrations of γ-irradiated sodium alginate on nitrate reductase activity (A) and carbonic anhydrase activity (B) of C. roseus L.
amino acid synthesis leading to the improved alkaloid content in the leaves. The growth promoting effect of γ-irradiated sodium alginate on alkaloids production in case of opium poppy has been reported by Khan et al. (2011).
CONCLUSION
Fig. 7 Effect of different concentrations of γ-irradiated sodium alginate on total alkaloid content of C. roseus L.
Based on the results extracted out of this study, it can be concluded that γ-irradiated sodium alginate has promotive effect on plant growth physiological and biochemical activities of plants and on the alkaloids pro-
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duction of C. roseus L. Treatment T4 (80 ppm of ISA), in the form of foliar spray, might presumably be considered important for maximizing the productivity and quality of the crop. However, further investigations are required to comprehend the mechanism and mode of action of alginate-derived oligomers on plants.
Acknowledgements The Bhabha Atomic Research Centre (BARC), Mumbai, Maharashtra, India, is highly acknowledged for its help in providing the γ-irradiated sodium alginate material for this investigation.
References Afaq S H, Tajuddin, Siddiqui M M H. 1994. Standardization of Herbal Drugs. Publication Division, Aligarh Muslim University, Aligarh, India. Akimoto C, Aoyagi H, Tanaka H. 1999. Endogenous elicitor-like effect of alginate on physiological activities of plant cells. Applied Microbiology and Biotechnology, 52, 429-436. Anthony J, Gabapathy A, Coothan K V, Streenivasan P P, Arjuna R, Palaninathan V. 2007. Beneficial effects of sulphated polysaccharides from Saragassum wightii against mitochondrial alterations induced by cyclosporine A in rat kidney. Molecular Nutrition and Food Research, 51, 14131422. Dwivedi R S, Randhawa N S. 1974. Evaluation of rapid test for the hidden hunger of zinc in plants. Plant and Soil, 40, 445451. Hien N Q, Nagasawa N, Tham L X, Yoshii F, Dang V H, Mitomo H, Makuuchi K, Kume T. 2000. Growth promotion of plants with depolymerised alginates by irradiation. Radiation Physics and Chemistry, 59, 97-101. Hu X, Jiang X, Hwang H, Liu S, Guan H. 2004. Promotive effects of alginate-derived oligosaccharide on maize seed germination. Journal of Applied Phycology, 16, 73-76. Idrees M, Naeem M, Khan M M A. 2010. The superiority of cv. ‘rosea’ over cv. ‘alba’ of periwinkle (Catharanthus roseus L.) in alkaloid production and other physiological attributes. Turkish Journal of Biology, 32, 81-88. Jamsheer M K. 2010. Response of beetroot (Beta vulgaris L.) to the application of phosphorus and gamma-irradiated sodium alginate. MSc thesis, Aligarh Muslim University, Aligarh, India. Jaworski E J. 1971. Nitrate reductase assay in intact plant tissues. Biochemical and Biophysical Research Communications, 43, 1247-1279. Khan Z H, Khan M M A, Aftab T, Idrees M, Naeem M,
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Moinuddin. 2011. Influence of alginate oligosaccharides on growth, yield and alkaloid production of opium poppy (Papaver somniferum L.). Frontiers of Agriculture in China, 5, 122-127. Kume T, Nagasawa N, Yoshii F. 2002. Utilization of carbohydrates by radiation processing. Radiation Physics and Chemistry, 63, 625-627. Lichtenthaler H K, Buschmann C. 2001. Chlorophylls and carotenoids: measurement and characterization by UV-VIS spectroscopy. In: Wrolstad R E, ed., Current Protocols in Food Analytical Chemistry. John Wiley and Sons, New York. pp. F4.3.1-F4.3.8. Luan L Q, Ha V T, Nagasawa N, Kume T, Yoshii F, Nakanishi T M. 2005. Biological effect of irradiated chitosan on plants in vitro. Biotechnology and Applied Biochemistry, 41, 49-57. Luan L Q, Hien N Q, Nagasawa N, Kume T, Yoshii F, Nakanishi T M. 2003. Biological effect of radiation-degraded alginate on flower plants in tissue culture. Biotechnology and Applied Biochemistry, 38, 283-288. Luan L Q, Nagasawa N, Ha V T T, Hien N Q, Nakanishi T M. 2009. Enhancement of plant growth stimulation activity of irradiated alginate by fractionation. Radiation Physics and Chemistry, 78, 796-799. Mollah M Z I, Khan M A, Khan R A. 2009. Effect of gamma irradiated sodium alginate on red amaranth (Amaranthus cruentus L.) as growth promoter. Radiation Physics and Chemistry, 78, 61-64. Nagasawa N, Mitomo H, Yoshii F, Kume T. 2000. Rradiation induced degradation of sodium alginate. Polymer Degradation and Stability, 69, 279-285. Natsume M, Kamao Y, Hirayan M, Adachi J. 1994. Isolation and characterization of alginate derived oligosaccharides with root growth promoting activities. Carbohydrate Research, 258, 187-197. Nwafor S V, Akah P A, Okali C O. 2001. Potential of plant products as anticancer agents. Journal of Natural Remedy, 1, 75-87. Qureshi A H. 2010. Effect of nitrogen and gamma-irradiated sodium alginate on the efficiency of beetroot (Beta vulgaris L.). MSc thesis, Aligarh Muslim University, Aligarh, India. Sarfaraz A, Naeem M, Nasir S, Idrees M, Aftab T, Hashmi N, Khan M M A, Moinuddin, Varshney L. 2011. An evaluation of the effects of irradiated sodium alginate on the growth, physiological activities and essential oil production of fennel (Foeniculum vulgare Mill.). Journal of Medicinal Plant Research, 5, 15-21. Singh D V, Maithy A, Verma R K, Gupta M M, Kumar S. 2000. Simultaneous determination of Catharanthus alkaloids using reserved phase high performance liquid chromatography. Journal of Liquid Chromatography & Related Technologies,
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Utilizing the γ-Irradiated Sodium Alginate as a Plant Growth Promoter for Enhancing the Growth, Physiological Activities
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23, 601-607. Thama L X, Nagasawab N, Matsuhashib S, Ishiokab N S, Itob T,
58, 203-203. Watson D J. 1947. Comparative physiological studies on the
Kume T. 2001. Effect of radiation-degraded chitosan on plants stressed with vanadium. Radiation Physics and Chemistry,
growth of the field crops. Annals of Botany, 11, 42-76. Yonemoto Y, Tanaka H, Yamashita T, Kitabatake N, Ishida Y,
61, 171-175. Tomoda Y, Umemura K, Adachi T. 1994. Promotion of barley
Kimura A, Murata K. 1993. Promotion of germination and shoot elongation of some plants by alginate oilgomers
root elongation under hypoxic conditions by alginate lyaselysate (A.L.L.). Bioscience, Biotechnology and Biochemistry,
prepared with bacterial alginate lyase. Journal of Fermentation and Bioengineering, 75, 68-70. (Managing editor WANG Ning)
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August 2011
2011, 10(8): 1213-1221
Utilizing the γ-Irradiated Sodium Alginate as a Plant Growth Promoter for Enhancing the Growth, Physiological Activities, and Alkaloids Production in Catharanthus roseus L. Mohd Idrees1, Mohd Naeem1, Masidur Alam1, Tariq Aftab1, Nadeem Hashmi1, Mohd Masroor Akhtar Khan1, Moinuddin1 and Lalit Varshney2 1 2
Department of Botany, Aligarh Muslim University, Aligarh 202002, India Bhabha Atomic Research Centre, Mumbai 400085, India
Abstract Sodium alginate is a polysaccharide that is largely obtained from the brown algae (Sargassum sp.). It has been used as a wonderful growth promoting substance in its depolymerized form for various plants. The aim of this study was to find out the effects of various concentrations of γ-irradiated sodium alginate (ISA), viz., deionized water (control, T 0), 20 (T 1), 40 (T2), 60 (T3), 80 (T4), and 100 ppm (T5) on the agricultural performance of Catharanthus roseus L. (Rosea) in terms of growth attributes, photosynthesis, physiological activities, and alkaloid production. The present work revealed that ISA applied as leaf-sprays at concentrations from 20 to 100 ppm might improve growth, photosynthesis, physiological activities, and alkaloid production in C. roseus L. significantly. Of the various ISA concentrations, 80 ppm proved to be the best one compared to other concentrations applied. Key words: γ-irradiated sodium alginate, plant growth promoter, chlorophyll and carotenoids content, carbonic anhydrase and nitrate reductase activities, growth attributes, photosynthesis
INTRODUCTION Alginates occupy a prominent position among natural polysaccharides available as structural part of the brown algae Sargassum (Anthony et al. 2007) in large amounts. Sodium alginate (SA), a new multifunctional marine bioactive material derived from the brown algae, consist of homopolymeric poly-β-(1 4) D-mannuronic acid residues and poly-α-(1 4) L-gluronic acid residues. Depolymerised sodium alginate, known as oligo-alginate, is prepared by acid hydrolysis or enzymatic degradation of sodium alginate (Hien et al. 2000). Applying ionizing radiation to degrade natural bioactive agents
and then using them as growth promoting substances is a novel emerging technology to exploit the full genetic potential of crops in terms of growth, yield, and quality. Polysaccharides, such as sodium alginate, have been used as wonderful growth promoting substances in their depolymerized form regarding various plants (Nagasawa et al. 2000). The SA irradiated by gamma rays has growth promoting activities like other plant growth promoters and acts as bio-fertilizer (Mollah et al. 2009). Depolymerised sodium alginate showed various biological effects on plants including enhanced seed germination, shoot elongation, and root growth (Yonemoto et al. 1993; Natsume et al. 1994; Hu et al. 2004). It also acts as the endogenous elicitor to pro-
Received 2 August, 2010 Accepted 16 November, 2010 Correspondence Mohd Naeem, Ph D, Mobile: +91-9719341207, E-mail: [email protected]
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60112-0
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mote the production of certain enzymes and plant growth (Akimoto et al. 1999; Khan et al. 2011; Sarfaraz et al. 2011). Catharanthus roseus L., a member of Apocyanacea family, has gained commercial importance due to the anticancer properties of its alkaloids (Nwafor et al. 2001). It is identified by two common varieties, namely, Rosea and Alba, found in India. It has several alkaloids which are distributed in all the parts of the plant. The physiologically important and antineoplastic alkaloids, vinblastine and vincristine are mainly present in the leaves, while the antihypertensive alkaloids such as ajmalicine, serpentine, and reserpine are found in roots (Singh et al. 2000). The variety Rosea proved superior over Alba in overall growth performance and alkaloids production in our previous study (Idrees et al. 2010). Taking into consideration, the importance of irradiated sodium alginate (ISA) as a plant growth promoter and the medicinal value of C. roseus L., the present study was undertaken to find out the effect of foliar spray of depolymerised sodium alginate on the agricultural performance of C. roseus L. in terms of growth, photosynthesis, physiological activities, and alkaloids production.
MATERIALS AND METHODS Preparation of aqueous solution of sodium alginate Sodium alginate was purchased from Sigma-Aldrich, USA. Sodium alginate was kept in water for swelling over night at room temperature to obtain 4% aquous solution. Then the solution was stired for several hours untill completely dissolved. The samples of sodium alginate were irradiated by γ rays using Co-60 as a source at 520 kilo Gray (kGy) in Gamma Radiation Chamber, Bhabha Atomic Research Centre (BARC), Mumbai, Maharashtra, India. The solid sodium alginate, thus prepared, was sealed in a glass tube with atmospheric air. A 20 ppm concentration was prepared by dissolving 2 mg of the ISA in 100 mL DDW. Different concentrations of the ISA, viz., 20, 40, 60, 80, and 100 ppm were finally prepared using double distilled water.
Mohd Idrees et al.
Plant materials and growth conditions Healthy seeds of periwinkle obtained from Indian Agricultural Research Institute, New Delhi, India, were surface sterilized by incubating in 0.02% HgCl2 solution for 5 min with frequent shaking. Later, the seeds were washed with de-ionized water. The seeds were sown in earthen pots (25 cm diameter and 25 cm height) and filled with 5 kg of homogenous mixture of the field soil.
Experimental design, growth and yield analyses The experiment was conducted on C. roseus L. according to simple randomized block design in the natural conditions of net house at the Botany Department, A.M.U., Aligarh U.P., India (27°52´N latitude, 78°51´E longitude, and 187.45 m altitude). Foliar spray of the γ-irradiated sodium alginate was applied to the plants at the stage of 2-3 true leaves. Total five sprays of the ISA were carried out using a hand sprayer. The treatments applied were: control (deionised water) (T0), 20 (T1), 40 (T2), 60 (T3), 80 (T4), and 100 ppm ISA (T5). Growth and biochemical attributes of the crop were determined at 90 d after sowing (DAS). Analysis of the crop was performed in terms of growth and other physiological attributes and total alkaloids content. At 90 DAS, four plants from each treatment pot were harvested and their roots were washed carefully with tap water to remove all adhering foreign particles. Water adhering to the roots was removed with a blotting paper and the fresh weights of plants were recorded thereafter. The plants were dried at 80°C for 24 h using a hot air oven and the dry weights of the plants were recorded subsequently. Each treatment was replicated four times and each replicate had three plants. Thus, each treatment consisted of 12 pots, and each pot having a single healthy plant. Plants were grown under naturally illuminated environmental conditions. Before transplanting, uniform basal doses of nitrogen, phosphorus, and potassium were given at the rate of 15, 25, and 25 kg ha -1, respectively, in the form of urea, potassium dihydrogen orthophosphate and muriate of potash. The plants were sufficiently watered as needed. The height of the plants was measured with the help
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Utilizing the γ-Irradiated Sodium Alginate as a Plant Growth Promoter for Enhancing the Growth, Physiological Activities
of a meter scale. Leaf-area index (LAI) was determined using the following formula described by Watson (1947): LAI=Leaf-area per plant/Area occupied per plant
Net photosynthetic rate, stomatal conductance, and water use efficiency Net photosynthetic rate, stomatal conductance, and water use efficiency were measured on sunny days at light saturation intensity between 1 100 and 1 200 h on youngest fully expanded periwinkle leaves using the infra red gas analyzer (IRGA), Li-Cor 6400 Portable Photosynthesis System (Lincoln, Nebraska, USA). The atmospheric conditions during the measurements were photosynthetically active radiation (PAR) (1 016±6 l) μmol m-2 s-1, relative humidity (60 ± 3)%, atmospheric temperature (38 ± 5)°C, and atmospheric CO2 concentration 360 μmol mol-1. The ratio of atmospheric CO2 to intercellular CO2 concentration was constant. The leaves of the periwinkle plants were enclosed in a gas exchange chamber for the photosynthetic measurements. These measurements were recorded three times for each treatment.
Total chlorophyll and carotenoids content Total chlorophyll and carotenoids content were estimated in the youngest fully developed leaves using the method of Lichtenthaler and Buschmann (2001). The fresh tissues from interveinal leaf area were ground using a mortar and pestle with 80% acetone. The optical density (OD) of the solution was read at 662 and 645 nm for chlorophyll a and b contents, respectively, and at 470 nm for carotenoids content, using a spectrophotometer (Shimadzu UV-1700, Tokoyo, Japan). The contents of photosynthetic pigments were expressed as mg g-1 FW.
Nitrate reductase (NR) activity Nitrate reductase (E.C. 1.7.1.1) activity was estimated by the intact tissue assay method developed by Jaworski (1971), which is based on the reduction of nitrate to nitrite according to the following biochemical reaction:
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The amount of nitrite formed was determined spectrophotometrically. Fresh chopped leaves, weighing 200 mg were transferred to a plastic vial. Each vial contained 2.5 mL phosphate buffer (pH 7.5), 0.5 mL potassium nitrate solution, and 2.5 mL of 5% isopropanol. After incubation for 2 h at 30°C, 0.4 mL of the contents were transferred from the vial to the test tube. To it, 0.3 mL each of 1% sulphanilamide and 0.02% N-(1-naphthyl) ethylenediamine dihydrochloride (NED-HCL) solution was added. After waiting for 20 min at room temperature for maximum colour development, the contents were diluted to a volume of 5 mL with distilled water. The OD of the contents was recorded at 540 nm using the spectrophotometer. NR activity was expressed as nano moles of nitrite produced per g fresh weight leaf tissue per hour (nmol NO2- g-1 FW h-1).
Carbonic anhydrase (CA) activity The carbonic anhydrase (E.C. 4.2.1.1) activity in the youngest fully developed leaves was determined using the method described by Dwivedi and Randhawa (1974). 200 mg of fresh leaf tissue was transferred to Petri plates, followed by incubation of the leaf tissue in 10 mL of 0.2 mol L-1 cystein hydrochloride solution for 20 min at 4°C. Thereafter, 4 mL of 0.2 mol L-1 sodium bicarbonate solution and 0.2 mL of 0.02% bromothymol blue was added to the homogenate. The reaction mixture was titrated against 0.05 N HCl using methyl red as indicator. The activity of CA was expressed as micromoles of CO2 produced per kg of fresh leaf tissue per second (μmol CO2 kg-1 FW s-1).
Total alkaloid content in leaves Total alkaloid content was estimated in the dry leafpowder as described by Afaq et al. (1994). 500 mg of fine powder of leaves was taken in a 100-mL round bottom reflux flask. To this, 50 mL of ethyl alcohol was added and the mixture was refluxed for 6 h. The mixture was then filtered, and followed by addition of 50 mL of diluted HCl. Later, the mixture was transferred to a separating funnel, to which 50 mL of diethyl ether was added. The contents were shaken for 15-20 min. The upper diethyl ether layer was discarded and the
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lower water layer was decanted in a beaker and was made slightly basic by adding ammonia solution. Subequently, the mixture was again transferred into a separating funnel adding 50 mL of diethyl ether. The contents were shaken for 15-20 min. The lower layer was discarded and the upper diethyl ether layer was decanted. To the decant, anhydrous sodium carbonate was added. The mixture was decanted and transferred to a pre-weighed dry porcelain dish and then it was evaporated till dryness. The weight of this porcelain dish was then taken again. Total alkaloids were expressed as percent alkaloid content in the dry leaves.
Statistical analysis The data were analysed statistically with SPSS-17 statistical software (SPSS Inc., Chicago, IL, USA) according to simple randomized block design. Mean values were statistically compared by Duncan’s multiple range test (DMRT) at P<0.05% level.
RESULTS AND DISCUSSION Data presented in Figs. 1-3 indicated that all the treatments had strong promotive effects on all growth parameters studied. Treatment T1 showed the poorest results for most of the parameters studied (Figs. 1-3). Of the five ISA concentrations, 80 ppm resulted in the
Mohd Idrees et al.
best values compared to the other ISA concentrations. This concentration (80 ppm ISA) exhibited 24.48, 24.57, 34.48, 21.94, and 16.05% higher values of plant height, leaf-area index, shoot fresh weight, root fresh weight, and root dry weight, respectively, in comparison to the control (Figs. 1-3). Treatment T5 did not further increase the values of the growth attributes studied; hitherto it significantly improved the above mentioned growth attributes compared to the control (Figs. 1-3). In this regard, previous studies have shown that a range of concentrations of radiation degraded sodium alginate depend upon the source and unit (kGy) of irradiation for a particular plant (Tomoda et al. 1994). The oligomers produced by depolymerisation of alginates have been reported to stimulate plant growth, promotion of germination, and shoot elongation in plants. Among different concentrations of radiation degraded oligomers of sodium alginate (irradiated at 520 kGy), 80 ppm concentration exhibited promotive effects on all the growth parameters studied (Figs. 1-3). In line with the present results, significant improvement in plant growth attributes by the application of radiation-derived oligosaccharides of alginate has earlier been reported. Hien et al. (2000) reported that γ-irradiated sodium alginate at concentrations of 20-100 ppm promoted the productivity of various crops. The results are in agreement with the findings of Hien et al. (2000), Thama et al. (2001), Kume et al. (2002), Luan et al. (2003, 2005, 2009), Khan et al. (2011), Jamsheer (2010),
Fig. 1 Effect of different concentrations of γ-irradiated sodium alginate on plant height (A) and leaf-area index (B) of C. roseus L. Bars showing the same letter are not significantly different at P=0.05 as determined by Duncan’s multiple range test. Error bars mean SE. The same as below.
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Utilizing the γ-Irradiated Sodium Alginate as a Plant Growth Promoter for Enhancing the Growth, Physiological Activities
Qureshi (2010), and Sarfaraz et al. (2011) in the case of various crops. The values of physiological and biochemical parameters were significantly increased in T4 treated plants (Figs. 4-6). The depolymerised sodium alginate significantly stimulated the photosynthetic parameters in the treated plants as depicted in Fig. 4. Among all the treatments applied, T4 exhibited the best results regarding photosynthetic parameters. The plants that received
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the treatment T4 registered 19.6, 20.1, and 20.0% higher values for net-photosynthetic rate, stomatal conductance, and water use efficiency, respectively, as compared to the control (Fig. 4). For the photosynthetic pigments viz., total chlorophyll and carotenoids content, application of ISA at 80 ppm always proved optimum (the best), giving 21.90 and 8.54% higher values for these pigments, respectively (Fig. 5). Presumably, ISA-treated plants might trap more sun-
Fig. 2 Effect of different concentrations of γ-irradiated sodium alginate on shoot fresh (A) and dry weights (B) of C. roseus L.
Fig. 3 Effect of different concentrations of γ-irradiated sodium alginate on root fresh (A) and dry weights (B) of C. roseus L.
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Fig. 4 Effect of different concentrations of γ-irradiated sodium alginate on net photosynthetic rate (A), stomatal conductance (B) and water use efficiency (C) of C. roseus L.
light to increase the rate of photosynthesis as compared to the control plants. The ISA-improved contents of photosynthetic pigments might presumably result in the increased photosynthetic rate in this study
Mohd Idrees et al.
and in our previous studies as well (Khan et al. 2011; Sarfaraz et al. 2011). Hien et al. (2000) also observed significant enhancement in net-photosynthesis and CO2 assimilation as a result of application of depolymerised SA. In the present study, ISA-sprayed leaves exhibited greater CA activity (18.23%) than the control (Fig. 6). Such a response of the plants to the applied ISA was expected because spray-treatment of ISA also increased the stomatal conductance (Fig. 4) that might have facilitated the diffusion of carbon dioxide into the stomata. In turn, the CO2 might have been acted upon by the CA. Finally, the CO2 could be reduced by ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) enzyme in the stroma region of chloroplast. In this connection, our results are in accordance with the findings of Tomoda et al. (1994), Natsume et al. (1994), Kume et al. (2002), and Luan et al. (2003), who reported that foliar spray of γ-irradiated sodium alginate enhanced the enzyme activities in plants. Luan et al. (2003) also suggested a key role of γ-irradiated sodium alginate in enhancing the biological activity of the plants. Nitrate reductase is the key enzyme in nitrogen metabolism and is responsible for initiation of nitrate assimilation and hence that of protein synthesis. Fig. 6 indicated that an increase in NR activity (16.47% higher than the control) by the application of γ-irradiated sodium alginate might have exerted a pivotal role in enhancing photosynthetic rate. Thus, the ultimate culmination of enhancement of NR activity might have increased the overall growth of ISA-treated plants in this study. Ultimately, these results might justify the increase in plant fresh and dry matter contents as a result of foliar application of γ-irradiated sodium alginate via improvement in the biochemical and physiological parameters studied in the ISA-treated plants (Figs. 2 and 3). The irradiated sodium alginate spray was also effective in increasing total alkaloid content compared to the water sprayed plants (control). Plants treated with spray of γ-irradiated sodium alginate at a concentration of 80 ppm (T4) registered an increase in the total alkaloid content by 14.81% compared to the control (Fig. 7). The increase in alkaloid content owing to foliar application of ISA might be ascribed to the expected increase in the leaf nitrogen content that might have promoted
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Utilizing the γ-Irradiated Sodium Alginate as a Plant Growth Promoter for Enhancing the Growth, Physiological Activities
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Fig. 5 Effect of different concentrations of γ-irradiated sodium alginate on total chlorophyll (A) and carotenoids contents (B) of C. roseus L.
Fig. 6 Effect of different concentrations of γ-irradiated sodium alginate on nitrate reductase activity (A) and carbonic anhydrase activity (B) of C. roseus L.
amino acid synthesis leading to the improved alkaloid content in the leaves. The growth promoting effect of γ-irradiated sodium alginate on alkaloids production in case of opium poppy has been reported by Khan et al. (2011).
CONCLUSION
Fig. 7 Effect of different concentrations of γ-irradiated sodium alginate on total alkaloid content of C. roseus L.
Based on the results extracted out of this study, it can be concluded that γ-irradiated sodium alginate has promotive effect on plant growth physiological and biochemical activities of plants and on the alkaloids pro-
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duction of C. roseus L. Treatment T4 (80 ppm of ISA), in the form of foliar spray, might presumably be considered important for maximizing the productivity and quality of the crop. However, further investigations are required to comprehend the mechanism and mode of action of alginate-derived oligomers on plants.
Acknowledgements The Bhabha Atomic Research Centre (BARC), Mumbai, Maharashtra, India, is highly acknowledged for its help in providing the γ-irradiated sodium alginate material for this investigation.
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