Radiolytically degraded sodium alginate enhances plant growth, physiological activities and alkaloids production in Catharanthus roseus L.

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Radiolytically degraded sodium alginate enhances plant growth, physiological activities and alkaloids production in Catharanthus roseus L. M. Naeem a,*, Tariq Aftab a, Abid A. Ansari b, Mohd Idrees c, Akbar Ali a, M. Masroor A. Khan a, Moin Uddin d, Lalit Varshney e a

Plant Physiology Section, Department of Botany, Aligarh Muslim University, Aligarh, 202 002, India Department of Biology, Faculty of Science, University of Tabuk, Tabuk, 71491, Saudi Arabia c Mathematics and Sciences Unit, College of Arts and Applied Sciences, Dhofar University, P. O. Box 2509, Salalah, 211, Oman d Women's College, Botany Section, Aligarh Muslim University, Aligarh, 202 002, India e Radiation Technology Development Division, Bhabha Atomic Research Centre, Mumbai, 400085, India b

article info

abstract

Article history:

Catharanthus roseus (L.) G. Don (Family Apocynaceae) is a medicinal plant that produces

Received 23 April 2015

indole alkaloids used in cancer chemotherapy. The anticancerous alkaloids, viz. vinblastine

Received in revised form

and vincristine, are mainly present in the leaves of C. roseus. High demand and low yield of

26 May 2015

these alkaloids in the plant has led to explore the alternative means for their production.

Accepted 21 July 2015

Gamma irradiated sodium alginate (ISA) has proved as a plant growth promoting substance

Available online xxx

for various medicinal and agricultural crops. A pot culture experiment was carried out to explore the effect of ISA on plant growth, physiological activities and production of anti-

Keywords:

cancer alkaloids (vinblastine and vincristine) in C. roseus at 120 and 150 days after planting

Catharanthus roseus

(DAP). Foliar application of ISA (0, 20, 40, 60, 80 and 100 mg L
1) significantly improved the

Vinblastine

performance of C. roseus. 80 mg L
1 of ISA enhanced the leaf-yield by 25.3 and 30.2% and

Vincristine and vindoline alkaloids

the herbage-yield by 29.4 and 34.4% at 120 and 150 DAP, respectively, as compared to the

Irradiated sodium alginate

control. The spray treatment of ISA at 80 mg L
1 improved the yield of vinblastine by 66.7 and 71.4% and of vincristine by 67.6 and 75.6% at 120 and 150 DAP, respectively, in comparison to the control. As compared to control, the application of ISA at 80 mg L
1 resulted in the maximum swell in the content and yield of vindoline, increasing them by 18.9 and 20.8% and by 81.8 and 87.2% at 120 and 150 DAP, respectively. Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Corresponding author. E-mail address: [email protected] (M. Naeem). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/j.jrras.2015.07.005 1687-8507/Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Naeem, M., et al., Radiolytically degraded sodium alginate enhances plant growth, physiological activities and alkaloids production in Catharanthus roseus L., Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.07.005

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Introduction

Sodium alginate, an eco-friendly polysaccharide of brown algae, has frequently been used as plant growth promoting substance in its gamma-irradiated form. Gamma-rays irradiation with cobalt-60 degrades the natural polysaccharides (chitosan, carrageenan and sodium alginate) into smaller oligomers with comparatively low molecular weight. In fact, foliar application of the degraded oligomers has been proved to stimulate various biological and physiological activities, including plant growth (Aftab et al., 2011; Hien et al., 2000; Khan, Khan, Aftab, Idrees, & Naeem, 2011; Naeem et al., 2012a, 2012b; Sarfaraz et al., 2011), seed germination (Hien et al., 2000), shoot elongation (Hien et al., 2000; Natsume, Kamao, Hirayan, & Adachi, 1994), root growth (Iwasaki & Matsubara, 2006), flower production, antimicrobial activity, amelioration of heavy metal stress, synthesis of phytoalexins, etc. (Darvill et al., 1992; Hu, Jiang, Hwang, Liu, & Guan, 2004; Kume, Nagasawa, & Yoshii, 2002; Luan et al., 2003). Irradiated sodium alginate (ISA) has growth promoting activities like other plant growth promoters and acts as bio-fertilizer (Mollah, Khan, & Khan, 2009). Hence, the use of depolymerized sodium alginate (and of other marine polysaccharides) in promoting plant growth and thereby, augmenting the desired active constituents in medicinal and aromatic plants is inexpensive and safe (Aftab et al., 2011, 2013, 2014; Ali et al. 2014; Idrees et al. 2012; Naeem et al., 2012a, 2012b). It is need of the hour to maximize the production of desired alkaloids of medicinally important plants in view of their massive demand worldwide. Catharanthus roseus (L.) G. Don (commonly known as ‘Sadabahar’ or ‘Periwinkle’, family Apocynaceae) is a medicinal plant that produces chemotherapeutically important alkaloids, viz. vinblastine and vincristine, which inhibit the growth of cancer cells, hindering the formation of mitotic apparatus during cell division (Gueritte & Fahy, 2005). Further, vinblastine could help in increasing the chance of surviving childhood leukemia, while vincristine might be used to treat Hodgkins' disease (Gueritte & Fahy, 2005). However, the content of these useful indole alkaloids in C. roseus leaves is in traces. In addition, L-tryptophan decarboxylase (TDC, EC 4.1.1.28) is the key enzyme, which is involved in the early stages of indole alkaloid biosynthesis (Facchini, Huber-Allanach, & Tari, 2000); it catalyzes the formation of tryptamine from tryptophan and, therefore, plays important role in terpenoid indole alkaloids biosynthesis. Keeping in view the importance of irradiated sodium alginate act as a plant growth promoter and low production of anticancerous alkaloids of this medicinal herb, this study was conduct to find out the effect of ISA on plant growth, herbage yield, activity of TDC enzyme and content and yield of total and therapeutically important alkaloids production of C. roseus.

2.

Materials and methods

2.1.

Plant materials and growth conditions

Department, Aligarh Muslim University, Aligarh (27 520 N latitude, 78 510 E longitude, and 187.45 m altitude) India. Healthy periwinkle seedlings of equal size were obtained from Woodbine Nursery, Civil Lines, Aligarh. The seedlings were then transplanted to earthen pots. Prior to transplantation, each pot (25 cm diameter  25 cm height) was filled with 5 kg of homogenous mixture of soil and farmyard manure (4:1). Physical and chemical characteristics of the soil were: texture sandy loam, pH (1:2) 7.5, E.C. (1:2) 0.46 dSm-1, available N, P and K 102.0, 7.8 and 145.6 mg kg
1 of soil, respectively. A uniform recommended basal dose of N, P and K (15:25:25 kg ha
1, respectively) was applied in the form of urea, single superphosphate and muriate of potash, respectively, at the time of transplanting the seedlings. The experiment was conducted in a simple randomized design. Each treatment was replicated five times and each replicate had three plants. Thus, each treatment consisted of 15 pots, and each pot contained a single healthy plant. The pots were sufficiently watered as required.

2.2. Experimental design and the analysis of growth, yield and quality Foliar spray of different concentrations of ISA was applied at 15 days interval when the plants were at 2e3 true leaf stage. Totally, five sprays of ISA were applied to the crop using a hand sprayer. The control plants were sprayed with double distilled water only. The applied treatments were consisted of 0, 20, 40, 60, 80 and 100 mg L
1 of ISA. Different aqueous concentrations of ISA were finally prepared using double distilled water for the spray treatments on plants. Plants were sampled for all physiological and biochemical parameters carried out at 120 and 150 DAP. All yield and quality attributes were also measured on the same dates. At sampling (120 and 150 DAP), five plants of each treatment 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 blotting paper. Then, the plant fresh and dry weights were measured using an electronic balance.

2.3. Irradiation procedure and GPC (Gel permeation Chromatography) analysis Sodium alginate was purchased from SigmaeAldrich, USA. The samples of sodium alginate were irradiated (Cobalt-60, GC-5000) in a Gamma Radiation Chamber (BRIT, Bhabha Atomic Research Centre, Mumbai, India) to 520 kGy gamma radiation dose at a dose rate of 2.4 kGy h
1. GPC of sodium alginate samples were done on Hitachi EMerck HPLC/GPC system using RI detector. The average molecular weight of the un-irradiated sodium alginate samples were estimated to be about 6,95,131. The detailed methodology has been described in our previous study (Naeem et al., 2012b).

2.4.

The pot experiment was conducted in naturally illuminated environmental conditions of the net house at the Botany

Scanning electron microscopy (SEM) analysis

The morphology structure of the sodium alginate samples were examined using the Scanning Electron Microscope (Philips XL 30 ESEM, Jeol, Japan). The samples were coated

Please cite this article in press as: Naeem, M., et al., Radiolytically degraded sodium alginate enhances plant growth, physiological activities and alkaloids production in Catharanthus roseus L., Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.07.005

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with gold. Scanning electron microscopy and elemental analysis was performed for un-irradiated as well as irradiated sodium alginate samples (Fig. 1a & b) at University Sophisticated Instruments Facility, Aligarh Muslim University, Aligarh, India.

2.5.

Determination of growth attributes

The growth attributes viz. number of leaves per plant, average leaf-area and fresh and dry weight of plants were determined at 120 and 150 DAP. Plants from each treatment were uprooted carefully followed by recording the number of leaves and fresh weight per plant. Plants were washed and then dried in a hot-air oven at 80  C for 24 h prior for measuring the plant dry weight. Only 10% of the randomly selected leaves of each sample (consisting of five plants) were used to determine the leaf area using graph paper sheet (Watson, 1958). The average area per leaf, thus determined, was multiplied with the total number of leaves to measure the total leaf area per plant.

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was recorded at 662, 645 and 470 nm to determine chlorophyll a, chlorophyll b and total carotenoid content, respectively, using a spectrophotometer (Shimadzu UV-1700, Tokoyo, Japan). Total chlorophyll content was assessed by adding together the content of chlorophyll a and b. The content of each photosynthetic pigment was expressed as mg g
1 leaf FW.

2.6.3.

Activity of nitrate reductase (NR)

2.6.

Determination of physiological attributes

Activity of nitrate reductase (E.C. 1.6.6.1) was estimated in the youngest fully developed leaves by the intact tissue assay method developed by Jaworski (1971). Fresh chopped leaves, weighing 200 mg, were transferred to plastic vials. Each vial contained 2.5 mL phosphate buffer (pH 7.5), 0.5 mL potassium nitrate solution and 2.5 mL of 5% isopropanol. The vials, containing the reaction mixture, were incubated for two hours at 30  C. After incubation, 1% sulphanilamide and 0.02% N-(1Naphthyl) ethylenediamine dihydrochloride (NED-HCL) were added. The test tubes were kept for 20 min at room temperature for maximum color development. The OD of the content was recorded at 540 nm. Activity of NR was expressed as
1 FW h
1. nM NO
2 g

2.6.1.

Net photosynthetic rate and stomatal conductance

2.6.4.

Net photosynthetic rate and stomatal conductance were determined using intact leaves (the randomly selected youngest fully expanded leaves) from the five replicates. Measurements were made on sunny days at 1100 h using an Infra Red Gas Analyzer (IRGA, Li-Cor 6400 Portable Photosynthesis System Lincoln, Nebraska, USA) at 120 and 150 DAP.

2.6.2.

Total contents of chlorophyll and carotenoid

Total content of chlorophyll and carotenoid in the leaves was estimated using the method of Lichtenthaler and Buschmann (2001). The fresh tissue from the interveinal area of leaf was grinded with 100% acetone using mortarpestle. The optical density (OD) of the pigment solution

Activity of carbonic anhydrase (CA)

The activity of carbonic anhydrase (E.C. 4.2.1.1) was measured in the fresh leaves selected randomly, using the method described by Dwivedi and Randhawa (1974). Two hundred mg of the leaves (chopped leaf-pieces) were transferred to Petri plates. The leaf pieces were dipped in 10 mL of 0.2 M cystein hydrochloride solution for 20 min at 4  C. The solution adhering to leaf pieces was removed with the help of a blotting paper, followed by immediately transferring them to a test tube containing 4 mL of phosphate buffer (pH 6.8). To it, 4 mL of 0.2 M sodium bicarbonate solution and 0.2 mL of 0.022% bromothymol blue were added. The reaction mixture was titrated against 0.05 N HCl using methyl red as indicator. The enzyme activity was expressed as mM CO2 kg
1 leaf FW s
1.

Fig. 1 e SEM of un-irradiated sodium alginate (a) and SEM of irradiated sodium alginate (b).

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2.6.5.

Activity of tryptophan decarboxylase (TDC)

The activity of tryptophan decarboxylase (E.C. 4.1.1.28) was carried out according to the method of Islas, Loyola-Vargas, and Miranda-Ham (1994). Frozen transformed roots (1000 mg) were pulverized in a cold mortar to fine powder and homogenized with 1.25 mL of 0.1 M HEPES (pH 7.5), containing 3 mM, dithiotreitol, 5 mM EDTA and 200 mg of polyvinylpolypyrrolidone. The extract was filtered through four layer of cheesecloth and then it was centrifuged at 18,000  g for 30 min. The resulting supernatant was used as a source of enzyme for the TDC activity. The protein content of the enzyme extract was determined as described by Peterson (1977), using albumin as standard. The activity of TDC enzyme was expressed as n mol mint
1 mg
1 protein.

2.6.6.

Yield and quality attributes

Leaf-yield was recorded by weighing all plant leaves using an electronic balance. Herbage-yield was measured weighing the total plant biomass excluding roots.

2.6.6.1. Total alkaloid content in leaves and roots. Total alkaloid content was estimated in leaves and roots as described by Afaq, Tajuddin, and Siddiqui (1994). The leaves and roots were dried in a hot-air oven at 80  C for twenty four hours. The samples were powdered and passed through a 72 mesh. Five hundred mg of the powdered sample was taken in a 100 mL round bottom reflux flask. To it, a known volume of ethyl alcohol was added. Then the mixture was refluxed for 6 h. Thereafter, it was filtered, followed by adding 50 mL of dilute HCl, and then the filtrate was transferred to a separating funnel, to which 50 mL of diethyl ether was added. The mixture was shaken for 15e20 min. The upper diethyl ether layer was discarded and the lower water layer was decanted to a beaker. The content, collected in beaker, was made slightly basic by adding ammonia solution. The decanted content was again transferred into a separating funnel with 50 mL of diethyl ether, followed by decanting the content again. To the final decant, anhydrous sodium carbonate was added. Then, the mixture was decanted in a pre-weighed dry porcelain dish, followed by evaporating the content till dryness; lastly, the dried content was weighed. Total alkaloid content in leaves/ roots was calculated using the following formula: Total alkaloid contentð%Þ ¼

WA
WE  100 WR

where, WE ¼ Weight of empty porcelain dish (g) WA ¼ Weight of porcelain dish after evaporation (g) WR ¼ Weight of the dried powder (g)

2.6.6.2. Content of vincristine, vinblastine and vindoline alkaloids 2.6.6.2.1. Plant material and sample extraction. Preparation of sample extraction and the chromatographic condition of high-performance liquid chromatography (HPLC) instrument were accomplished through the method of Uniyal, Bala, Mathur, and Kulkarni (2001). Freshly harvested leaves were oven dried at 80  C for 24 h and then grinded to fine powder.

A volume (30 mL) of 90% ethanol was added to 5 g of leaf powder; it was left over night and then filtered. The residue was again extracted with 90% ethanol (3  30 mL) at room temperature (27  C), and the pooled alcoholic extract was filtered and concentrated in vacuo at 40  C. The dried residue was re-dissolved in ethanol (10 mL), diluted with water (10 mL) and then acidified with 3% hydrochloric acid (10 mL). This was then extracted with hexane (3  30 mL), the hexane extract was discarded and the aqueous portion of the content was cooled to 10  C; it was basified with ammonium hydroxide to pH 8.5 and then was extracted with chloroform (3  30 mL). The combined chloroform extract was washed with water, evaporated to dryness and re-dissolved in 1 mL of chloroform. After that, it was passed through a silica Sep-Pak cartridge (Waters), pre-saturated with chloroform, and it was washed successively with 5 mL each of chloroform and chloroformmethanol mixture (9:1, v/v) and dried over anhydrous sodium sulfate before being evaporated to dryness. The residue obtained was dried to constant weight in order to determine the total alkaloid content. An aliquot (10 mg) of the crude alkaloid was dissolved in 1 mL of methanol, and 10 mL of it was subjected to HPLC analysis.

2.6.6.2.2. HPLC procedure. Chromatographic analysis was carried out using HPLC (Shimadzu, Japan; LC-10A). Solvents were filtered by using a millipore system and analysis was performed on a m Bondapak C18 reversed-phase column, 10 mm (30 cm  3.9 mm I.D.). A constant flow rate of 0.6 mL min
1 was used during analysis. The composition of mobile phase was optimized by using acetonitrile: 0.1 M phosphate buffer: glacial acetic acid (38:62:0.3); pH 4.14, flow rate 0.6 mL min
1, column temperature 26  C, detector wave length 254 nm. For standard, stock solutions of vinblastine, vincristine and vindoline were prepared dissolving 1 mg of each in 1 mL of methanol. The solutions were subjected and the retention time (Rt) for vinblastine (Rt-5 min), vincristine (Rt-4 min) and vindoline (Rt-10 min) were noticed. 2.7.

Statistical analysis

The data were analyzed statistically using SPSS-17 statistical software (SPSS Inc., Chicago, IL, USA) according to simple randomized design. Means were compared using Duncan's Multiple Range Test (DMRT) at p < 0.05. Least significant difference (LSD) was calculated and also employed in the Tables and Figures.

3.

Results

The data shown in this study indicates that all the ISA treatments had strong promotive effect on all the growth, yield and quality attributes and physiological and biochemical parameters. Of the six ISA concentrations, 80 mg L
1 proved to be the best compared to other concentrations. The present work revealed that ISA, applied as leaf-sprays at 20e100 mg L
1 concentrations, improved the plant growth attributes, physiological and biochemical parameters, and yield and content of alkaloids of C. roseus significantly. Application of 100 mg L
1 of ISA did not further improve the growth attributes, but it

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significantly enhanced the above studied attributes in comparison to the control (Tables 1e5; Figs. 2e7).

3.1.

Growth attributes

Foliar application of different concentrations of ISA significantly (p  0.05) improved growth attributes of C. roseus at 120 and 150 DAP studied in sequence. There was a progressive increase in values with the increase in ISA concentration up to 100 mg L
1 (Tables 1 and 2). The maximum values of growth characteristics were attained at 120 DAP with 80 mg L
1 of ISA. As compared to the control, application of 80 mg L
1 of ISA increased the number of leaves by 15.2 and 22.9%, average leaf-area by 12.3 and 12.8%, plant fresh weight by 34.5 and 40.1%, and plant dry weight by 38.1 and 43.2% at 120 and 150 DAP, respectively (Tables 1 and 2).

3.2.

Physiological and biochemical attributes

The application of depolymerized form of sodium alginate significantly enhanced the photosynthetic parameters (net photosynthetic rate and stomatal conductance) in the present study. A significant increase in net photosynthetic rate and stomatal conductance was recorded at 120 and 150 DAP with the application ISA at 80 mg L
1. Compared to the control, application of the 80 mg L
1 of ISA accelerated the rate of net photosynthetic rate by 17.0 and 20.6% at 120 and 150 DAP, respectively (Fig. 2A). 80 mg L
1 of ISA also increased the stomatal conductance significantly, exceeding the control by 11.8 and 14.5% at 100 and 120 DAP, respectively (Fig. 2B). Depolymerized sodium alginate significantly increased the photosynthetic pigments (total chlorophyll and carotenoid content) in the treated plants as depicted in Table 3, with 80 mg L
1 of ISA proving the best. As compared to the control, application of ISA at 80 mg L
1 ISA enhanced the total chlorophyll content by 23.5 and 23.8%, respectively at 120 and 150 DAP (Table 3). Total carotenoid content were significantly increased with the increasing levels of ISA (Table 3). 80 mg L
1 of ISA proved the best, improving the carotenoid content by 8.4 and 8.8%, at 120 and 150 DAP, respectively, as compared to the control (Table 3).

Table 1 e Effect of six foliar concentrations of irradiated sodium alginate (ISA) (0, 20, 40, 60, 80 and 100 mg L¡1) on number of leaves, average leaf area and leaf yield per plant of Catharanthus roseus L. at 120 and 150 DAP. Means within a column followed by the same letter(s) are not significantly different (p ≤ 0.05). Treatments (mg L
1)

ISA ISA ISA ISA ISA ISA

0 20 40 60 80 100

LSD at 5%

Number of leaves per plant

Average leaf area (cm2)

120 DAP

150 DAP

120 DAP

150 DAP

204.8f 214.0e 220.5d 224.8c 236.0a 228.5b

242.0f 253.5e 275.0d 280.5c 297.3a 289.8b

10.10e 10.52d 10.78c 11.10b 11.34a 11.16b

12.30e 12.82d 13.19c 13.50b 13.86a 13.45b

4.30

4.82

0.15

0.16

Table 2 e Effect of six foliar concentrations of irradiated sodium alginate (ISA) (0, 20, 40, 60, 80 and 100 mg L¡1) on fresh and dry weights per plant of Catharanthus roseus L. at 120 and 150 DAP. Means within a column followed by the same letter(s) are not significantly different (p ≤ 0.05). Treatments (mg L
1)

ISA ISA ISA ISA ISA ISA

0 20 40 60 80 100

LSD at 5%

Fresh weight per plant (g)

Dry weight per plant (g)

120 DAP

150 DAP

120 DAP

150 DAP

62.14f 65.70e 68.46d 78.19c 83.57a 81.10b

70.54f 72.16e 78.20d 82.36c 98.84a 96.70b

15.24f 15.90e 16.72d 18.30c 21.05a 20.70b

16.85f 18.10e 18.98d 20.45c 24.18a 22.70b

2.35

2.10

0.16

0.18

Table 3 e Effect of six foliar concentrations of irradiated sodium alginate (ISA) (0, 20, 40, 60, 80 and 100 mg L¡1) on total chlorophyll content and total carotenoid content in fresh leaves of Catharanthus roseus L. 120 and 150 DAP. Means within a column followed by the same letter(s) are not significantly different (p ≤ 0.05). Treatments (mg L
1)

ISA ISA ISA ISA ISA ISA

Total chlorophyll content (mg g
1 FW)

Total carotenoid content (mg g
1 FW)

120 DAP

120 DAP

f

0 20 40 60 80 100

LSD at 5%

150 DAP f

c

150 DAP

1.020 1.130e 1.145d 1.170c 1.260a 1.235b

1.138 1.175e 1.350d 1.372c 1.409a 1.365b

0.190 0.193c 0.200b 0.202b 0.206a 0.202b

0.214c 0.216c 0.221b 0.225b 0.232a 0.226b

0.034

0.042

0.003

0.005

Nitrate reductase (NR) activity was significantly affected by the application of ISA. Foliar spray of 80 mg L
1 ISA resulted in the highest values of NR activity at both the growth stages. The activity of the enzyme increased to the maximum extent at 150 DAP (Table 4). As compared to the control, 80 mg L
1 of ISA resulted in 16.1 and 19.3% increase in NR activity at 120 and 150 DAP, respectively (Table 4). Carbonic anhydrase activity was positively affected by the application of ISA. Foliar application of 80 mg L
1 ISA exhibited the highest values of CA activity at the both the growth stages. The activity of the enzyme increased to the maximum extent at 150 DAP (Table 4). Compared to the control (water spray treatment), 80 mg L
1 of ISA resulted in 16.2 and 17.7% increase in CA activity, at 120 and 150 DAP, respectively (Table 4). Activity of tryptophan decarboxylase (TDC) was significantly enhanced by the application of ISA at both the stages. The activity of the enzyme was maximum at 150 DAP. The ISA concentration of 80 mg L
1 proved the best, improving the activity of TDC enzyme by 17.4 and 23. 6%, over the control at 120 and 150 DAP, respectively (Table 4).

3.3.

Yield and quality attributes

Application of ISA at 80 mg L
1 surpassed the control regarding leaf-yield and herbage-yield by 25.3 and 30.2% and

Please cite this article in press as: Naeem, M., et al., Radiolytically degraded sodium alginate enhances plant growth, physiological activities and alkaloids production in Catharanthus roseus L., Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.07.005

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Table 4 e Effect of six foliar concentrations of irradiated sodium alginate (ISA) (0, 20, 40, 60, 80 and 100 mg L¡1) on activities of nitrate reductase (NR), carbonic anhydrase (CA) and tryptophan decarboxylase (TDC) of Catharanthus roseus L. 120 and 150 DAP. Means within a column followed by the same letter(s) are not significantly different (p ≤ 0.05). Treatments (mg L
1)

ISA ISA ISA ISA ISA ISA

Nitrate reductase activity
1 [nM NO
(FW) h
1] 2 g

Carbonic anhydrase activity [mol (CO2) kg
1 (FW) s
1]

120 DAP

120 DAP

120 DAP

150 DAP

221.0f 229.2e 241.4d 248.2c 256.5a 249.5b

230.6f 240.3e 250.5d 265.0c 275.0a 260.9b

5.31e 5.38e 5.63d 5.80c 6.17a 5.95b

5.35f 5.46e 5.58d 5.72c 6.29a 5.84b

0.14

0.10

0 20 40 60 80 100

LSD at 5%

5.0

4.5

Table 5 e Effect of six foliar concentrations of irradiated sodium alginate (ISA) (0, 20, 40, 60, 80 and 100 mg L¡1) on leaf-yield and herbage-yield per plant of Catharanthus roseus L. at 120 and 150 DAP. Means within a column followed by the same letter(s) are not significantly different (p ≤ 0.05). Treatments (mg L
1)

Leaf-yield per plant (g) 120 DAP

ISA ISA ISA ISA ISA ISA

150 DAP

f

0 20 40 60 80 100

LSD at 5%

Herbage-yield per plant (g) 120 DAP

f

150 DAP

f

52.40 56.50e 60.86d 62.80c 65.68a 63.90b

56.24 59.12e 64.48d 68.26c 73.24a 70.74b

57.54 60.70e 64.19d 67.40c 74.43a 70.76b

60.54f 62.16e 65.36d 70.24c 81.33a 76.20b

4.30

4.82

0.15

0.16

by 29.4 and 34.3% at 120 and 150 DAP, respectively (Table 5). Application of ISA increased the content and yield of alkaloids when compared to the un-treated plants. As presented in Fig. 2, the foliar spray of ISA at 80 mg L
1 resulted in the maximum content and yield of leaf alkaloid, outshining the

Tryptophan decarboxylase activity (nm min
1 mg
1 protein) 120 DAP

150 DAP

10.2 10.0 10.6 11.2 12.0 10.5

11.5 11.3 11.8 12.6 14.2 14.0

0.002

0.002

control by 12.1 and 14.2% in alkaloids content and by 22.9 and 29.5% in alkaloids yield at 120 and 150 DAP (Fig. 3A and B). Application of ISA also increased the content and yield of root alkaloids at both the growth stages. ISA at 80 mg L
1 exceeded the control by 7.14 and 7.36% in root alkaloids content and by 58.1 and by 63.9% in root alkaloids yield at 120 and 150 DAP, respectively (Fig. 4A and B). As compared to the control, there was observed no increase in the active components particularly in the content of vinblastine and vincristine, when ISA concentrations were applied to the crop foliage (Figs. 5A and 6A). However, the yield of vinblastine and vincristine was significantly accelerated by the application of ISA. Foliar spray of ISA at 80 mg L
1 considerably increased the yield of vinblastine and vincristine, exceeding the control by 66.7 and 71.4% regarding vinblastine and by 67.6 and 75.6% regarding vincristine at 120 and 150 DAP, respectively (Figs. 5B and 6B). Foliar spray of ISA at 80 mg L
1 increased the content and yield of vindoline too, surpassing the control by 18.9 and 20.8% in vindoline content and by 81.8 and 87.2% in vindoline yield at 120 and 150 DAP, respectively (Fig. 7A and B).

20

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Stomatal conductance (m mol m-2 s-1)

Net photosynthetic rate [(μ mol(CO2) m-2 s-1)]

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Irradiated sodium alginate concentrations (mg L-1)

Fig. 2 e Effect of six concentrations of irradiated sodium alginate (0, 20, 40, 60, 80 and 100 mg L¡1) on net photosynthetic rate (A), stomatal conductance (B) of Catharanthus roseus L. studied at 120 and 150 DAP. Means within a column followed by the same letter(s) are not significantly different (p ≤ 0.05). Bars (┬) represent the LSD at 5%. Please cite this article in press as: Naeem, M., et al., Radiolytically degraded sodium alginate enhances plant growth, physiological activities and alkaloids production in Catharanthus roseus L., Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.07.005

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Leaf alkaloid content (%)

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Leaf alkaloid yield per plant (μg)

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Irradiated sodium alginate concentrations (mg L-1)

Irradiated sodium alginate concentrations (mg L-1)

Fig. 3 e Effect of six concentrations of irradiated sodium alginate (0, 20, 40, 60, 80 and 100 mg L¡1) on leaf alkaloid content (A) and leaf alkaloid yield (B) of Catharanthus roseus L. studied at 120 and 150 DAP. Means within a column followed by the same letter(s) are not significantly different (p ≤ 0.05). Bars (┬) represent the LSD at 5%.

Discussion

Gel permeation chromatography (GPC) of the un-irradiated and irradiated sodium alginate samples were carried out as reported previously (Naeem et al., 2012b). GPC study revealed the formation of low molecular weight fractions in irradiated sodium alginate sample, showing oligomers of 5,95,000 molecular weight, which might be responsible for plant growth promotion in this study. Foliar application of ISA at 80 mg L
1 proved most effective, resulting in the highest values of growth attributes, herbage yield and the content and yield of alkaloids of C. roseus. In this study, oligomers derived from sodium alginate behaved like endogenous growth elicitor and functioned as signal molecules to trigger the synthesis of different enzymes and activate various plant

Root alkaloid content (%)

2.0

A

120 DAP 150 DAP

c

d

responses, exploiting the gene expression as observed in the case of Cd stress conditions (Ma, Li, Bu, & Li, 2010). Confirming our results, the gamma-degraded natural polysaccharides, viz. sodium alginate, carrageenan and chitosan, applied in the form of foliar sprays, have been reported to enhance plant growth in general (Aftab et al., 2011, 2013, 2014; Hien et al., 2000; Hu et al., 2004; Naeem et al., 2012a, 2012b; Khan et al., 2011; Kume et al., 2002; Luan et al., 2003; Relleve et al., 2000; Sarfaraz et al., 2011). Leaf-applied ISA also enhanced the leaf-area in this study (Table 1), which might provide increased opportunity for light harvesting, leading to the accumulation of enhanced plant dry matter compared to the control (Table 2). In the present study, application of radiation-derived oligosaccharides of sodium alginate resulted in significant improvement in plant growth attributes. The present results

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Root alkaloid yield per plant (μg)

4.

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Irradiated sodium alginate concentrations (mg L-1)

Fig. 4 e Effect of six concentrations of irradiated sodium alginate (0, 20, 40, 60, 80 and 100 mg L¡1) on root alkaloid content (A) and root alkaloid yield (B) of Catharanthus roseus L. studied at 120 and 150 DAP. Means within a column followed by the same letter(s) are not significantly different (p ≤ 0.05). Bars (┬) represent the LSD at 5%. Please cite this article in press as: Naeem, M., et al., Radiolytically degraded sodium alginate enhances plant growth, physiological activities and alkaloids production in Catharanthus roseus L., Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.07.005

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0.025

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a

a Vinblastine content (%)

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0.015

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Vinblastine yield per plant (μg)

120 DAP 150 DAP

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Irradiated sodium alginate concentrations (mg L-1)

Irradiated sodium alginate concentrations (mg L-1)

Fig. 5 e Effect of six concentrations of irradiated sodium alginate (0, 20, 40, 60, 80 and 100 mg L¡1) on vinblastine content (A) and vinblastine yield (B) of Catharanthus roseus L. studied at 120 and 150 DAP. Means within a column followed by the same letter(s) are not significantly different (p ≤ 0.05). Bars (┬) represent the LSD at 5%.

are in conformity with the earlier findings that report the positive effect of degraded oligomers of polysaccharides on the growth and development of various crops including mint (Mentha arvensis L.) (Naeem et al., 2012b, 2014), artemisia (Artemisia annua L.) (Aftab et al., 2011), opium (Papaver somniferum L.) (Khan et al., 2011), fennel (Foeniculum valgare Mill.) (Sarfaraz et al., 2011), red amaranth (Amaranthus cruentus L.) (Mollah et al., 2009), barley (Hordeum vulgare L.) (Tomoda, Umemura, & Adachi, 1994) and potato (Solanum tuberosum L.) (Relleve et al., 2005). It is considered that there is specific structural and size requirement of the oligosaccharides for inducing the physiological processes in plants (Darvill et al., 1992). However, the phenomenon by which the radiolytically degraded polysaccharides stimulate the growth and development processes in plants still needs further investigations.

In this regard, Relleve et al. (2000) suggested that a certain molecular weight of oligomers of degraded carrageenan was required to get optimum growth effects on rice plants. In this direction, the biological activity of the irradiated k-carrageenan (generated by different gamma-ray doses) has been studied in potato tissue culture (Relleve et al., 2000). Seemingly, the ISA-mediated increase in leaf area (Table 1) could result in trapping more sunlight and utilizing additional CO2 to increase the rate of photosynthesis in comparison to the control. Also, the enhanced photosynthetic rate in this study could have resulted due to ISA-mediated augmentation in the leaf-chlorophyll content (Table 3). The increase in photosynthetic rate due to application of irradiated sodium alginate and k-carrageenan has been studied previously regarding several medicinal plants (Aftab et al., 2011, 2013,

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Vincristine content (%)

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0.002 2

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Fig. 6 e Effect of six concentrations of irradiated sodium alginate (0, 20, 40, 60, 80 and 100 mg L¡1) on vincristine content (A) and vincristine yield (B) of Catharanthus roseus L. studied at 120 and 150 DAP. Means within a column followed by the same letter(s) are not significantly different (p ≤ 0.05). Bars (┬) represent the LSD at 5%. Please cite this article in press as: Naeem, M., et al., Radiolytically degraded sodium alginate enhances plant growth, physiological activities and alkaloids production in Catharanthus roseus L., Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.07.005

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Vindoline content (%)

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Vindoline yield per plant (μg)

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Irradiated sodium alginate concentrations (mg L-1)

Fig. 7 e Effect of six concentrations of irradiated sodium alginate (0, 20, 40, 60, 80 and 100 mg L¡1) on vindoline content (A) and vindoline yield (B) of Catharanthus roseus L. studied at 120 and 150 DAP. Means within a column followed by the same letter(s) are not significantly different (p ≤ 0.05). Bars (┬) represent the LSD at 5%.

2014; Khan et al., 2011; Naeem et al., 2012a, 2012b, 2015; Sarfaraz et al., 2011). In support of our study, the ISA application has also been reported to induce cell signaling, leading to stimulation of various physiological processes in various plants, including ISA-mediated improved content of photosynthetic pigments and enhanced net photosynthetic rate (Farmer, Thomas, Michael, & Clarence, 1991). Due to growth promoting effect of ISA, application of ISA could result in improvement in the growth of plant root and augmentation in shoot elongation, which led to enhancement in plant productivity and improvement in physiological parameters (ElRehim, 2006; Naeem et al., 2012a, 2014). This study also records a significant increase in the activities of NR and CA as a result of ISA application (Table 4). In this regard, our findings support those studies that claim the synthesis of certain enzymes in the tissue culture of certain plants as a result of application of irradiated polysaccharides (Akimoto, Aoyagi, & Tanaka, 1999; Patier et al., 1995). The ISA-mediated improvement in physiological parameters (Tables 3 and 4; Fig. 2) might also justify the increase in plant fresh and dry matter contents in C. roseus plants in the present study (Table 2). Leaf-applied ISA also increased the TDC activity positively at both the stages (Table 3). To be noted, it is documented that TDC enzyme played a role in enhancing the biosynthesis of terpenoid indole-alkaloids (TIA) (Facchini et al., 2000; Molchan, Romashko, & Yurin, 2012). The ISA-stimulated increase in the growth and other physiological parameters studied might possibly culminate in maximization of the leaf-yield and herbage-yield of the plants (Table 5). Expectedly, the improved dry matter production (Table 2) and herbage-yield (Table 5) in ISA-treated plants might result due to enhanced water and nutrient uptake from the soil, followed by smooth translocation of photosynthates and other metabolites to the sinks as argued by Khan et al. (2007). As compared to water spray treatment (control), ISA application was also effective in increasing the total alkaloids

content. The increase in alkaloids content (Fig. 3) owing to application of ISA might be ascribed to the increase in the leaf nitrogen content that might have promoted amino acid synthesis leading to the improved alkaloid content in the leaves as suggested by Naeem et al. (2011). In this regard, previous studies have shown that a range of concentrations of ISA depend upon the source and unit (kGy) of irradiation for a particular plant (Relleve et al., 2005). To support our results, the growth promoting effect of ISA on alkaloids production in the case of opium poppy and C. roseus has earlier been reported by Khan et al. (2011) and Idrees et al. (2011), respectively. As compared to the control, there was observed no increase in the content of vinblastine and vincristine (Figs. 5A and 6A). However, ISA increased the overall production (yield) of vinblastine and vincristine (Fig. 5B and 6B) due to increased production of plant biomass (Table 5). Apart from this, the content and yield of vindoline was also increased in ISA-treated plants (Fig. 4A and B). Dimeric compounds such as vincristine and vinblastine are produced by the biochemical coupling of smaller indole alkaloids such as vindoline and catharanthine. Categorically, the ISA behaved like PGRs, the exogenous application of which might evoke the intrinsic genetic potential of the plant that may result in improved enzyme activities, uptake of nutrients, enhanced photosynthesis and improved translocation of photosynthates and other metabolites to the reproductive parts (Naeem et al., 2014).

5.

Conclusion

The optimum leaf applied-concentration (80 mg L
1) of irradiated sodium alginate could be employed to increase the growth attributes, physiological activities, herbage yield, and content and yield of alkaloids of Catharanthus roseus. Optimized concentration of ISA considerably increased the yield of

Please cite this article in press as: Naeem, M., et al., Radiolytically degraded sodium alginate enhances plant growth, physiological activities and alkaloids production in Catharanthus roseus L., Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.07.005

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vincristine, vinblastine and vindoline in C. roseus plants. We hope that the application of radiation-processed sodium alginate might be applied as plant growth promoter in future to achieve the desired quality of several medicinal and aromatic plants. However, further investigations are required to comprehend the mechanism and mode of action of sodium alginate-derived oligomers with regard to yield and quality of medicinal and the agricultural crops.

Acknowledgment The financial support by the Council of Science and Technology, Lucknow, UP, India, to the awardee of Young Scientist (Dr. M. Naeem) (Project no. CST/1235) is gratefully acknowledged.

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Please cite this article in press as: Naeem, M., et al., Radiolytically degraded sodium alginate enhances plant growth, physiological activities and alkaloids production in Catharanthus roseus L., Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.07.005