International Journal of Agricultural Science and Research (IJASR) ISSN(P): 2250-0057; ISSN(E): 2321-0087 Vol.

4, Issue 1, Feb 2014, 113-122 TJPRC Pvt. Ltd.

ENHANCING THE GROWTH, YIELD AND PRODUCTION OF ESSENTIAL OIL AND CITRAL IN LEMONGRASS BY THE APPLICATION OF TRIACONTANOL
ZEBA H. KHAN, F. MOHAMMAD & M MASROOR A. KHAN Department of Botany, Plant Physiology Section, Aligarh Muslim University, Aligarh

ABSTRACT
Cymbopogon flexuosus (Steud.) Wats. (lemongrass) is an important source of citral that is used for the preparation of ionones and artificial perfumes. Keeping in mind, the importance of this plant, a pot experiment was carried out according to a factorial randomized design to study the effect of four levels of foliar spray of triacontanol (TRIA) on the performance of two varieties (Krishna and Neema) of lemongrass. Four levels of spray (0, 10-7, 10-6 and 10-5 M TRIA) were applied each at 75, 90 and 105 days after sowing (DAS). The foliar application of 10-6 M TRIA proved best for all characteristics studied, including essential oil and citral content particularly of Krishna. It increased the essential oil content and yield by 42.0, 110.8% respectively and citral content and yield by 10.8 and 134.1% respectively in Krishna and Neema at 120 DAS. However, higher concentration of TRIA (10-5 M) proved deleterious.

KEYWORDS: Triacontanol, Cymbopogon Flexuosus, Essential Oil, Citral Content, Plant Growth Regulator INTRODUCTION
Lemongrass is cultivated for its essential oil which is used in perfumery, medicinal, cosmetic and pharmaceutical industries (Singh et al., 1996; Akhila, 2006). The essential oil has insect repellent and anti-cancerous properties and is also used in aromatherapy (Fatima, 2002; Sharma et al., 2009; Choudhury et al., 2012). Except from these medicinal properties, this plant has soil binding capacity (Farooqi et al., 2000). Citral, an important constituent of lemongrass oil, is most widely used in aroma chemicals in the world, as it is the starting material for the preparation of important ionones: -ionone and -ionone. The former is used as a flavor, cosmetics and perfumes, and the latter for synthesis of vitamin A (Pengelly, 2004). Plant growth regulators have worldwide application for optimizing the yield by modifying the growth and development of plants (Hernandez, 1997; Jaleel, 2006; 2007). Out of various PGRs, TRIA (a long fatty primary alcohol chain, CH3 (CH2)28CH2OH), has proved to be potent plant growth promoting substance for many agricultural and horticultural crops (Ries, 1991; Idrees et al., 2010; Naeem et al., 2010). TRIA has been reported to increase growth and yield (Aftab et al., 2010; Idrees et al., 2010), dry matter accumulation of leaves (Borowaski, 1992), photosynthetic pigment content (Kumaravelu et al., 2000) and CO2 assimilation rate (Blamowski et al., 1998) among others. Keeping the medicinal properties of lemongrass and the role of TRIA in mind, the present experiment was designed to find out whether the exogenous application of TRIA could increase the growth, yield and essential oil component of two lemongrass varieties namely Krishna and Neema.

MATERIALS AND METHODS

Plant Material and Growth Conditions The slips of Krishna and Neema were procured from the Central Institute of Medicinal and Aromatic Plants, Lucknow, India. They were surface-sterilized with 0.2% HgCl2 solution for 5 min with frequent shaking, followed by

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repeated washings with deionized water to remove the adhered HgCl2 solution to the surface of plant-slips. These surface sterilized plant-slips were then grown in earthen pots (25 cm diameter 25 cm height) containing 5 kg of homogenous mixture of soil and organic manure in the ratio of 4:1. A basal fertilizer dose consisting of 15 kg N + 15 kg P2O5 + 15 kg K2O/ha was given uniformly prior to sowing. The soil was maintained at proper moisture to ensure better growth of the plants. Experimental Setup and Growth Analysis The experiment was conducted in the naturally-illuminated environmental conditions in a net house of the Department of Botany, Aligarh Muslim University, Aligarh (27 53N latitude, 78 51E longitude, and 187.45 m altitude). The pots were arranged according to a factorial randomized design. Foliar treatments comprised one variant and varieties, the other. The foliar treatments comprised four TRIA concentrations (0, 107, 106 and 105 M). Varieties Krishna and Neema each treatment was replicated four times and each replicate-pot contained one plants. Physical and chemical properties of the soil used in the experiment were determined according to Jackson, 1973. The soil was sandy loam with pH 7.8, organic matter 0.35% and electrical conductivity 0.5 mhos cm-1. The initial content of soil N, P2O5 and K2O was 98.4, 7.1 and 141.8 mg kg-1 soils respectively. Plants were sprayed three times with TRIA concentration. The first spray was carried out at 75 DAS; the second at 90 and third spray at 105 DAS. Control plants were sprayed with deionized water. Performance of the crop was assessed in terms of growth attributes, physiological and biochemical parameters and yield and quality characters. Growth, physiological and biochemical attributes were determined at 120 DAS and yield and quality characters at 150 DAS. Plant samples from were washed carefully with tap water to remove all adhering soil particles, followed by surface drying thereafter using blotting paper. Later, the growth attributes viz. shoot length per plant, root length per plant, leaves per plant, tillers per plant and shoot fresh weight per plant and root fresh weight per plant were recorded. Plant samples were dried at 80C for 24 h using a hot-air oven and then weighed to obtain shoot and root dry weight on per plant basis.

PHYSIOLOGICAL AND BIOCHEMICAL ANALYSES

Estimation of Total Chlorophyll and Carotenoid Contents Total chlorophyll and carotenoid contents in fresh leaves were estimated using the method of Lichtenthaler and Buschmann, (2001). The fresh leaf tissue was grinded using mortar-pestle containing 80% acetone. The optical density (OD) of the pigment-extract was recorded at 662 and 645 nm for estimation of chlorophyll a and b, respectively and at 470 nm (for carotenoid content) using a spectrophotometer (Shimadzu UV-1700, Tokyo, Japan). The photosynthetic pigments were expressed as mg g-1 FW. Nitrate Reductase (Nr) Activity The NR (E.C. 1.6.6.1) activity was estimated in the intact tissue by the method given by Jaworski, (1971). 200 mg fresh chopped leaves were transferred to a plastic vial. The reaction mixture, containing 2.5 mL phosphate buffer (pH 7.5), 0.5 mL 0.2 M potassium nitrate solution and 2.5 mL 5% isopropanol, was incubated for 2 h in dark at 30 oC. Into a test tube containing 0.4 mL the incubated mixture and, 0.3 mL each of 1% sulphanilamide and 0.02% N-(1-naphthyl) ethylene diaminedihydro chloride (NED-HCl) was added. The test tubes were kept for 20 min at room temperature for maximum colour development. The OD of colored solution was recorded at 540 nm using the spectrophotometer. The NR activity was expressed as nM NO-2 g-1 leaf (FW) h-1.

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Carbonic Anhydrase (CA) Activity The activity of CA (E.C. 4.2.1.1) was determined in the fresh leaves using the method of Dwivedi and Randhawa, (1974). 200 mg fresh leaf tissue was transferred on to Petri plates, followed by incubation of the leaf tissue in 10 mL 0.2 M cystein hydrochloride solution for 20 min at 4C. Thereafter, 4 mL 0.2 M sodium bicarbonate solution and 0.2 mL 0.02% bromothymol blue were added to the homogenate. The reaction mixture was titrated against 0.05 N HCl using methyl red as indicator. CA activity was expressed as M CO 2 kg-1 leaf (FW) s-1. Estimation of Leaf Nutrient Contents Leaf samples were digested for the estimation of leaf-N, -P and -K contents. The leaves were dried in a hot air oven at 80C for 24 h. The dried leaves were grinded using mortar-pestle, followed by passing the content through a 72 mesh to get a fine leaf-powder. 100 mg leaf-powder was carefully transferred in to a digestion tube. To it, 2 mL concentrated sulphuric acid analytical reagent grade was added. The mixture was heated on a temperature-controlled Kjeldahl assembly at 80C for about 2 h and then cooled for about 15 min at room temperature. To it, 0.5 mL of 30% hydrogen peroxide (H2O2) was added drop by drop, followed by heating the content gently. This step was repeated until the content of the digestion tube turned colorless. The peroxide-digested leaf-material, thus prepared, was used to estimate per cent content of N, P and K in the leaves on dry weight basis. Leaf-N content was estimated according to the method of Lindner (1944) with a slight modification made by Novozamsky et al., (1983). A 10 mL aliquot was poured into a 50 mL volumetric flask, followed by additions of 2 mL of 2.5 N sodium hydroxide and 1 mL of 10% sodium silicate solution in order to neutralize the excessive acid and prevent turbidity, respectively. A 5 mL this solution was poured into a 10 mL graduated test tube and then a 0.5 mL of Nesslers reagent was added. Regarding estimation of leaf-N content, the optical density of the solution was recorded at 525 nm, using the spectrophotometer. The method of Fiske and Subba Row, (1925); with a slight modification introduced by Rorison et al., (1993), was used to estimate the leaf-P content. A 5 mL peroxide-digested leaf material was poured into a 10 mL graduated test tube, followed by additions of 1 mL molybdic acid (2.5%) and 0.4 mL 1-amino-2-naphthol-4-sulphonic acid. The content was kept at room temperature for colour development, followed by making the volume up to 10 mL with double distilled water. The OD of the solution was recorded at 620 nm using the spectrophotometer. Leaf K content was determined according to Hald, (1947) with the help of a flame-photometer (AIMIL, C150, India), using a specific filter for K emission spectrum. The peroxide digested material was discharged through an atomizer in the form of a fine mist into a chamber, where it was drawn into the flame. Combustion of the element (K) produced light of a particular wavelength [ max for K = 767 nm (violet)]. The light produced was passed through the appropriate filter to impinge upon a photoelectric cell that activated a galvanometer leading to a digital display of the K content in the leaf as per the emission spectrum constructed. Isolation of Essential Oil 50 g chopped leaf pieces were hydro-distilled to extract the essential oil. The extraction of essential oil was carried out according to Guenther, (1955) by hydro-distillation procedure using a Clevengers appar atus (Borosil, India). The volume of the extracted essential oil was determined accordingly. The oil samples were dehydrated over anhydrous sodium sulphate and stored in the sealed glass vials at 4oC for further analyses by gas liquid chromatography.

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Compositional of Essential Oil The components of essential oil were analyzed by GLC (Nucon 5700, New Delhi, India) equipped with at-1000 stainless steel column, a flame ionization detector and an integrator. N was used as a carrier gas. The flow rates of N, H and O were 0.5, 0.5 and 5 mL s-1, respectively. The GLC apparatus was run with the following specifications: detector temperature 250C; oven temperature 160C; injector temperature 250C; sample size 2 L. The identification of the active constituents was made on the basis of retention time and their quantification was carried out comparing the experimental peaks with those obtained from the reference standards. Statistical Analysis The data were analyzed statistically according to factorial randomized design using SPSS-17 statistical software (SPSS Inc., Chicago, IL, USA). The least significant difference (LSD) was computed for the significant data at P < 0.05.

RESULTS
The favorable effects of the foliar spray of TRIA were noted on most of the growth parameters studied particularly of variety Krishna. The foliar spray of TRIA at 10-6 M proved best. It enhanced parameters of Krishna namely shoot length per plant by 16.1%, root length per plant by 30.1%, leaves per plant by 41.4%, tillers per plant 32.2%, shoot fresh mass per plant by 48.1%, root fresh mass per plant by 47.5%, shoot dry mass per plant by 57.9%, root dry mass per plant by 51.6% over the water-sprayed treatment (Figures 1and 2). All the biochemical parameters were found to be accelerated by the foliar spray of TRIA especially of Krishna. The exogenous application TRIA significantly increased parameters of Krishna namely chlorophyll by 53.6%, carotenoid content by 21.1%, nitrate reductase activity by 15.8%, carbonic anhydrase activity by 27.2%, leaf N content by 19.8%, leaf P content by 23.8% and leaf K content by 10.1% over the water-sprayed treatment (Figures 3 and 4). Among treatments of TRIA, 10-6 M proved best in for enhancing yield and content of essential oil particularly of Krishna (Figure5). In terms of percentage, it increased the essential oil content and oil yield by of Krishna 42.0% and 110.8% respectively over the water sprayed treatment. The foliar application of TRIA also increased the citral content (active constituent of essential oil) and citral yield. Foliar treatment 10-6 M TRIA increased the citral content and citral yield of Krishna by 10.8 % and 134.1% respectively over the water-sprayed treatment (Figure 5).

DISCUSSIONS
Growth Parameters The improvement in growth parameters, namely shoot length per plant, root length per plant, leaves per plant, tillers per plant, shoot fresh mass per plant , root fresh mass per plant, shoot dry mass per plant, root dry mass per plant, by foliar spray of TRIA compared with the water-sprayed treatment is a noteworthy observation (Figures 1 and 2). The increase in growth parameters may be ascribed to TRIA mediated activation of a secondary messenger which moves rapidly throughout the plant resulting in stimulation of growth and uptake of water that cause cell enlargement, cell proliferation, accumulation of amino acids and increase in protein content (Khan et al., 2007; Naeem et al., 2009; Taiz and Zeiger, 2010). The enhancement in these parameters might be culminated into increased shoot and root length, number of leaves and tillers and ultimately fresh and dry mass of plants. These results corroborate the finding of Ries, 1991; Idrees et al., 2010 and Naeem et al., 2010.

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Biochemical Parameters The enhancement in biochemical parameters, namely chlorophyll content, carotenoid content, nitrate reductase activity, carbonic anhydrase activity, leaf N, -P, -K content by the exogenous application of TRIA in comparison with the water sprayed treatment (Figures 3 and 4) is not far to seek. The growth hormone TRIA has been reported to improve the number and size of chloroplast (Ivanov and Angelov, 1997; Chen et al., 2003; Muthuchelian et al., 2003; Naeem et al., 2010).This improvement in chloroplast might be responsible for increase in number of chlorophyll and carotenoid molecules of treated plants (Figure 3) hence increase in photosynthetic carbon dioxide assimilation. An improvement in these parameters was also reported by Trewavas and Gilory, 1991. These results coincide with those of Naeem et al., (2011); Hashmi et al., (2011) who reported stimulation of chlorophyll synthesis by the exogenous application of 10-6 M of TRIA in Mentha arvensis and Ocimum basilicum respectively. A probable reason for the enhanced nitrate reductase and carbonic anhydrase activity in treated plants (figure 3) may be due to the influence of TRIA on de-novo synthesis of these enzymes as reported by Okabe et al., 1980 for carbonic anhydrase. The results are in accordance with the finding of other workers including Naeem et al., (2011); Hashmi et al., (2011). The enhancement in leaf nutrients may be related to compositional or chemical changes in membrane of plants leading to alterations in nutrient concentration (Figure 4). These results conformity with Chaudhary et al., (2006); Naeem et al., (2011) and Hashmi et al., (2011) on Capsicum annuum, Mentha arvensis and Ocimum basilicum respectively. Yield and Quality Characteristics Leaf applied TRIA not only benefitted growth and physiological attributes as mentioned earlier but also improved the content and yield of essential oil (Figure 5) This may be explained on the basis of cumulative direct or indirect role of photosynthetic pigments, enzyme activities and nutrient content among others, in photosynthesis, partitioning of photosynthates and other metabolites and synthesis of secondary metabolites. Thus, the improved values for these parameters might be culminated into higher values for fresh matter, content and yield of essential oil and content and yield of citral of treated plants. The positive role of TRIA on the active constituent was also substantiated by our GLC analysis. Our finding broadly corroborate the findings of Srivastava and Sharma, (1991); Naeem et al., (2011) on other aromatic plants.

CONCLUSIONS
Exogenous Application of all the concentrations of TRIA improves overall performance of lemongrass as compared to the control plant. While the optimum concentration of phytohormone (TRIA 10 -6 M) increases plant growth, physio-biochemical and quality attributes (yield of essential oil and the active constituents of oil). Further, TRIA promoted a significant production of citral of the essential oil.

ACKNOWLEDGEMENTS
We are grateful to the Chairman, Department of Botany, Aligarh Muslim University, Aligarh, for providing research facilities. We are also grateful for non-net UGC fellowship for financial support for research work.

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APPENDCIES

Figure 1: Effect of Four Levels of Triacontanol (TRIA) on the Length of Shoot (A) and Root (B), Number of Leaves (C) and Tillers (D) of Two Varieties of Cymbopogon flexuosus at 120 DAP. Each Value Represents the Mean of Four Replicates with Error Bars Shows Standard Error

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Figure 2: Effect of Four Levels of Triacontanol (TRIA) on Fresh Weights Shoot (E) and Root (F), Dry Weights Shoot (G) and Root (H) of Two Varieties of Cymbopogon flexuosus at 120 DAP. Each Value Represents the Mean of Four Replicates with Error Bars Shows Standard Error

Figure 3: Effect of Four Levels of Triacontanol (TRIA) on Total Chlorophyll Content (I) and Carotenoids Content (J), Carbonic Anhydrase Activity (K) and Nitrate Reductase Activity (L) of Two Varities of Cymbopogon flexuosus at 120 DAP. Each Value Represents the Mean of Four Replicates with Error Bars Shows Standard Error

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Figure 4: Effect of Four Levels of Triacontanol (TRIA) on Leaf-Nitrogen Content (M),-Phosphorus (N) and Potassium Content (O) of Two Varieties of Cymbopogon flexuosus at 120 DAP. Each Value Represents the Mean of Four Replicates with Error Bars Shows Standard Error

Figure 5: Effect of Four Levels of Triacontanol (TRIA) on Essential Oil Content (P) and Oil Yield (Q), Citral Content (R) and Citral Yield (S) of Cymbopogon flexuosus at 120 DAP. Each Value Represents the Mean of Four Replicates with Error Bars Shows Standard Error