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Industrial Crops and Products 55 (2014) 272–279

Contents lists available at ScienceDirect

Industrial Crops and Products

jo u r n al homep age: www.elsev ier .com/ locate / indcrop

mploying depolymerised sodium alginate, triacontanol and8-homobrassinolide in enhancing physiological activities,roduction of essential oil and active components in Mentha arvensis L

. Naeema,∗, Mohd. Idreesa, Tariq Aftaba,d, M. Masidur Alama, M. Masroor A. Khana,oin Uddinb, Lalit Varshneyc

Plant Physiology Section, Department of Botany, Aligarh Muslim University, Aligarh 202002, IndiaWomen’s College, Botany Section, Aligarh Muslim University, Aligarh 202002, IndiaRadiation Technology Development Division, ISOMED, Bhabha Atomic Research Centre, Mumbai 400085, IndiaDepartment of Botany, Jamia Hamdard (Hamdard University), New Delhi, 110062, India

r t i c l e i n f o

rticle history:eceived 9 November 2013eceived in revised form 28 January 2014ccepted 30 January 2014vailable online 16 March 2014

eywords:int

ctive constituentshotosynthesis8-Homobrassinolideriacontanol

a b s t r a c t

There is immense need of enhancing the content and yield of active constituents of medicinally importantplants in view of their massive demand worldwide. Various phytohormones have proved effective inthis regard. Gamma-rays irradiated sodium alginate (ISA), triacontanol (TRIA) and 28-homobrassinolide(HBR) have also proven as potent plant growth promoting substances for a number of agricultural andhorticultural crops. Considering the medicinal importance, a pot experiment was conducted to explorethe individual as well as combined effect of best foliar doses of ISA, TRIA and HBR on growth, yieldand quality of mint (Mentha arvensis L.). The spray of ISA, TRIA and HBR, applied alone on plants, waspositively effective. However, the effect of their combined application was much pronounced as comparedto that of their individual application; it improved most of the plant growth attributes, physiologicaland biochemical parameters, herbage yield and the content and yield of active constituents of mintsignificantly studied at 100 and 120 DAP. Of the seven spray-treatments [(i) Control, (ii) 100 ppm ISA,(iii) 10−6 M TRIA, (iv) 10−7 M HBR, (v) 100 ppm ISA + 10−6 M TRIA, (vi) 100 ppm ISA + 10−7 M HBR, (vii)100 ppm ISA + 10−6 M TRIA + 10−7 M HBR], 100 ppm ISA + 10−6 M TRIA + 10−7 M HBR proved to be the best.The combined application resulted in the highest content and yield of essential oil (EO) over the control

by 42.1 and 43.9% and 114.0 and 121.7% at 100 and 120 DAP, respectively. This combined treatment ofplant growth regulators (PGRs) also proved the best, increasing the content of menthol, l-menthone,isomenthone and menthyl acetate by 7.5 and 6.2%, 31.8 and 32.1%, 25.8 and 21.7%, and 40.1 and 37.2%,respectively, over the respective control at 100 and 120 DAP. As compared to the control, it increased theper plant yields of menthol, l-menthone, isomenthone and menthyl acetate by 129.9 and 135.5%, 190.0

4.3%,

and 194.4%, 162.5 and 16

. Introduction

Gamma-rays irradiation with cobalt-60 degrades the naturalolysaccharides, such as chitosan, carrageenan and sodium algi-ate, into smaller oligomers with comparatively low moleculareight. Oligomers, obtained from radiolytically degraded polysac-

harides including those of irradiated sodium alginate (ISA), have

alid applications in the field of agriculture, as plant growth pro-oters (Hien et al., 2000; Kume et al., 2002). Application of the

egraded polysaccharides (oligomers) on plants promotes various

∗ Corresponding author. Tel.: +91 9719341207.E-mail addresses: naeem phd@yahoo.co.in, naeemgaur@gmail.com,

asroorbot@gmail.com (M. Naeem), alam281@gmail.com (M. Masidur Alam).

926-6690/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.indcrop.2014.01.052

and by 225.0 and 187.5% at 100 and 120 DAP, respectively.© 2014 Elsevier B.V. All rights reserved.

biological and physiological activities, including plant growth ingeneral (Hien et al., 2000; Aftab et al., 2011; Khan et al., 2011;Sarfaraz et al., 2011; Naeem et al., 2012a,b), seed germination (Hienet al., 2000), shoot elongation (Natsume et al., 1994; Hien et al.,2000), root growth (Iwasaki and Matsubara, 2000), flower pro-duction, antimicrobial activity, amelioration of heavy metal stress,phytoalexin induction, etc. (Darvill et al., 1992; Kume et al., 2002;Luan et al., 2003; Hu et al., 2004).

Triacontanol (TRIA), a long chain primary alcohol (C30H61OH),has been realized as a potent plant growth promoting substance forvarious agricultural and horticultural crops. It increases the rate of

several biochemical and physiological processes (Ries and Houtz,1983; Ries, 1991; Naeem et al., 2009, 2012c) and, thereby, improvesthe plant growth as well as yield and quality characteristics of thecrops (Ries, 1985).

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Recently, brassinosteroids (BRs) have emerged as a new group ofrowth promoting phytohormones. 28-Homobrassinolide (HBR) isne of the several BRs, the role of which in enhancing growth, pro-uctivity and quality of plants, via improving various physiologicalrocesses, has been established both under stress and normal con-itions (Khripach et al., 2000; Arora et al., 2011; Zhang et al., 2008;wamy and Rao, 2011; Sharma et al., 2011; Naeem et al., 2012d).

Out of a large number of EO bearing plants, mint (Mentha arven-is L.) constitutes most important source of therapeutic agents usedn the alternative systems of medicine (The Wealth of India, 1992).urther, mint oil has wide applications in pharmaceutical, agro-hemical and flavoring industries worldwide (Misra et al., 2000;assou et al., 2004). Keeping the importance and increasing demandf mint EO in mind, this study was conducted to find out the bestoncentration effect (Naeem et al., 2011, 2012a,d) of ISA, TRIA andBR applied alone or in combination in order to enhance the pro-uctivity, physiological activities and production of EO of mint.here is no information regarding the combined effect of ISA, TRIAnd HBR application on this medicinally important crop till date.

. Materials and methods

.1. Plant materials and growth conditions

The pot experiment was conducted in the natural conditions ofhe net house at Botany Department, Aligarh Muslim University,ligarh, India. Prior to transplanting, each pot was filled with 5 kgomogenous mixture of soil and organic (cowdung) manure (4:1).hysico-chemical characteristics of the experimental soil mixture4 parts soil:1 part cowdung manure) were: texture-sandy loam,H (1:2) 7.5, E.C. (1:2) 0.48 dSm−1, available N, P and K 102.4, 7.8nd 145.9 mg kg−1 of soil, respectively. A uniform recommendedasal dose of N, P and K (25:11:21 mg kg−1 soil, respectively) waspplied in the form of urea, single superphosphate and muriate ofotash, respectively, at the time of planting. The experiment wasonducted in randomized block design using earthen pots (25 cmiameter × 25 cm height). Each treatment was replicated five times.ach pot contained a single healthy plant. The pots were watereds and when required.

The best foliar dose of each of the PGRs used was selected on theasis of earlier findings of our study (Naeem et al., 2011, 2012a,d).he individual and combined application of optimized concentra-ions of ISA (100 ppm), TRIA (10−6 M) and HBR (10−7 M) was carriedut at 10 days interval when the plants were at 2–3 true leavestage to find out the most excellent response of mint crop. Totalve foliar sprays of ISA, TRIA and HBR were applied to the cropsing a hand sprayer. Un-irradiated sodium alginate was not tested

n the present study as the chemical gave equal effect with that ofontrol (Naeem et al., 2012a). Seven spray treatments [(i) Control,ii) 100 ppm ISA, (iii) 10−6 M TRIA, (iv) 10−7 M HBR, (v) 100 ppmSA + 10−6 M TRIA, (vi) 100 ppm ISA + 10−7 M HBR, (vii) 100 ppmSA + 10−6 M TRIA + 10−7 M HBR] were applied, using distilled waters absolute control. The crop was harvested at 100 and 120 daysfter planting.

.2. Irradiation and gel permeation chromatography (GPC)nalysis

Solid form of sodium alginate (Sigma Aldrich, USA) was sealed in glass tube with atmospheric air. The samples of sodium alginateere irradiated in a Gamma Chamber (Cobalt-60, GC-5000) made

y BRIT, Mumbai, India. The samples were irradiated to 520 kGyamma radiation dose at a dose rate of 2.4 kGy/h. GPC of sodiumlginate samples were also done accordingly (Naeem et al., 2012a).ifferent aqueous concentrations of irradiated sodium alginate

Products 55 (2014) 272–279 273

(ISA) were finally prepared using double distilled water as spraytreatments.

2.3. Determination of growth attributes

The growth attributes viz. plant height, leaf-area, leaf-yield perplant and fresh and dry weights of plant were determined at 100and 120 DAP. All the leaves of the plant were weighed to determineleaf-yield per plant. Five plants of each treatment were uprootedcarefully followed by measuring the height and fresh weight ofplant. The plants were dried in a hot-air oven at 80 ◦C for 24 h priorto recording the plant dry weight. Only 10% of the total leaves ofeach sample (consisting of five plants) were used to determine theleaf area using graph paper sheet (Watson, 1958). The mean areaper leaf, thus determined, was multiplied with the total number ofleaves to measure the total leaf area per plant.

2.4. Determination of physiological attributes

2.4.1. Estimation of total chlorophyll and carotenoids contentsTotal content of leaf chlorophyll and carotenoids was estimated

using the method of Lichtenthaler and Buschmann (2001). The freshtissue from the interveinal area of leaf was grinded with 100% ace-tone using mortar-pestle. The optical density (OD) of the pigmentsolution was recorded at 662, 645 and 470 nm to determine chloro-phyll a, chlorophyll b and total carotenoids content, respectively,using a spectrophotometer (Shimadzu UV-1700, Tokoyo, Japan).Total chlorophyll content was assessed by adding the contents ofchlorophyll a and b. The content of photosynthetic pigments wasexpressed as mg g−1 leaf FW.

2.4.2. Determination of net photosynthetic rate and stomatalconductance

Net photosynthetic rate and stomatal conductance of theyoungest fully expanded leaves were measured in five replicateson sunny days at 1100 h using an Infra Red Gas Analyzer (IRGA, Li-Cor 6400 Portable Photosynthesis System Lincoln, Nebraska, USA)at 100 and 120 DAP.

2.4.3. Determination of carbonic anhydrase (CA) activityThe activity of carbonic anhydrase (E.C. 4.2.1.1) was measured

in the fresh leaves, using the method described by Dwivedi andRandhawa (1974). Two hundred mg of fresh leaf (chopped leaf-pieces) were transferred to Petri plates. The leaf pieces were dippedin 10 mL of 0.2 M cystein hydrochloride solution for 20 min at 4 ◦C.The solution adhering at the cut surfaces of the leaf pieces wasthen removed with the help of a blotting paper followed by theirtransfer immediately to a test tube containing 4 mL of phosphatebuffer of pH 6.8. To it, 4 mL of 0.2 M sodium bicarbonate solution and0.2 mL of 0.022% bromothymol blue were added. The reaction mix-ture was titrated against 0.05 N HCl using methyl red as indicator.The enzyme activity was expressed as �M CO2 kg−1 leaf FW s−1.

2.4.4. Total phenol contentTotal phenol content was estimated by the method described

by Sadasivam and Manickam (2008). Five hundred mg of the leaveswere grinded with 10 times volume of 80% ethanol, using mortar-pestle. The homogenate was centrifuged at 10,000 rpm (10,062 × g)for 10 min at 4 ◦C. The supernatant was evaporated to dryness,adding 5 mL of double distilled water (DDW) thereafter. Later,0.5 mL of Folin-Ciocalteau Reagent and 2 mL of 20% Na2CO3 solu-

tion were added to each test tube. The OD of the solution, thusobtained, was measured at 650 nm against a reagent blank. Usingthe standard curve, the content of total phenols in the test sampleswas determined as mg phenol per 100 g of dry leaves.

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.5. Yield and quality parameters

Herbage yield of the crop was measured by weighing the totaliomass per plant excluding the roots. The EO of mint was extractednd determined gravimetrically according to Guenther (1972).resh leaves were collected from each treatment pot. Later, theyere chopped together. Sufficient quantity of chopped leaf-piecesas taken for EO estimation. The EO content in the leaves was

xtracted by distillation method for 3 h, using a Clevenger’s appa-atus.

The active constituents of the EO, namely, menthol, l-methone,somenthone and menthyl acetate, were analyzed using Gas Liquidhromatography (GLC, Nucon 5700, New Delhi, India) equippedith an AT-1000 stainless steel column, a flame ionization detector

nd an integrator. Nitrogen was used as the carrier gas. The flowates of nitrogen, hydrogen and oxygen were maintained at 0.5,.5 and 5 mL s−1, respectively. The temperature schedule of GLCas as follows: detector temperature, 250 ◦C; oven temperature,

60 ◦C; injector temperature, 250 ◦C. The sample size was 2 �L forll the measurements. The identification of the active constituentsas based on retention time of the particular constituent in theLC column. The active constituents were quantified in per centontent, comparing their peaks with the peaks obtained from theeference standards reported by Adams (2007).

.5.1. Determination of specific gravity of essential oilThe specific gravity of EO was determined at 25 ◦C with a ‘spe-

ific gravity bottle’ according to Afaq et al. (1994).

.5.2. Determination of refractive index of essential oilThe refractive index of the EO was determined according to

enkins et al. (1967) employing an Abbe’s Refractometer (Sipcon,ew Delhi, India). Two to three drops of oil were placed on theouble prism, clamping the prisms together firmly. The instru-ent was adjusted until the border line between light and dark

alves of the view-field exactly coinciding with the cross hairs ofhe telescope. The refractive index of EO was noted directly fromhe graduated scale. The mean of three readings was designated asefractive index of EO. The refractive index of the EO was expresseds ND

24◦; where, ND

24◦denotes the index of the light refraction for

he ‘D’ line (sodium light) measured at 24◦.

.6. Statistical analysis

The data were analyzed statistically using SPSS-17 statisticaloftware (SPSS Inc., Chicago, IL, USA). Means were compared usinguncan’s Multiple Range Test (DMRT) at P ≤ 0.05. Standard erroras also employed in this regard.

. Results

The present study indicated that the spray of ISA, TRIA and HBR,hich was individually applied to plants, improved the perfor-ance of mint crop significantly. However, combined application

f these PGRs was much effective compared to their individualpplication carried out as leaf-sprays. It improved most of thelant growth attributes, physiological and biochemical parame-ers, and content and yield of EO; it also enhanced the levels ofctive constituents of mint significantly. However, specific gravitynd refractive index of EO were not affected by applying the PGRs

ither alone or in combination both at 100 and 120 DAP (Tables 1–3nd Figs. 1 and 2). Of the seven spray treatments, the combination ofhe three PGRs (100 ppm ISA + 10−6 M TRIA + 10−7 M HBR) provedo be the best one compared to other treatments.

d Products 55 (2014) 272–279

3.1. Growth attributes

As compared to the control, the effect of the spray treatmentswas positively significant on height, leaf-area, leaf-yield and freshand dry weights of plant both at 100 and 120 DAP. Foliar spray of ISAgave significantly enhanced value when it was applied either withTRIA and/or HBR. Of the spray treatments, the combined applica-tion of the PGRs proved to be most excellent as compared to othertreatments (Table 1). Combined application of the PGRs (100 ppmISA + 10−6 M TRIA + 10−7 M HBR) enhanced the plant height, leaf-area, leaf-yield, and fresh and dry weights of plant by 65.6 and66.4%, 46.1 and 47.1%, 67.6 and 71.6%, 63.0 and 66.9%, and by 69.7and 72.9% at 100 and 120 DAP, respectively, when compared to thecontrol plants (Table 1).

3.2. Physiological and biochemical attributes

All physiological and biochemical attributes were significantlyaffected by the individual and combined application of ISA, TRIAand HBR at both the growth stages. However, specific gravity andrefractive index of EO were not significantly affected at either ofthe growth stages (Table 2). Compared to the control, the com-bined application of ISA, TRIA and HBR accelerated the rate ofphotosynthesis and stomatal conductance by 38.8 and 39.6% andby 26.0 and 31.2%, respectively, at 100 and 120 DAP (Fig. 1A and B).The combined spray treatment also improved the chlorophyll andcarotenoids contents significantly. It enhanced the total contentof chlorophyll and carotenoids by 31.1 and 31.7% and by 18.2 and18.4% at 100 and 120 DAP, respectively, compared to the control(Fig. 1C and D).

In the present study, the leaves treated with combination of ISA,TRIA and HBR, improved the CA activity by 30.2 and 31.7% comparedto the control at 100 and 120 DAP, respectively (Fig. 2A). Combinedapplication of ISA, TRIA and HBR also improved the level of leaf-phenolic content by 11.0 and 12.5% over the control at 100 and 120DAP, respectively (Fig. 2B).

3.3. Yield and quality attributes

Promotive effect was observed on yield and quality of mintby PGR treatments. However, the combined application of PGRs(100 ppm ISA + 10−6 M TRIA + 10−7 M HBR) proved the best for theyield and quality attributes. Of the various selected concentrations,the combined application of ISA, TRIA and HBR at 100 ppm, 10−6 Mand 10−7 M, respectively, enhanced the herbage yield of the cropsignificantly, giving 62.5 and 68.6% higher values at 100 and 120DAP, respectively, over the control (Table 2). The promotive effectof combined application of ISA, TRIA and HBR was also observedon the content and yield of EO in comparison to the water sprayedplants. It resulted in the highest content and yield of EO, over thecontrol by 42.1 and 43.9% and 114.0 and 121.7% at 100 and 120DAP, respectively (Table 2). However, specific gravity and refrac-tive index of EO were not significantly improved by individual orcombined application of ISA, TRIA and HBR at either of the growthstages (Table 2).

In response to ISA, TRIA and HBR treatments, there was observeda progressive increase in the content and yield of active componentsas compared to the control (Table 3). Combined spray of ISA, TRIAand HBR significantly enhanced the active constituents of EO overthe control. Of the various selected concentrations, the combinedPGRs treatment (100 ppm ISA+ 10−6 M TRIA + 10−7 M HBR) provedthe best and significantly increased the contents of menthol, l-

menthone, isomenthone and menthyl acetate by 7.5 and 6.2%, 31.8and 32.1%, 25.8 and 21.7%, and 40.1 and 37.2%, respectively, over therespective control at 100 and 120 DAP (Table 3). As compared to thecontrol, it increased the per plant yields of menthol, l-menthone,

M. Naeem et al. / Industrial Crops and Products 55 (2014) 272–279 275

Table 1Effect of foliar sprays of ISA, TRIA and HBR on growth attributes of mint (Mentha arvensis L.) at 100 and 120 DAP.

Growth attributes Treatment concentrations*

DAP Control ISA TRIA HBR ISA + TRIA ISA + HBR ISA + TRIA +HBR

Plant height(cm)

100 70.96 ± 1.10f 114.90 ± 1.10c 112.15 ± 1.17d 94.65 ± 1.19e 116.40 ± 1.15b 116.20 ± 1.20b 117.50 ± 1.24a

120 84.70 ± 1.11f 137.64 ± 1.19c 134.45 ± 1.05d 116.40 ± 1.15e 138.90 ± 1.20b 138.84 ± 1.28b 140.94 ± 1.20a

Leaf-area perplant (cm2)

100 2970.4 ± 20.17f 4171.9 ± 23.28d 4129.0 ± 13.4e 4296.0 ± 16.4c 4320.0 ± 12.40b 4325.0 ± 14.32b 4340.11 ± 12.74a

120 4725.8 ± 17.90f 6845.8 ± 15.72e 6862.8 ± 15.0c 6850.0 ± 16.4d 6879.2 ± 14.62b 6879.4 ± 14.60b 6951.6 ± 12.36a

Leaf-yield perplant (g)

100 14.25 ± 0.162f 20.70 ± 0.162e 23.45 ± 0.132c 22.95 ± 0.130d 23.70 ± 0.125b 23.60 ± 0.148b 23.89 ± 0.152a

120 27.20 ± 0.230f 40.38 ± 0.216e 45.34 ± 0.231c 45.00 ± 0.230d 45.88 ± 0.219b 45.58 ± 0.224b 46.67 ± 0.230a

Fresh weightper plant (g)

100 53.48 ± 1.10e 84.85 ± 1.21c 79.68 ± 1.02d 79.46 ± 1.10d 86.24 ± 1.30b 86.08 ± 1.45b 87.16 ± 1.42a

120 65.42 ± 1.63f 106.40 ± 1.60c 97.12 ± 1.09d 93.10 ± 2.19e 108.24 ± 1.40b 108.14 ± 1.38b 109.16 ± 1.40a

Dry weight perplant (g)

100 12.16 ± 0.205e 19.10 ± 0.318c 18.62 ± 0.407d 18.60 ± 0.417d 20.12 ± 0.412b 19.70 ± 0.375b 20.64 ± 0.362a

120 14.52 ± 0.262d 23.98 ± 0.332c 24.26 ± 0.338c 23.90 ± 0.440c 24.70 ± 0.435b 24.60 ± 0.370b 25.10 ± 0.354a

Means within a column followed by the same letter(s) are not significantly different (P ≤ 0.05). Means of five replicates ± SE.* Treatment concentrations: IAS 100 ppm, TRIA 10−6 M, HBR 10−7 M.

Table 2Effect of foliar sprays of ISA, TRIA and HBR on yield and quality attributes of mint (Mentha arvensis L.) at 100 and 120 DAP.

Yield and qualityattributes

Treatment concentrations*

DAP Control ISA TRIA HBR ISA+TRIA ISA + HBR ISA + TRIA + HBR

Herbage yieldper plant (g)

100 35.84 ± 0.16e 55.90 ± 0.14d 56.68 ± 0.225c 56.62 ± 0.14c 57.16 ± 0.220b 56.90 ± 0.205b 58.24 ± 0.210a

120 51.45 ± 0.26f 81.16 ± 0.26e 86.19 ± 0.202c 82.98 ± 0.25d 86.54 ± 0.215b 86.46 ± 0.210b 86.76 ± 0.216a

Essentialoil-content (%)

100 0.646 ± 0.001f 0.884 ± 0.002e 0.902 ± 0.001d 0.885 ± 0.004e 0.914 ± 0.002b 0.911 ± 0.002c 0.918 ± 0.002a

120 0.949 ± 0.009f 1.348 ± 0.002c 1.229 ± 0.002e 1.190 ± 0.002d 1.356 ± 0.002b 1.349 ± 0.001c 1.366 ± 0.001a

Essential oil-yieldper plant (mL)

100 0.258 ± 0.001f 0.501 ± 0.001e 0.542 ± 0.003c 0.526 ± 0.002d 0.548 ± 0.002b 0.545 ± 0.002b 0.552 ± 0.003a

120 0.460 ± 0.001f 0.950 ± 0.001e 0.998 ± 0.004c 0.970 ± 0.002d 1.014 ± 0.002b 1.010 ± 0.002b 1.020 ± 0.002a

Specific gravity ofessential oil (g/cm3)

100 0.892 ± 0.001a 0.894 ± 0.001a 0.894 ± 0.001a 0.892 ± 0.001a 0.894 ± 0.001a 0.894 ± 0.001a 0.892 ± 0.001a

120 0.892 ± 0.001a 0.895 ± 0.001a 0.894 ± 0.001a 0.893 ± 0.001a 0.895 ± 0.001a 0.892 ± 0.001a 0.894 ± 0.001a

Refractive index ofessential oil

100 1.460 ± 0.001a 1.464 ± 0.001a 1.462 ± 0.001a 1.462 ± 0.001a 1.460 ± 0.001a 1.462 ± 0.001a 1.462 ± 0.001a

120 1.462 ± 0.001a 1.465 ± 0.001a 1.465 ± 0.001a 1.463 ± 0.001a 1.462 ± 0.001a 1.462 ± 0.001a 1.462 ± 0.001a

Means within a column followed by the same letter(s) are not significantly different (P ≤ 0.05). Means of five replicates ± SE.* Treatment concentrations: IAS 100 ppm, TRIA 10−6 M, HBR 10−7 M.

Table 3Effect of foliar sprays of ISA, TRIA and HBR on active constituents and its yield of mint (Mentha arvensis L.) at 100 and 120 DAP.

Content and yieldof activeconstituents

Treatment concentrations*

DAP Control ISA TRIA HBR ISA + TRIA ISA + HBR ISA + TRIA + HBR

Mentholcontent (%)

100 80.20 ± 0.024d 80.24 ± 0.020d 82.76 ± 0.023c 85.70 ± 0.01b 82.78 ± 0.02c 85.75 ± 0.02b 86.24 ± 0.01a

120 81.50 ± 0.020d 81.54 ± 0.032d 82.10 ± 0.029c 86.26 ± 0.02 b 82.12 ± 0.02c 86.20 ± 0.02b 86.56 ± 0.02a

Menthol yieldper plant (mL)

100 0.207 ± 0.003g 0.402 ± 0.004f 0.448 ± 0.003e 0.450 ± 0.003d 0.454 ± 0.001c 0.459 ± 0.003b 0.476 ± 0.002a

120 0.375 ± 0.003f 0.775 ± 0.004e 0.819 ± 0.005d 0.837 ± 0.005c 0.833 ± 0.002c 0.871 ± 0.003b 0.883 ± 0.002a

l-Menthonecontent (%)

100 3.90 ± 0.020g 5.09 ± 0.030c 4.80 ± 0.034e 4.30 ± 0.010f 5.12 ± 0.028b 4.86 ± 0.021d 5.14 ± 0.030a

120 3.92 ± 0.020f 4.90 ± 0.031c 4.20 ± 0.026e 4.52 ± 0.021d 4.95 ± 0.024b 4.95 ± 0.029b 5.18 ± 0.027a

l-Menthone yieldper plant (mL)

100 0.010 ± 0.001d 0.026 ± 0.004b 0.026 ± 0.002b 0.023 ± 0.002c 0.026 ± 0.001b 0.026 ± 0.002b 0.029 ± 0.002a

120 0.018 ± 0.001e 0.047 ± 0.003c 0.042 ± 0.002d 0.044 ± 0.002d 0.050 ± 0.002b 0.050 ± 0.001b 0.053 ± 0.002a

Isomenthonecontent (%)

100 2.98 ± 0.010e 3.29 ± 0.021d 3.57 ± 0.029c 2.99 ± 0.012e 3.58 ± 0.024b 3.60 ± 0.019b 3.75 ± 0.022a

120 3.00 ± 0.010e 3.30 ± 0.020d 3.47 ± 0.029c 3.02 ± 0.020e 3.58 ± 0.020b 3.60 ± 0.023b 3.65 ± 0.020a

Isomenthone yieldper plant (mL)

100 0.008 ± 0.001f 0.016 ± 0.004d 0.019 ± 0.002c 0.016 ± 0.002e 0.020 ± 0.001b 0.019 ± 0.002b 0.021 ± 0.002a

120 0.014 ± 0.002f 0.031 ± 0.003d 0.035 ± 0.003c 0.016 ± 0.003e 0.035 ± 0.002b 0.036 ± 0.003b 0.037 ± 0.003a

Menthyl acetatecontent (%)

100 1.62 ± 0.010e 1.85 ± 0.020d 2.16 ± 0.020c 1.85 ± 0.012d 2.23 ± 0.020b 1.85 ± 0.021d 2.27 ± 0.018a

120 1.64 ± 0.012f 1.80 ± 0.021e 2.12 ± 0.029c 1.82 ± 0.010e 2.24 ± 0.020b 1.94 ± 0.021d 2.25 ± 0.015a

Menthyl acetateyield per plant (mL)

100 0.004 ± 0.001d 0.009 ± 0.002c 0.011 ± 0.002b 0.009 ± 0.002b 0.012 ± 0.001ab 0.010 ± 0.001b 0.013 ± 0.001a

120 0.008 ± 0.001e 0.017 ± 0.002d 0.021 ± 0.001b 0.018 ± 0.001c 0.023 ± 0.001a 0.018 ± 0.001c 0.023 ± 0.001a

Means within a column followed by the same letter(s) are not significantly different (P ≤ 0.05). Means of five replicates ± SE.* Treatment concentrations: IAS 100 ppm, TRIA 10−6 M, HBR 10−7 M.

276 M. Naeem et al. / Industrial Crops and Products 55 (2014) 272–279N

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Fig. 1. Effect of foliar sprays of ISA, TRIA and HBR on net photosynthetic rate (A) and stomatal conductance (B), total chlorophyll content (C) and total carotenoids content(D) of mint (Mentha arvensis L.) studied at 100 and 120 DAP. Means within a column followed by the same letter(s) are not significantly different (P ≤ 0.05). Error bars (�)show SE.

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Treatm ents [ISA (100 ppm), TRIA (1 0-6 M), HBR (1 0-7 M)]

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Control ISA TRIA HBRISA+TRIA

ISA+HBRISA+TRIA+HBR

(A) (B)

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Fig. 2. Effect of foliar sprays of ISA, TRIA and HBR on carbonic anhydrase activity (A) and total phenolic content (B) of mint (Mentha arvensis L.) studied at 100 and 120 DAP.Means within a column followed by the same letter(s) are not significantly different (P ≤ 0.05). Error bars (�) show SE.

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somenthone and menthyl acetate by 129.9 and 135.5%, 190.0 and94.4%, 162.5 and 164.3%, and by 225.0 and 187.5% at 100 and 120AP, respectively (Table 3).

. Discussion

Like plant growth regulators, which improve the plant defensey acting as signaling molecules, AO (alginate oligosaccharides)esemble with an endogenous growth elicitor that functions as aignal to trigger the synthesis of different enzymes and activate var-ous responses exploiting the gene expression (Ma et al., 2010). Itas already been reported that polysaccharides such as sodium algi-ate, carrageenan and chitosan, in their depolymerized form, areffective in promotion of germination and shoot elongation (Hient al., 2000; Mollah et al., 2009; Aftab et al., 2011; Khan et al., 2011;arfaraz et al., 2011; Naeem et al., 2012a,b; Idrees et al., 2012; Aftabt al., 2013). In the present study, the application of ISA enhancedhe leaf-area, which might obviously provide increased opportunityor light harvesting leading to the accumulation of enhanced plantry matter, compared to the control (Table 1). The present studyhowed significant improvement in plant growth attributes by thepplication of radiation-derived oligomers of sodium alginate (ISA).

There is enough evidence regarding the growth-promotingffect of TRIA (Sharma et al., 2006; Khan et al., 2007, 2009; Naeemt al., 2009, 2011, 2012c) and HBR (Swamy and Rao, 2006, 2008,009, 2011; Talaat and Abdallah, 2011; Naeem et al., 2012d),

ndicating PGR-mediated increase in plant height and dry mat-er accumulation in various plants. Improvement in shoot heightnd leaf area index might contribute to the enhanced values ofry weight of TRIA and HBR treated plants in the present studyTables 1 and 2). However, when TRIA was applied with ISA andBR on plants, it proved much effective in this regard (Table 1). It

s noteworthy to mention here that the combined application ofSA, TRIA and HBR proved much effective in comparision to theirndiviual application in this regard (Table 1).

Foliage of the plants sprayed with the PGRs employed elevatedhe content of both chlorophyll and carotenoids in comparison toontrol plants (Fig. 1). In addition, the enhancement in the chloro-hyll content might have resulted in increased photosynthetic rate.he increase in photosynthetic rate due to application of ISA, haseen indicated previously by several workers (Aftab et al., 2011;han et al., 2011; Sarfaraz et al., 2011; Naeem et al., 2012a). The ISAas also been reported to induce cell signaling, leading to stimula-ion of various physiological processes in various plants, includingSA-mediated improved content of photosynthetic pigments andnhanced net photosynthetic rate (Naeem et al., 2012a; Aftab et al.,013). In view of growth promoting effect of ISA, its applicationould result in enhanced growth of plant root and a shoot and,hereby, might bring result in augmented plant productivity andmproved physiological parameters (El-Rehim, 2006).

Earlier studies have revealed an increase in the rate of both CO2xation and photosynthesis in different plants as a result of TRIApplication (Srivastava and Sharma, 1990; Misra and Srivastava,991; Srivastava and Sharma, 1991; Ivanov and Angelov, 1997;hen et al., 2003; Muthuchelian et al., 2003; Naeem et al., 2009,011). Further, in this study, increased photosynthesis was par-llel with the elevated levels of leaf chlorophyll and carotenoidsontents (Fig. 1C and D).

The application of 28-homobrassinolide also resulted in ele-ated carbon dioxide fixation as compared to the untreated controllants (Fig. 1). Besides, the exogenous application of HBR (100 �M)

nd 24-epibrassinolide (EBL) (3 �M) resulted in a significantncrease in the CO2 fixation in geranium (Swamy and Rao, 2008,009). In line with our studies, there are several reports regardinghe positive effects of brassinosteroids on chlorophyll levels in

Products 55 (2014) 272–279 277

plants (Alam et al., 2007; Sharma et al., 2011; Naeem et al.,2012d).

Carbonic anhydrase activity was positively affected by the ISAalone as well as by a combination of ISA, TRIA and HBR. The activ-ity of the enzyme increased to the maximum extent at 120 DAP(Fig. 2). In this regard, our findings are similar to those that claimthe synthesis of certain enzymes in tissue culture studies followingaddition of alginate derived oligomers (Akimoto et al., 1999).

Present study revealed that the leaves treated with TRIAimproved the CA activity considerably at both sampling stages(Fig. 2A). The enhancement of CA activity due to TRIA applicationmight also be ascribed to the de novo synthesis of CA, which mightinvolve the genes associated with its transcription and translationin the cell (Okabe et al., 1980).

In addition, the enhancement of CA activity in the HBR-treatedplants might have presumably been responsible for the enhancedrate of CO2 fixation that, accordingly, could have been responsiblefor significant increase in the fresh and dry weights of HBR-treatedplants (Table 1).

The ISA alone increased the level of leaf phenolic content at boththe sampling stages (Fig. 2B). However, combined effect of ISA withTRIA and HBR was much pronounced in increasing the level of phe-nolic content in the leaves at 100 as well as 120 DAP (Fig. 2B). Thepositive results obtained in this regard in response to ISA applica-tion might be ascribed to such a specific role of oligomers obtainedby irradiation with Co-60 gamma rays.

In addition, TRIA application alone improved the leaf-phenoliccontent at both the sampling stages. However, TRIA, applied withISA and HBR, increased the total phenolic content maximally in thisstudy (Fig. 2B). The leaf phenolic content reflects the free radicalscavenging capability of the plant that may help the plant to main-tain the normal growth at later growth stages, at which frequentproduction of free radicals takes place, inducing the bad effects ofaging (Dimitrios, 2006). The significant effect of TRIA on phenolcontent has also been reported by Kumaravelu et al. (2000) andNaeem et al. (2011) regarding green gram and mint respectively.

The significant increase in the above mentioned yield param-eters of the ISA treated plants might possibly culminate inmaximization of the leaf-yield and herbage-yield of the mint plantemployed (Tables 1 and 2). Presumably, the improved content andyield of EO in TRIA treated plants could be due to the enhanced ratesof photosynthesis and improved translocation of photosynthatesand other metabolites to the reproductive organs as indicated bythe photosynthetic model for oil production in Mentha piperitaL. proposed by Clark and Menary (1980) and later supported bySrivastava and Sharma (1991). Thus, the significant increase in theabove mentioned yield parameters of the TRIA treated plants mightpossibly culminate in maximization of the leaf-yield, herbage yieldand the EO content of mint plant in the present study.

The positive role of TRIA in increasing growth, yield and qual-ity together with improving the physiological processes in variousmedicinal plants including Artemisia annua L. (Shukla et al., 1992;Aftab et al., 2010), Coriandrum sativum L. (Idrees et al., 2010), Cym-bopogon flexuosus Steud, Watts. (Misra and Srivastava, 1991), M.arvensis L. (Srivastava and Sharma, 1991; Naeem et al., 2011) andPapaver somniferum L. (Srivastava and Sharma, 1990) has earlierbeen reported.

The effect of TRIA on EO yield might be mediated through theTRIA-improved growth and metabolism as reported in this study(Table 2). TRIA might enhance the intrinsic genetic potential of theplants to produce additional production of EO. Additionally, sec-ondary metabolism might contribute to elevate the levels of EO

in mint crop. Thus, this study might provide more insight to therole of the PGRs employed in the secondary metabolism of mint.Ries and Houtz (1983) suggested that TRIA, like other plant hor-mones might activate enzymes or alter a membrane, which could

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rigger a cascading effect resulting in increased metabolism andhe enhanced accumulation of various critical intermediate com-ounds. The increase in the EO content in lavender, spearmint,

apanese mint, and coriander as a result of application of EBL,threl, gibberellic acid and TRIA has been reported in this contextSrivastava and Sharma, 1991; Youssef and Talaat, 1998; Singh et al.,999; Swamy and Rao, 2009; Idrees et al., 2010).

Moreover, there was reported a significant increase in EO accu-ulation in Japanese mint (M. arvensis L.) due to the application of

hlormequat chloride by Farooqi and Sharma (1988). Likewise, thepirostane analogs of brassinosteroids were found to increase theroduction of leaves as well as to elevate the levels of EO in hydro-onically grown mint (Maia et al., 2004). According to Swamy andao (2009), EBL, a brassinosteroid, caused a significant increase inhe content and yield of EO in geranium plant when it was appliedith 3 �M concentration of EBL. The improved content and yield

f EO in HBR-treated plants in the present study could perhapse ascribed to the enhanced rates of photosynthesis (Fig. 1b) asointed out by Swamy and Rao (2008, 2009) in the case of geranium.

Furthermore, it might be postulated that the positive effect ofBR on mint EO yield might be attributed to the HBR-improvedverall plant growth and metabolism as revealed by our studyTable 2). Thus, HBR-enhanced plant growth, photosynthesis andhe overall plant metabolism might have accounted significantly forO accumulation in the present study. Our results also corroboratehe findings of Shukla and Farooqi (1990), Srivastava and Sharma1991), Khan et al. (2007), Aftab et al. (2010), Idrees et al. (2010,011) and Naeem et al. (2012d) regarding various medicinal plantshich were positively influenced by different plant growth regula-

ors. A positive effect of HBR on mint EO yield and its componentsmenthol, menthone, isomenthone and menthyl acetate) has beeneported by Naeem et al. (2012d) also. However, combined effectf ISA with TRIA and HBR was much effective in increasing the levelf content and yield of EO in the fresh leaves at 100 as well as 120AP (Table 2).

. Conclusion

The combined application of the tested PGRs could significantlymprove the growth attributes, physiological activities, herbageield and content and yield of EO and those of its components.urther, the present study indicated that the combined applica-ion of the PGRs employed was positively significant to increase theesired production of mint EO compared to the individual applica-ion of applied treatments. We hope that tested combination maye applied in future to achieve the desired quality of several medic-

nal and aromatic plants. Further, this technique may be safelydopted for boosting up the growth, yield and quality of medicinalnd other crop plants.

cknowledgements

Financial support by the Science and Engineering Researchouncil, Department of Science & Technology, Government of India,ew Delhi, for the award of Young Scientist to Dr. Mu. Naeem

Project No. SR/FT/LS-003/2008) is gratefully acknowledged.

eferences

dams, P.R., 2007. Identification of Essential Oil Components by Gas Chromatogra-phy/Mass Spectrometry. Allured Publishing Corporation, Carol Stream, IL, USA.

faq, S.H., Tajuddin, Siddiqui, M.M.H., 1994. Standardization of Herbal Drugs. A.M.U.

Press, Aligarh.

ftab, T., Khan, M.M.A., Idrees, M., Naeem, M., Singh, M., Ram, M., 2010. Stimulationof crop productivity, photosynthesis and artemisinin production in Artemisiaannua L. by triacontanol and gibberellic acid application. J. Plant Interact. 4,273–281.

d Products 55 (2014) 272–279

Aftab, T., Khan, M.M.A., Idrees, M., Naeem, M., Moinuddin, Hashmi, N., Varshney,L., 2011. Enhancing the growth, photosynthetic capacity and artemisinin con-tent in Artemisia annua L. by irradiated sodium alginate. Radiat. Phys. Chem. 80,833–836.

Aftab, T., Naeem, M., Idrees, M., Khan, M.M.A., Moinuddin, Varshney, L., 2013. Cumu-lative role of irradiated sodium alginate and nitrogen fertilizer on growth,biochemical processes and artemisinin production in Artemisia annua. Ind. CropProd. 50, 874–881.

Akimoto, C., Aoyagi, H., Tanaka, H., 1999. Endogenous elicitor-like effect of algi-nate on physiological activities of plant cells. Appl. Microbiol. Biotechnol. 52,429–436.

Alam, M.M., Hayat, S., Ali, B., Ahmad, A., 2007. Effect of 28-homobrassinolidetreatment on nickel toxicity in Brassica juncea. Photosynthetica 45,139–142.

Arora, N., Bhardwaj, R., Sharma, P., Arora, H.K., 2011. Effects of 28-homobrassinolideon growth, lipid peroxidation and antioxidative enzyme activities in seedlingsof Zea mays L. under salinity stress. Acta Physiol. Plant. 30, 833–839.

Chen, X., Yuan, H., Chen, R., Zhu, L., He, G., 2003. Biochemical and photochemicalchanges in response to triacontanol in rice (Oryza sativa L.). Plant Growth Regul.40, 249–256.

Clark, R.J., Menary, R.C., 1980. Environmental effects on peppermint (Mentha piperitaL.). II: Effects of temperature on photosynthesis, photorespiration and dark res-piration in peppermint with reference to oil composition. Aust. J. Plant Physiol.7, 693–697.

Darvill, A., Auger, C., Bergmann, C., Carlson, R.W., Cheong, J.J., Eberhard, S., Hahn,M.G., Lo, V.M., Marfa, V., Meyer, B., Mohnen, D., OíNeill, M.A., Spiro, M.D., Hal-beek, H., York, W.S., Albersheim, P., 1992. Oligosaccharins–oligosaccharides thatregulate growth, development and defense response in plants. Glycobiology 2,181–198.

Dimitrios, B., 2006. Sources of natural phenolic antioxidants. Trends Food Sci. Tech-nol. 17, 505–512.

Dwivedi, R.S., Randhawa, N.S., 1974. Evaluation of rapid test for the hidden hungerof zinc in plants. Plant Soil 40, 445–451.

El-Rehim, A.H.A., 2006. Characterization and possible agricultural application ofpolyacrylamide/sodium alginate crosslinked hydrogels prepared by ionizingradiation. J. Appl. Polym. Sci. 101, 3572–3580.

Farooqi, A.H.A., Sharma, S., 1988. Effect of growth retardants on growth and essentialoil content in Japanese mint. Plant Growth Regul. 7, 39–45.

Guenther, E., 1972. The Essential Oils: History-Origin in Plants Production-Analysis,vol. 1. Robert E. Krieger Publishing Company, Huntington, New York.

Hien, N.Q., Nagasawa, N., Tham, L.X., Yoshii, F., Dang, H.V., Mitomo, H., Makuuchi, K.,Kume, T., 2000. Growth promotion of plants with depolymerised alginates byirradiation. Radiat. Phys. Chem. 59, 97–101.

Hu, X., Jiang, X., Hwang, H., Liu, S., Guan, H., 2004. Promotive effects of alginate-derived oligosaccharide on maize seed germination. J. Appl. Phycol. 16, 73–76.

Idrees, M., Khan, M.M.A., Aftab, T., Naeem, M., 2010. Synergistic effects of gibberellicacid and triacontanol on growth, physiology, enzyme activities and essentialoil content of Coriandrum sativum L. Asian Aust. J. Plant Sci. Biotechnol. 4, 24–29.

Idrees, M., Naeem, M., Alam, M., Aftab, T., Hashmi, N., Khan, M.M.A., Moinuddin,Varshney, L., 2011. Utilizing the gamma irradiated sodium alginate as a plantgrowth promoter for enhancing the growth, physiological activities and alka-loids production in Catharanthus roseus L. Agric. Sci. China 10, 1213–1221.

Idrees, M., Nasir, S., Naeem, M., Aftab, T., Khan, M.M.A., Moinuddin, Varshney,L., 2012. Gamma irradiated sodium alginate induced modulation of phospho-enolpyruvate carboxylase and production of essential oil and citral content oflemongrass. Ind. Crop Prod. 40, 62–68.

Ivanov, A.G., Angelov, M.N., 1997. Photosynthesis response to triacontanol correlateswith increased dynamics of mesophyll protoplast and chloroplast membranes.Plant Growth Regul. 21, 145–152.

Iwasaki, K., Matsubara, Y., 2000. Purification of alginate oligosaccharides with rootgrowth-promoting activity toward lettuce. Biosci. Biotechnol. Biochem. 64,1067–1070.

Jenkins, G.L., Knevel, A.M., Digangi, F.E., 1967. Quantitative Pharmaceutical Chem-istry, 6th ed. McGraw Hill Book Company, London.

Khan, R., Khan, M.M.A., Singh, M., Nasir, S., Naeem, M., Siddiqui, M.H., Mohammad,F., 2007. Gibberellic acid and triacontanol can ameliorate the optimum yieldand morphine production in opium poppy (Papaver somniferum L.). Acta Agric.Scand. Sect. B: Soil Plant Sci. 57, 307–312.

Khan, M.M.A., Bhardwaj, G., Naeem, M., Moinuddin, Mohammad, F., Singh, M., Nasir,S., Idrees, M., 2009. Response of tomato (Lycopersicon esculentum Mill.) to appli-cation of potassium and triacontanol. Acta Hort. (ISHS) 823, 199–207.

Khan, Z.A., Khan, M.M.A., Aftab, T., Idrees, M., Naeem, M., 2011. Influence of algi-nate oligosaccharides on growth, yield and alkaloid production of opium poppy(Papaver somniferum L.). Front. Agric. China 5, 122–127.

Khripach, V., Zhabinskii, V., Groot, A.D., 2000. Twenty years of brassinosteroids:steroidal plant hormones warrant better crops for the XXI century. Annal. Bot.86, 441–447.

Kumaravelu, G., David, L.V., Ramanujam, M.P., 2000. Triacontanol-induced changesin the growth, photosynthetic pigments, cell metabolites, flowering and yield ofgreen gram. Biol. Planta 43, 287–290.

Kume, T., Nagasawa, N., Yoshii, F., 2002. Utilization of carbohydrates by radiationprocessing. Radiat. Phys. Chem. 63, 625–627.

Lichtenthaler, H.K., Buschmann, C., 2001. Chlorophylls and Carotenoids: Measure-ment and Characterization by UV–Vis Spectroscopy. John Wiley and Sons, NewYork, pp. F4.3.1–F4.3.8 (Curr. Prot. Food Anal. Chem.).

ps and

L

M

M

M

M

M

M

N

N

N

N

N

N

N

O

R

R

R

S

on the growth and chemical constituents of lavender plant. Ann. Agric. Sci.

M. Naeem et al. / Industrial Cro

uan, L.Q., Hien, N.Q., Nagasawa, N., Kume, T., Yoshii, F., Nakanishi, T.M., 2003. Bio-logical effect of radiation-degraded alginate on flower plants in tissue culture.Biotechnol. Appl. Biochem. 38, 283–288.

a, L.J., Li, X.M., Bu, N., Li, N., 2010. An alginate-derived oligosaccharide enhancedwheat tolerance to cadmium stress. Plant Growth Regul. 62, 71–76.

aia, N.B., Bovi, O.A., Zullo, M.A.T., Perecin, M.B., Granja, N.P., Carmello, Q.A.C.,Robaina, C., Coll, F., 2004. Hydroponic cultivation of mint and vetiver withspirostane analogues of brassinosteroids. Acta Hortic. (ISHS) 644, 55–59.

isra, A., Srivastava, N.K., 1991. Effects of the triacontanol formulations “Miraculan”on photosynthesis, growth, nutrient uptake, and essential oil yield of lemongrass(Cymbopogon flexuosus) Steud, Watts. Plant Growth Regul. 10, 57–63.

isra, P.N., Hasan, S.A., Kumar, S., 2000. Cultivation of Aromatic Plants in India, Isted. Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India.

ollah, M.Z.I., Khan, M.A., Khan, R.A., 2009. Effect of gamma irradiated sodium algi-nate on red amaranth (Amaranthus cruentus L.) as growth promoter. Radiat. Phys.Chem. 78, 61–64.

uthuchelian, K., Velayutham, M., Nedunchezhian, N., 2003. Ameliorating effectof triacontanol on acidic mist-treated Erythrina variegata seedlings: changes ingrowth and photosynthetic activities. Plant Sci. 165, 1253–1257.

aeem, M., Khan, M.M.A., Moinuddin, Siddiqui, M.H., 2009. Triacontanol stimu-lates nitrogen-fixation, enzyme activities, photosynthesis, crop productivity andquality of hyacinth bean (Lablab purpureus L.). Sci. Hort. 121, 389–396.

aeem, M., Khan, M.M.A., Moinuddin, Idrees, M., Aftab, T., 2011. Triacontanol-mediated regulation of growth and other physiological attributes, activeconstituents and yield of Mentha arvensis L. Plant Growth Regul. 65, 195–206.

aeem, M., Idrees, M., Aftab, T., Khan, M.M.A., Moinuddin, Varshney, L., 2012a. Irradi-ated sodium alginate improves plant growth, physiological activities, and activeconstituents in Mentha arvensis L. J. Appl. Pharm. Sci. 2, 28–35.

aeem, M., Idrees, M., Aftab, T., Khan, M.M.A., Moinuddin, Varshney, L., 2012b.Depolymerised carrageenan enhances physiological activities and menthol pro-duction in Mentha arvensis L. Carbohydr. Polym. 87, 1211–1218.

aeem, M., Khan, M.M.A., Moinuddin, 2012c. Triacontanol: a potent plant growthregulator in agriculture. J. Plant Interact. 7, 129–142.

aeem, M., Idrees, M., Alam, M., Aftab, T., Khan, M.M.A., Moinuddin, 2012d.Brassinosteroid-mediated enrichment in yield attributes, active constituentsand essential oil production in Mentha arvensis L. Russ. Agric. Sci. 38, 106–114.

atsume, M., Kamao, Y., Hirayan, M., Adachi, J., 1994. Isolation and characteriza-tion of alginate derived oligosaccharides with root growth promoting activities.Carbohydr. Res. 258, 187–197.

kabe, K., Lindlar, A., Tsuzuki, M., Miyachi, S., 1980. Effects of carbonic anhy-drase on ribulose 1,5-biphosphate carboxylase and oxygenenase. FEBS Lett. 114,142–144.

ies, S.K., 1985. Regulation of plant growth with triacontanol. Crit. Rev. Plant Sci. 2,239–285.

ies, S., 1991. Triacontanol and its second messenger 9-�-L(+)-adenosine as plant

growth substances. Plant Physiol. 95, 986–989.

ies, S., Houtz, R., 1983. Triacontanol as a plant growth regulator. HortSci. 18,654–662.

adasivam, S., Manickam, A., 2008. Biochemical Methods, 3rd ed. New Age Interna-tional (P) Ltd. Publishers, New Delhi.

Products 55 (2014) 272–279 279

Sarfaraz, A., Naeem, M., Nasir, S., Idrees, M., Atab, T., Hashmi, N., Khan, M.M.A.,Moinuddin, Varshney, L., 2011. An evaluation of the effects of irradiated sodiumalginate on the growth, physiological activity and essential oil production offennel (Foeniculum vulgare Mill.). J. Med. Plant. Res. 5, 15–21.

Sharma, K., Kaur, H., Thind, S.K., 2006. Kinetin and triacontanol effects on leaf char-acteristics, nitrate reductase activity, nodulation and yield in soybean Glycinemax (L.) Merrill under reduced light intensity. Environ. Ecol. 24, 426–429.

Sharma, I., Pati, P.K., Bhardwaj, R., 2011. Effect of 28-homobrassinolide onantioxidant defence system in Raphanus sativus L. under chromium toxicity.Ecotoxicology 20, 862–874.

Shukla, A., Farooqi, A.H.A., 1990. Utilization of plant growth regulators in aromaticplant production. Curr. Res. Med. Arom. Plants 12, 152–157.

Shukla, A., Farooqi, A.H.A., Shukla, Y.N., Sharma, S., 1992. Effect of triacontanol andchlormequat on growth, plant hormones and artemisinin yield in Artemisiaannua L. Plant Growth Regul. 11, 165–171.

Singh, P., Srivastava, N.K., Mishra, A., Sharma, S., 1999. Influence of ethrel andgibberellic acid on carbon metabolism, growth, essential oil accumulation inspearmint (Mentha spicata). Phtosynthetica 36, 509–517.

Srivastava, N.K., Sharma, S., 1990. Effect of triacontanol on photosynthesis, alka-loid content and growth in opium poppy (Papaver somniferum L.). Plant GrowthRegul. 9, 65–71.

Srivastava, N.K., Sharma, S., 1991. Effect of triacontanol on photosynthetic char-acters and essential oil accumulation in Japanese mint (Mentha arvensis L.).Photosynthetica 25, 55–60.

Swamy, K.N., Rao, S.S.R., 2006. Influence of brassinosteroids on rooting and growthof geranium (Pelargonium sp.) stem cuttings. Asian J. Plant Sci. 5, 619–622.

Swamy, K.N., Rao, S.S.R., 2008. Effect of 28-homobrassinolide on growth, photosyn-thesis, and essential oil content of geranium [Pelargonium graveolens (L.) Herit].Am. J. Plant Physiol. 3, 173–179.

Swamy, K.N., Rao, S.S.R., 2009. Effect of 24-epibrassinolide on growth, photosynthe-sis, and essential oil content of Pelargonium graveolens (L.) Herit. Russ. J. PlantPhysiol. 56, 616–620.

Swamy, K.N., Rao, S.S.R., 2011. Effect of brassinosteroids on the performance ofColeus (Coleus forskohlii). J. Herb. Spice Med. Plant. 17, 12–20.

Talaat, N.B., Abdallah, A.M., 2011. Effect of 28-homobrassinolide and 24-epibrassinolide on the growth, productivity and nutritional value of two fababean (Vicia faba L.) cultivars. Arch. Agron. Soil Sci. 56, 649–669.

Tassou, C.C., Nychas, G.J.E., Skandamis, P.N., 2004. Herbs and spices and antimicro-bials. In: Peter, K.V. (Ed.), Handbook of Herbs and Spices. Woodhead PublishingLimited, Abington Hall, Abington, Cambridge CB1 6AH, England, pp. 35–53.

The Wealth of India, 1992. In: Ambusta, C.S. (Ed.), Raw Materials, vol. 2. Publicationand Information Directorate, CSIR, New Delhi, pp. 349–352.

Watson, D.J., 1958. The dependence of net assimilation rate on leaf-area index. Ann.Bot. 22, 37–54.

Youssef, A.A., Talaat, I.M., 1998. Physiological effect of brassinosteroids and kinetin

(Cairo) 43, 261–272.Zhang, M., Zhai, Z., Tian, X., Duan, L., Li, Z., 2008. Brassinolide alleviated the adverse

effect of water deficits on photosynthesis and the antioxidant of soybean (Glycinemax L.). Plant Growth Regul. 56, 257–264.