INTRODUCTION

1. Aromatic Plants :
The term aromatic comes from the word aroma meaning fragrance or flavour. Aromatic
plants, also known as herbs and spices, have been used from times immemorial for their
preservative and medicinal properties, in addition to enhancing aroma and flavour of foods
(Chang et al. 2000; Li et al. 2006; Piccaglia et al. 1993). Aromatic plants contain odorous,
volatile, hydrophobic and highly concentrated compounds called essential oils (or volatile or
ethereal oils). The term essential oil is concomitant to fragrance or perfumes because these
fragrances are oily in nature and they represent the essence or the active constituents of the
plants. These are obtained from various parts of the plant such as flowers, buds, seeds,
leaves, twigs, bark, wood, fruits and roots (Brenes et al. 2010; Negi et al. 2012). The
essential oils are complex mixtures of secondary metabolites consisting of low-boiling-point
phenylpropenes and terpenes (Greathead et al. 2003). Essential oils are the most important
raw materials of the fragrance and aroma industry. They are also used in the food and
pharmaceutical industries due to their therapeutic, antimicrobial and antioxidant activities.
Nevertheless, they have biological activities that make them able to be used as herbicides,
pesticides and anticancer compounds (Mahmoud and Croteau 2002; Abrahim et al. 2003;
Burfield and Reekie 2005; El Tamer 2005). The oils are usually extracted by steam
distillation while currently the use of supercritical carbon dioxide has become increasingly
popular. Essential oils of aromatic plants also play an important role in aromatherapy. This
form of complementary and alternative medicine is used for the prevention and treatment of
certain disorders. Two different modes of action have been suggested: One is directly related
to the pharmacological effects of essential oils. The other relates more to the positive
influence of aromatic substances on the limbic system of the brain via the olfactory system.
The aroma therapy for management of pain, anxiety, depression, hypertension etc. is also
very useful (Yim et al. 2009; Setzer 2009; Hur et al. 2012; Tillet and Ames 2010; Lee et
al. 2011; Smith et al. 2011).

FIGURE 1: ACTIVITIES AND USES OF AROMATIC PLANTS

antimicrobial
activity
Anticoccidal activity
antioxidant activity
Main activities

Aromatic plants
,extracts, essential
oils
Uses
e.g

Pharmaceutical industry

medicines
Feed additives e.g., antioxidants,
Growth promoters

cosmetic industry
food industry

e.g. skin products

e.g.,flavours,fragrances

1.1Palmarosa(Cymbopogon martinii) :
Aromatic plants, basically Lemongrass and Palmarosa, have been the topic of study.
Lemongrass(Cymbopogon flexusous) belongs to the family Poaceae and is commonly cultivated
as culinary and medicinal herb because of their scent, resembling that of lemons (Soenarko and
S 1977).It contains an active compound called citrel responsible for the lemon flavour

Palmarosa (Cymbopogon martinii [Roxb.] Wats. Var. motia) is a perennial grass, widely
distributed in tropical and subtropical regions. It contains essential oil, whose main components
are geraniol and geranyl acetate (Khanuja et al. 2005). The essential oil from C. martinii is
widely used as a valuable component for perfumes, cosmetic and pharmaceutical products
(Akhila et al. 2009; Singh et al. 1996)and against the action of various bacteria, fungi and
microorganisms (Rajeswara et al. 2009; Duarte et al. 2005). The essential oil also has some of
the important properties such as insect repellent and is also used in aromatherapy (Fatima et al.
2002). Due to these applications, essential oil demand in the domestic and international market
has increased, which has stimulated its cultivation. Used extensively as a fragrance component in
cosmetics, perfumes and especially soaps due to excellent tenacity. Aroma therapists recommend
it as an oil to diffuse during flu epidemics. Its action against viral illnesses and against bacteria,
together with its pleasant smell makes it a great oil to use to disinfect a room. Palmarosa oil
blends well with cananga, geranium, oak moss, rosewood, amyris, sandalwood, guaiacwood,
cedarwood and floral oils.
C. martini oil is being distilled in India for more than 50 years. However, till very recently most
of this oil except for one plantation in Dehradun, was obtained from wild growth in Madhya
Pradesh, Maharashtra and Andhra Pradesh. During this period a number of other countries
including Guatemala and Brazil have taken up the production of quality Palmarosa Oil with the
result that production of Indian Palmarosa Oil has declined. From the past decade, a number of
agronomical experiments have been carried out with the result that a number of Palmarosa
plantations producing high grade oil have been developed, mainly in Karnataka, Uttar Pradesh
and Orissa. It is estimated that both from cultivated and natural source about 60-70 tonnes of oil
is now being produced annually in India.

FIG.2 : Palmarosa (Cymbopogon martinii) cultivar grown in Integral university garden

There are numerous reports in the literature concerning the effects of growth regulators on
growth and development of various aromatic plants (Sangwan et al. 2001). Most of these growth
substances have exhibited influences by modifying the growth characters. Gibberellins are
endogenous plant hormones, which improve the growth of plants. In aromatic plants, where
active principle is generally present in the leaves or flowers, gibberellins have been used to
improve growth and herb yield of aerial parts of the plants. Gibberellic acid (GA ) also
3

significantly enhanced the foliage yield of Cymbopogon jwarancusa (Ansari et al. 1988). Auxins
and cytokinins constitute other major groups of plant hormones. Auxins are well known for their
role in cell elongation and differentiation in the plants. Similarly, role of cytokinins in cell
division and differentiation in the plants is well established. Both these hormones have been used
for improvement of growth and development of aromatic plants. Indole acetic acid (IAA) also
stimulated leaf growth in C. Jwarancusa (Ansari et al. 1988). Effect of kinetin on herbage and
5

oil yield has been reported earlier in Mentha specie (Farooqi et al. 2003; Keltawi and Croteau
1987). The present study was under-taken to examine the effect of plant growth regulators on the
essential oil biosynthesis in Palmarosa and plant growth.
Biosynthesis of terpenoids is dependent on primary metabolism, e.g. photosynthesis and
oxidative pathways for carbon and energy supply (Sangwan et al. 2001). Therefore in this study,
effect of plant growth regulators on primary metabolic parameters like nitrate reductase (NR)
activity, chlorophyll content and protein content was also determined.
1.2 Plant Growth Regulators :
These are organic compounds either natural or synthetic, that modify or control one or more
specific, physiological process within a plant. They are also called plant growth substance. They
are required in very low concentration ranging from 10-6 molar to 10-5molar (Balakrishnan et al.
2011) with high application rates of 1M same compound often being herbicidal. Those
substances laborated by the plant are referred to as phytohormones whereas others are called as
synthetic plant hormones.
There are five major endogenous plant growth substances. These are auxin, gibberelin,
cytokines, abscisic acid and ethylene. Moreover, plants contain numerous other molecules that
are active in various aspects of growth and development, for example Brassinolide, phenolics,
salicylic acid, ethrel. Phytohormones occur in free or conjugated with sugars, amino acid and
possibly peptides. Free forms are considered to be biologically active while conjugates are
viewed as functioning in controlling levels of the more active free forms. There are no specific or
specialized glands that produce hormones. These are synthesised in the plant and act at any part
as their target. They are transported via xylem and/or phloem and by diffusion. They differ in
their effects (Mir et al. 2010).
1.2.1

Mechanism of action of plant growth regulators :

There are two general classes of hormones found in animal system-steroid and peptides both of
which probably also occur in plant system. This steroid class forms a hormone receptor complex
in the cytoplasm, which is then transported into nucleus where mRNA is synthesised, resulting in
a given response. The peptide class binds to a receptor at a plasma membrane forming hormone
6

receptor complex, which causes the synthesis of secondary messenger for a given response. Each
receptor is specific to one hormone (Arteca 1996).
AUXINS: The word auxin was derived from Greek word auxein meaning to grow. These are
organic compounds having a ring structure and a side chain. A number of auxin including indole
3-acetic acid(IAA), indole 3-butyric acid, phenyl acetic acid and 4-chloro-indole acetic acid have
been isolated from different parts of plant.
They are generally produced by the growing apices of the stem and roots, from where they
migrate polarly through phloem to the regions of their action. However, they may also be
produced in young leaves, seeds and fruits of higher plants. Typical IAA concentration ranges
from 0.01 to 3 mg per litre (www.sigmodder.com).
Auxin enhance a number of physiological proceeses in plants, including abscission of older
mature leaves and fruits, apical dominance, cell division, cell enlargement, distribution of growth
between primary and lateral root and shoot meristems, flowering and parthenocarpy, loosening of
cell wall, root inhibition, synthesis of ribonucleic acid(RNA), deoxyribonucleic acid(DNA)and
protein(Hanisak 1997; Walz et al. 2002; Debi et al. 2005;Yamazoe et al. 2005;Boohkorkaew
et al. 2008)

Indole-3 -acetic acid

GIBBERELIN: The word gibberelin was coined by Yabula and Sumiki(1938) after the genus
name of the fungus Gibberella fugikuroi from which the active factor was isolated. These are
tetracyclic diterpenoid acid. Over 136 naturally occurring gibberelin have been isplated from
plants, fungi, and bacteria(McMillian 2002). These are abbreviated as GA with a subscript such
as GA1, GA2, GA3 and so on. GA3, also known as gibberelic acid, is commonly available and
most widely used in plant physiological research (Noggle and Fritz 1986). It was isolated and
7

characterized by two groups, one headed by cross in England and one by Stodole in the
USA(Brian et al. 1954; Stodola et al. 1955).

Cytokinin (CYT): It is a phytohormone that participates in events in the course of whole plant
ontogeny, from fecundated ovule to senescence and death. It is present in processes such as cell
division, shoot initiation and growth, senescence delay and photomorfogenic development,
control of chloroplast division and growth, modulation of metabolism and morphogenesis in
response to environmental stimulus (Kieber 2002; Pozo et al. 2005).

Cytokinin
Abscisic acid (ABA): It has great importance in developmental processes and seed germination,
as the induction of seed dormancy, protein and lipid synthesis, tolerance to desiccation and
inhibition of the embryonic to vegetative development. In mature plants ABA acts on the
response to drought through stomata aperture. It also acts on adaptation to stress conditions like
low temperature, salinity, hypoxia and in response to pathogen attacks. In a general way ABA is
considered a hormone with inhibitory activity on growth (Nambara and Marion-Poll 2005;
Pozo et al. 2005).

Abscisic acid
Ethylene (ET): It is a phytohormone that acts on seedgermination, shoot and root growth, flower
development, flower and leaf senescence and abscission and fruit maturation. Its particularity is
the emission by diffusion as a gas. It is also associated with plant defense, acting on the induction
of xylem inclusions and phytoalexins synthesis.Ethylene production can be induced by factors
like drought, inundation, ozone exposure, or mechanical injury, which associate it with stress
responses (Taiz and Zeiger 2004; Adie et al. 2007).

Ethylene

AIMS AND OBJECTIVES

1.3

The following were the objectives of this study 10

 To study the effect of plant growth regulators (GA3, IAA, K, Ethrel) on growth and
essential oil yield in Palmarosa. Following parameters were investigated
Measurements of growth parameters like plant height, leaves area, tiller no and herb yield
Estimation of oil content
Nitrate reductase activity
Peroxidase activity
Protein estimation
Chlorophyll Estimation
Proline content determination
Oil extraction by hydro-distillation using clevanger apparatus.
Determination of oil composition by GLC/GC-MS.

11

REVIEW OF LITERATURE

2. LITERATURE REVIEW :
12

The oil of Cymbopogon martinii(Palmarosa) is one of the most important essential oil-bearing
herbaceous species of the Poaceae family because of its high geraniol content. The oil of
Cymbopogon martini is used as base for fine perfumery and is valued because of its geraniol
content. Tamuli et al(2012) studied the quality of essential oil in Palmarosa(Cymbopogon
martinii) and found that the oil from healthy leaves yielded 69.48% geraniol. Another major
component geranyl acetate was found at about 17.82% in the oil from healthy leaves. Besides the
perfumery value, the oil has a great wound healing effect. There are several other aromatic
grasses of the genus Cymbopogon, which are cultivated for their essential oils i.e. lemongrass
and citronella java. Lemongrass is one of the most widely grown essential oil plants in the
tropics and subtropics of India, Indonesia, Madagascar, and countries in Africa and South
America (Anaruma et al., 2010; Ganjewala and Luthra 2010; Weiss 1997). Lemongrass
biomass is steam distilled for the extraction of essential oil, a natural product with wide
application in the food and pharmaceutical industries, perfumery and cosmetics, and eco-friendly
pesticides (Ganjewala and Luthra 2010; Weiss 1997). Lemongrass oil has a pleasant and
refreshing aroma and antifungal and antibacterial properties (Anaruma et al. 2010; Guynot et
al. 2003; Pandey et al. 2003; Kumar et al. 2007, 2009;). However, lemongrass essential oil
production is currently limited to the tropics and subtropics. The lemon- like aroma is due to
presence of citral.
Farooqi et al.(2015) examined the response of gibberelic acid(GA3) on herbage yield, essential
oil content and oil composition in Palmarosa (Cymbopogon martinii) and reported that
Gibberellins are endogenous plant hormones, which improve the growth of plants. In aromatic
plants, where active principle is generally present in the leaves or flowers, gibberellins have been
used to improve growth and herb yield of aerial parts of the plants. Gibberellic acid (GA 3) also
significantly enhanced the foliage yield of Cymbopogon jwarancusa.

Plant growth was

improved by GA3 significantly, and the increase was maximum at 100 ppm concentration in
plant height, leaf area, tiller number and herbage yield. Chlorophyll content, protein content, NR
activity and oil content increased in the plants due to GA 3 treatment compared to untreated
plants, and the increase was maximum at 100 ppm concentration.
Farooqi et al.(2000) claimed that aromatic grasses have great potential as agro and social
forestry plants and for wasteland reclamation with their proven soil binding properties.
13

Maudu et al. (2011) conducted a study to determine the effect of gibberellins on sprouting of
cuttings and subsequent quality of bush tea (Athrixia phylicoides DC). The study was conducted
under a 40% shade net with light/shading as a gradient. Treatments consisted of gibberellins
(Progibb 40%) applied at various rates as follows: 0, 1, 2, 3 and 4%. For growth parameters such
as plant height, number of branches, leaf area and fresh biomass, best results were recorded at 3
and 4% gibberellin application rates. Chemical attributes in the form of total polyphenols, and
total antioxidants tended to decline with increasing gibberellin application rate. However, tannin
content peaked at 2% gibberellin application rate. Overall, gibberellin application improved bush
tea growth and chemical attributes.
Rabie(1996) stated that Gibberellic acids (GA3) are the most important natural growth
regulators in use. They are used to induce major changes in the growth required to increase the
quantity and quality of edible characters, chemical composition and yield criteria of Hibiscus
sabdariffa L. Plants.
El-Keltawi & Croteau(1987) explained that plant growth regulators that exert their effect at the
level of gibberelin metabolism might increase the accumulation of essential oil in plants. Growth
retardants such as phosphone D and chlormequat chloride(CCC) which influence gibberelin
metabolism have been shown to increase terpene formation resulting in increased essential oil
content of peppermint and sage.
Swain and Singh(2005) found that GA3 exerts profound effects on fundamental process of plant
growth and development in Mentha piperita. GA3 is widely regarded as a growth promoting
compound that positively regulates processes such as seed germination, stem elongation and leaf
expansion. GA3 application was reported to increase weight of aerial parts in Viola (Vlahos,
1991).
Bose et al.(2013) found that GA3 improved plant biomass and stolon yield, leaf area, branching
and leaf stem ratio and essential oil synthesis in Mentha arvensis L which is the main source of
menthol rich essential oil used commercially in various food, pharmaceutical and other
preparations.

14

Hazzoumi et al.(2014) studied the effect of gibberelic acid(GA3) on the content and composition
of essential oils of the aromatic plant Basil (Ocimum gratissimum L), especially on the main
compound methyl chavicol and its isomer (the trans-anethole). GA 3 caused a slight decrease in
the oil yield (0.2%), but it increased the diversity of compounds (17 molecules) with the
appearance of four new compounds (naphthalene, camphor, germacrene-D, and ledene) and
disappearance of ( cedrene, azulene). This variation also caused a very important decrease in
the main compound methyl chavicol(from 75.16% in the control to 70.7%) and increases its
isomer (trans-anethole).
Hassanpouraghdam et al.(2011) conducted a pot experiment to evaluate the effects of GA 3 on
growth characteristics, essential oil content and yield in French lavender(Lavandula officinalis
Chaix), which is an important multidisciplinary aromatic plant with great use in pharmaceutical,
fragrance and food industries. The results showed that highest amounts for volatile oil content
and yield belonged to 300 mgl -1(ppm) GA3 treatment. Chlorophyll content as well as leaves fresh
and dry weight attained their greatest quantities under 300ppm GA3 application. Tissue nitrogen
and phosphorus content were affected by treatments with their highest concentration possessed
by 300 and 100mgl-1 GA3 applications respectively. Potaasium content was not affected by the
treatments.
Kapoor and Kaul(2006) found that application of GA3 to Chenopodium ambrosiodes
L.var.anthelminticum(L.) resulted in an increase of approximately 25 and 33% in the volatile oil
content and yield of fruits. Vegetative growth was also increased.
Azadeh et al.(2013) investigated the effect of pretreatment with gibberelic acid(GA 3) on
germination indices in Secale montanum. The results showed that pretreatment with gibberelic
acid increased the germination percentage, germination rate, root length and shoot length. GA3 at
50ppm at 100C and 100ppm at 100C had the most positive effect on germination and growth
factors, respectively.
Kavina et al.(2011) investigated the effect of gibberelic acid in Mentha piperita Linn.
(pippermint, Family: Lamiaceae). GA3 enhanced the fresh and dry weights and stem length
increased the larger extent when compared with control.

15

Poyh and Ono (2006) observed in sage (Salvia officinalis) treated with 100 mg L-1 of gibberellic
acid (GA3) resulted in higher essential oil content compared to control plants.
Misra et al.(2009) investigated the changes in growth parameters, 14C0 2 and [U-14C]-sucrose
incorporation into the primary metabolic pools and essential oil were investigated in leaves and
stems of Catharanthus roseus L. treated with gibberellic acid (GA3). Compared to the control,
GA3 treatment induced significant phenotypic changes and a decrease in chlorophyll content,
carbon assimilation rate, and stomatal conductance.
Xu et al.(1998) confirmed that exogenously applied, gibberellin promoted stolon elongation and
inhibited tuber formation and increased fresh weight in potato.
Rohamare et al.(2013) carried out a field experiment to investigate the effects of foliar
application of gibberellic acid (GA3) on growth, yield and essential oil components of Ajwain
(Trachyspermum ammi L.) .The results revealed that foliar application of GA3 significantly
enhanced the vegetative characters i.e. plant height, no. of branches and leaf area of ajwain in
addition to photosynthetic pigments, total carbohydrate, essential oil percentage, essential oil
yield/plant and thymol content. Ajwain essential oil composition was mainly characterized by
presence of thymol, p-cymene and terpinene.
Zhang et al.(2005) verified that after GA3 application (14 M) there was a 400% increase in the
concentration of artemisinin compared to control plants.
Ethrel or Ethephon or Bromeflor is one of the most widely used plant growth regulator. It acts as
a growth retarding agent and causes dwarfism in plants. As a result, stress is produced in plants
and more secondary metabolites are produced. Srivastava et al. (2008) evaluated Ethrel (2chloroethylphosphonic acid) in field trials as a chemical hybridizing agent on four varieties of
palmarosa (Cymbopogon martinii): PRC-1, Tripta, Trishna, and Composite. Application of 500
g/ml Ethephon to foliage either as a mist or by wrapping Ethephon-soaked cotton around the
stem during the vegetative and anthesis stages initiated increases in growth and yield attributable
to heterosis. A positive correlation between Ethephon application and yield of dry matter and
Ethephon application and essential oil with enhanced geraniol levels was obtained,
suggesting Ethephon treatment

is

an

effective

chemical

hybridizing

agent

in

palmarosa. Ethephon also appeared to stimulate ethylene production in flowers of palmarosa,

resulting in male sterility and premature anther-petal senescence. Although many environmental
16

groups worry about toxicity resulting from use of growth hormones and fertilizers, the toxicity of
ethephon is actually very low, and any ethephon used on the plant is converted very quickly to
ethylene (Joint meeting of FAO panel of experts 1994).
Farooqi et al.(1988) found that Ethephon at 0.06% concentration significantly decreased growth
of Japanese mint but had no significant effect on oil content. Menthone content was significantly
increased by 0.06% ethephon.
Cho et al.(1988) found an increased production of purine alkaloids by Coffea arabica cells by
addition of ethrel.
Misra et al.(2009) investigated the changes in growth parameters, 14C02 and [U-14C]-sucrose
incorporation into the primary metabolic pools and essential oil were investigated in leaves and
stems of Catharanthus roseus L. treated with ethrel. Compared to the control, etherel treatments
induced significant phenotypic changes and a decrease in chlorophyll content, carbon
assimilation rate, and stomatal conductance. Treatment with etherel led to increased total
incorporation of CO2 into the leaves where as total incorporation from 14C sucrose was
decreased. When 14CO2 was fed, the incorporation into the ethanol soluble fraction, sugars,
organic acids, and essential oil was significantly higher in etherel treated leaves than in the
control. Thus the overall growth was stunted.
Chaturvedi et al.(2009) studied the effect of ethrel in Saussurea costus (Falc.). Ethepon was
applied as foliar sprays (25, 50, 100 ppm and 1.0 mM and 1.5 mM) to greenhouse-grown plants
of Saussurea costus (Falc.) Lipsch. seedlings. Under stress conditions, the growth of the ethepon
treated seedlings was measured by leaf area, root length and root dry weight. A significant
increment in root length was recorded for all treatments and showed a concentration-dependent
pattern excluding for 50 ppm PCB treatment where the results were non-significant at 5% P
level. Photosynthetic rate (A), transpiration rate (E) and stomatal conductance (gS) were found
decreased in all the treatments as compared to control.
JC et al.(2001) found that small quantities of 2- chloroethylphosphonic acid (Ethephon or
17

Ethrel) were added to suspension cultures of Camellia sinensis. Growth was reduced by 20%
but production of phenolics and flavans increased by 90 and 75%, respectively.
Farooqi et al.(2007) found that Ethrel at 50, 100, 250 and 500 mgL -1 in Chrysanthemum
cinerariaefolium produced a significant positive effect on pyrethrins level, decreased plant
height, while 50 and 100 mg ll significantly increased fresh and dry flower yield.

Wenjing Chen and Roman Houbowicz(2010) showed that soaking the seeds of lettuce
(Lactuca sativa L.) in the ethereal oils solutions did decrease the percentage of dead seeds in the
tested samples. Soaking of the seeds in the ethereal oils solutions also increased the percentage
of healthy ungerminated seeds. Treating the seeds with the concentrations of the ethereal oils
solutions, although had no effect on their germination, still shortened the length of seedlings.
Moreover, the used ethereal oils lowered the amount of identified Alternaria alternata and
Cladosporium sphaerospermum fungi on the seeds.
Poyh and Ono (2006) confirmed that application of ethrel on sage (Salvia officinalis), which is
degraded when in contact with plant cells producing ethylene, in concentrations of 50 and 100
mg L-1, resulted in a decrease in plant height, yet there was an increase of 38-42% on fresh and
dry mass of flowers in relation to control plants. On the other hand, high concentrations (250 and
500 mg L-1) had negative effects not only on plant height, but also on flower production.
Haque et al. (2007) concluded that single flower mass was not influenced by application of
ethrel in Chrysanthemum cinerariaefolium.
Amalou et al.(1991) treated rubber tree (Hevea brasiliensis) bark with chloro-2-ethyl
phosphonic acid (ethrel), an ethylene-releasing chemical, induced, after a lag period of 13 to 21
hours, a marked increase in the total adenine nucleotides (essentially ATP and ADP) of latex
cells. This rise in the latex adenylate pool was concomitant with a marked decrease in the [ATP]/
[ADP] ratio without significant changes in the adenylate energy charge.
Haque et al.(2007) stated that in Chrysanthemum cinerariaefolium piretrin production increased
31 and 44% in relation to control for 50 and 100 mg L-1 ethrel concentrations, respectively. It was
18

also observed that under ethrel treatment there was a higher incorporation of 14CO2 in piretrins.
According to the authors this result indicates that ethrel could influence the activity of enzymes
linked to the piretrin biosynthetic pathway.
2.1 ESSENTIAL OIL OF AROMATIC PLANTS :
A number of plants have the capacity of synthesizing and storing aromatic substances and
commonly referred to as secondary plant products. These chemicals are alkaloids, terpenoids,
steroids, phenolics, etc. Since ancient times, the plant terpenoids have been used for flavours,
fragrance, food preservatives, perfumes and cosmetics (Loomis & Croteau, et al.,1980). The
terpenoids are ubiquitous in higher plants. The aromatic plants synthesize and accumulate
substantial quantities of monoterpenes that constitute the odoriferous volatile part of their
essential oil. Substantial quantities of essential oils are found in plants belonging to families
Labiatae, Rutaceae, Compositae, Rosaceae, Pinaceae and Graminae(Croteau et al., 1989).
The ever increasing commercialization of the secondary metabolites of plants has prompted man
to cultivate the aromatic and medicinal plants on an increasingly large scale(Hussain et al.,
1989). Oils contained within plant cells are liberated through heat and pressure from various
parts of the plant matter; for example, the leaves, flowers, fruit, grass, roots, wood, bark, gums
and blossom. The extraction of essential oils from plant material can be achieved by various
methods, of which hydro-distillation, steam and steam/water distillation are the most common
method of extraction (Bowles, 2003; Margaris et al., 1982; Surburg & Panten, 2006). Other
methods include solvent extraction, aqueous infusion, cold or hot pressing, effleurage,
supercritical fluid extraction and phytonic process (Da Porto et al., 2009).
The feeding with aromatic herbs, spices and some dietary supplements can supply the body with
essential oils. There are a lot of specific dietary sources of essential oils, such as example orange
and citrus peel, caraway, dill; cherry, spearmint, caraway, spearmint, black pepper and
lemongrass. Thus, human exposure to essential oils through the diet or environment is
widespread. However, only little information is available on the estimation of essential oil intake.
In most cases, essential oils can be absorbed from the food matrix or as pure products and cross
the blood brain barrier easily. This later property is due to the lipophilic character of volatile
19

compounds and their small size. The action of essential oils begins by entering the human body
via three possible different ways including direct absorption through inhalation, ingestion or
diffusion through the skin tissue.
Chemically,

essential

oils

represent

mainly

the

mixture

of

structurally

related

monoterpenoids(the C10- carbon compounds) and their derivatives. Plants are adapted to store
large quantities of these compounds in their plant parts, the most important being the leaves,
flowers or fruits and roots. Lavender oil (containing linalool and its acetate ester) is obtained
from the flowers of Lavendula vera. Rose oil which is rich in geraniol and its derivative
citronellal is also produce in petals of rose flowers(Rosa damacena).
2.1.1 ESSENTIAL OIL BIOSYNTHESIS IN CYMBOPOGON SPECIES :
The essential oil of Cymbopogons, the aromatic grasses are mainly made up of terpenoids and
have a varied combination of monoterpenes in their essential oils (Rejenduru and Das et al.,
1983). It is well established that terpenoids synthesized from 5-C units of isopentyl
pyrophosphate(IPP) and its isomer dimethyl allyl pyrophosphate (DMAPP). These monomeric
building blocks are biosynthetic equivalent of Ruzickas classical isoprene unit.
Specific prenyl transferases ( prenyl pyrophosphate synthase) couple desired number of IPP units
to DMAPP onwards leading to homologous series (C 10 mono- terpenoids; C15 sesquiterpenoids,
etc.) of phenyl pyrophosphatases. The monoterpenes are predominantly the product of secondary
metabolisms of plants. Modern methods of separation and structure determination and the advent
of radioisotope and 14C labelled techniques have led to vary rapid advances in the knowledge of
the routes of biosynthesis of mono terpenes. The biosynthesis of terpenoids/essential oils takes
place through the mevalonate isoprenoid pathway (Croteau et al., 1987). The mevalonate
pathway of terpene biosynthesis in plant is outlined in (Fig. 3).
Besides the ubiquitous mevalonate pathway, there are now reports of non mevalonate pathway
referred to as Rohmer pathway or 1- deoxy-D-xylulose-5-phosphate (DOXP) pathway.
According to this scheme, DOXP pathway starts with DOXP synthase catalyzed formation of 1deoxy-D-xylulose-3-phosphate (DOXP) from glyceraldehydes-3-P and pyruvate (condensation
via TPP dependent decarboxylation pyruvate derived acetyl aldehyde-TPP). DOXP is
subsequently transformed to 2-C-methyl-D-erythrotol-4-phosphate by an intramolecular C-C
20

skeleton rearrangement involving DOXP reducto-keto-isomerase in a mechanism similar to ketol

acid reducto-isomerase (KAKI) operational in the biosynthesis of valine, leucine and isoleucine
(Litchenthaler et al., 1999). Both the enzyme catalyze a C-C skeleton rearrangement followed by
a NADPH- dependent reduction step. 2-C-methyl-D-erythritol-4-phosphate ultimately gets
metabolically transposed to IPP (Fig.3).
Mevalonate (MVA) is formed from glycolitically generated acetyl CoA. Subsequently,
hydroxymethyl gluataryl CoA (HMG CoA) synthetase catalyses the formation of HMG CoA. A
soluble enzyme HMG CoA reductase catalyses the reductive deacylation of HMG CoA into
mevalonate (MVA). Successive enzymatic phosphorylation and decarboxylation of MVA yield
isopentenyl pyrophosphate (IPP), the basic 5 carbon containing isoprenoid unit. The IPP is
isomerised to dimethylallyl pyrophosphate (DMAPP). The IPP and DMAPP then produce
geranyl pyrophosphate (GPP), a C10 compound under the catalytic action of GPP synthetase. The
C10 compound which arises from mevalonate is the precursor of monoterpenes.
In Cymbopogon species the principal interest is the biosynthetic steps whereby the monoterpenes
diverge from other terpenoid compounds and specifically the conversion of geranyl
pyrophosphate to the acyclic monoterpenes, citral, geranial, citronellol, citronellal, neral, etc.
(Fig.4). Geraniol undoubtedly arises in-vitro by hydrolysis of the corresponding pyrophosphates
(Perez et al., 1980). Use of 14C and 3H labelled precursors reveal that leaf blades of Cymbopogon
flexuosus converted Geraniol into citral trans, whereas nerol lost the hydrogen while being
converted into citral cis. Secondly, the citral trans is converted into citral cis and vice-versa.
There is no separate route for the biosynthesis of two aldehyde isomers (Akhila et al., 1985).

21

FIG
Pyruvate+glycerald
ehyde-3-phosphate

Sucrose

Acetyl CoA

1-deoxyxylulose-5
phosphate

Hydroxymethyl glutaryl
CoA
Mevalona
te
Pathway

Mevalonate (MVA)

Nonmevalonate
Pathway

Mevalonate
pyrophosphate
(MVAPP)

Isopentyl
pyrophosphate
(IPP)

Dimethylallyl
pyrophosphate
(DMAPP)

Geranyl
pyrophosphate
(GPP)
Monoterpenes

FIG.3: General Scheme of Monoterpene biosynthesis

Sangwan et al(1993a) showed that the enzyme catalyses oxidation of geraniol into citral. It also
oxidises nerol but at slower rates. The enzyme was found to be located in cytosol which also
contains alcohol dehydrogenase activity. Cymbopogon cultivars differing in amount of citral and
geraniol in their essential oils were screened for geraniol dehydrogenase activity to discern the
feasibility of its serving as biochemical marker for oil yield and quality. The enzyme activity had
22

a positive and significant association with citral to geraniol. The results are suggestive of a strong
relationship between the enzyme activity and essential oil quality in Cymbopogon cultivars
(Sangwan et al., 1993b).

Geranyl pyrophosphate
(GPP)
Geranyl
acetate

Gerani
ol

Citronell
ol

Citronell
yl
acetate

Citronell
al
Geranial

Citral trans
Neral
Citral cis

Nerol

FIG. 4: Metabolic Interconversion Of Monoterpenes in Cymbopogon

sp.
Many evidences including the diurnal and seasonal fluctuation in monoterpene content, and the
time course of the incorporation of labelled precursors into monoterpenes indicate that
monoterpenes are in a state of metabolic flux. The oil content of leaves of Cymbopogon
winterianus decreased with maturity and exhibited seasonal and diurnal changes. Maximum
accumulation of citral occurred during the day when temperature was highest (Singh et
al.,1979). The percentage of oil is maximum during October and November months (Chandra et
23

al., 1970). The oil obtained from the harvests in the month of September showed marked rise in
the aldehyde content (maximum 45%) over other months of the year (Malwatkar et al.,1984),
(Verma et al,.1985) reported that in Cymbopogon khasianus, the increase in the feeding time of
the labelled precursor 2-14C acetate resulted in the decrease in the radioactivity of citral with a
corresponding increase in hydrocarbons and/or undentified

products. This supports the

suggestion that monoterpenes do undergo turnover in plant.

2.2 PHYSIOLOGY OF ESSENTIAL OIL PRODUCTION :
One of the most important characteristics of oil accumulation is its dependence on the
developmental stage of the plant as well as its concerned organ, tissue and the cells. The origin of
the leaves from primordial, there expansion to full maturity and finally loss through senescence
are particularly important in aromatic grasses in which the leaves from the source of this
commercially valuable oil. A close coordination between leaf ontogeny and oil accumulation and
biogenesis has demonstrated in many aromatic plants. (Singh et al., 1989) reported that net
essential oil production is associated with early growth period in C. flexuosus. Maximum oil
content (1.18%) in the leaves of C. martiniihas the end of blooming while flowers and
inflorescence possesses much higher oil as compared to leaves (Akhila et al., 1987; Sangwan et
al., 1994).
Ontogeny also effects the oil composition, but only rarely, to a very substantial extent e.g.in C.
martinii geraniol increases from 65% to 81% till flowering stage and has been demonstrated to
be formed at the expense of geranyl acetate (Sangwan et al., 1984). However, C. flexuosus does
not exhibit much pronounced effects in its composition. Its main constituent citral attains highest
value at the leaf age of 20 days and remains almost constant thereafter (Singh et al., 1989). Even
during the later stages of leaf growth and development, substantial increase in the in-vitro
activity of geraniol dehydrogenase involved in citral generation has also been reported. The
enzyme level has been shown to be well correlated with citral geraniol ratio not only with
difference in specie but also with the developmental stages (Sangwan et al., 1993b; Singh et al.,
1990).
Narain and Gupta (1948) showed that the flowering tops of C. martinii contained more oil than
other parts. Subba Roy et al., 1948 pointed out that oil content remain maximum for 1 week to
10 days after initiation of flowering. Gupta et al., 1978 found that C. martinii foliage oil had the
24

highest percentage of geaniol. Saxena et al., 1978 had observed that geraniol content in the oil
increase from 64.8% at vegetative state to 81.4% at the flowering stage. Pareek et al., 1981
reported the maximum oil content in C. martinii at flowering initiation, but the oil yield was
highest at the flower open stage and early seed formation stage when the plant biomass was
highest. Oil quality is dependent on the harvesting time. Akhila et al., 1984 reported maximum
oil (1.18%) in the leaves towards the end of blooming.Sangwan et al., 1985 reported high oil
content in the flower as well as maximum oil in a plant at the flowering time. During leaf
ontogeny the amount of citronellal, geraniol and citronellol in the essential oil increased with leaf
expansion whereas the amount of geranyl acetate and citronellyl acetate decreased. As the leaves
matured, a significant decrease in the essential oil, citronellal and geraniol, content was observed
(Luthra et al., 1991).
Thus in general leaf ontogeny strongly influences the expression of essential oil metabolism and
this type of integration of the oil production with the preset developmental program of the tissue
is particularly interesting.
2.2.1 Site of Oil Production :
Plant volatile oils are synthesized, stored and released to environment by a variety of epidermal
or mesophyll structures, whose morphology tends to be the characteristic of the taxonomic
group. These structures on leaves, roots, stem, floral part and fruits include oil cells, secretory
glands, glandular hairs or trichomoes which synthesize and accumulate large quantities of these
compounds (Fahlen et al., 1997). These epidermal appendages (glandular trichomes, glandular
hairs, resin ducts, etc. occur in different plant parts from flowers to roots with special attributes
individually The studies concerning the site of terpenoid biosynthesis in particular, have been
elaborately discussed by Croteaus group (Croteau,et al., 1977). Micro hairs containing oil have
been found on axial epidermis of the leaf lamina of citronella (Cymbopogon winterianus) but
could not be located on the adaxial face (lruthayathas & Herath,et al., 1982). Shanker et al.,
1999 reported that in Mentha arvensist rich oil glands were present on both the leaf surfaces. The
location of oil holding hair is restricted to the interveinal region. An intriguing correlation
between one of the anatomical characters and chemical composition of essential oil of citronella
has also been projected (lruthayathas & Herath, 1975). Using specific water soluble stains,
LewinSohfl et al(1998) have recently found that specific oil cells, present in parenchymal
25

tissues, are the sites of citral accumulation in lemongrass (Cymbopogon citratus) and lemongrass
leaf surface does not contain glandular trichomes, such as those present in many other aromatic
plants.
Primary metabolic processes like photosynthesis, respiration, etc. influence growth and
development of the plant as well as biogenesis of secondary plant products (Hess et al., 1975).
This is because the initial precursors for essential oil or other secondary products are provided by
the primary metabolic processes. Thus, essential oil metabolism is by and large controlled by the
balance between photosynthesis and utilization of photosynthate for growth and differentiation.
The ultimate partitioning of the photo-synthetically fixed carbon is an important component of
physiological mechanism of essential oil production. Therefore, photosynthetic characteristics
and performance of the tissue, among other factors, are at the centre stage in making carbon
shareable or separable for the anabolism of the oil components.
Herath and Ormord (1979) studied the rate of photosynthesis in the leaves of Lemongrass (C.
flexuosus). In peppermint leaf discs, photosynthetic electron transport when inhibited by DCMU
and Paraquat resulted into depression in the oil mono-terpenoids suggesting that photosynthetic
NADPH production may be at atleast partially cater to the transformation of monoterpene
ketones to alcohols (Maffei & Codignola, 1990). Clark and Menary (1980) have hypothesized
that in Mentha piperita, the production and utilization of photosynthates controls oil production.
Role of mobilization of photosynthetically generated transient starch has also been found to be
very substantial in Cymbopogon species (Singh et al., 1991).
2.3 CHANGES IN PLANT METABOLISM UNDER PLANT GROWTH REGULATOR:
2.3.1 Peroxidase :
Peroxidase (EC 1.11.1.7) enzymes are widely distributed in higher plants and have been
suggested to play roles in growth, development and lignification. (Inn et al., 1981; Van &
Cairnes, 1982; Stitch & Ebermann, 1984). They utilize hydrogen peroxide to oxidize a wide
range of hydrogen donors, such as phenolic substances, cytochrome-C, ascorbic acid, etc.
(Scandalios, 1974). Increased peroxidase activity has been cited as an indicator of environmental
stress (Siegel & Galston, 1968). Mukherjee and Choudhari (1981) reported that water stress
26

promotes the oxidative metabolism of tissue cf Vigna seedlings which resulted in an increase in
the peroxidase activity. Among aromatic plants, peroxidase activity has been analysed in
Cymbopogon flexuosus in relation to micronutrients (Farooqi et al., 1985).
2.3.2 Nitrate reductasc (NR) activity :
There are considerable evidences that once nitrate is taken up by the plant, nitrate reductase is a
rate limiting enzyme in the conversion of nitrate to ammonia (Davies. & Ross, 1984). NR in
higher plants exists as metalloprotein with a molecular weight of 220-230 KDa. The NADH:NR
(EC 1.6.6.1.) which catalyses the reduction of NO 3 to NO2" utilizing NADH as reducing
equivalent, is the predominant form of NR in higher plants (Abdin et al., 1993). The NR is
regulated by various factors such as its own substrate (NO 3"), nitrogen metabolites, light,
temperature, moisture, oxygen, carbon dioxide and plant growth regulators (Abdin et al., 1993).
There is no information available on the NR activity in relation to water stress in Cymbopogon
species. However, Farooqi et al., (1985) studied NR activity in cymbopogon in relation to
rnicronutrient. They found that in iron deficient plant nitrate reductase activity was reduced.
Direct evidence is lacking on whether the lowering of NR activity in water stressed leaves
reflects a loss of enzyme activity, a decrease rate of enzyme synthesis or an increased rate of
enzyme degradation.

2.3.3 Proline :
Accumulation of proline is a widespread plant response to environmental stresses (Yancey et al.,
1982). Accumulation of proline increases osmotic pressure (OP) of cells (Kuznetsov et al.,
1999). The increase in proline content may be due to the enhancement of proteolysis, inhibition
of protein synthesis, a decrease in deamination activity and finally activation of de-novo
synthesis under stress (Kuznetsov et al., 1999). Proline accumulation under wafer deficit
stressed conditions is believed to relate to its de-novo synthesis (Good & Zaplachinski, 1994;
Rascio et al., 1994; Kishore et al., 1995; Sundaresan & Sudhakaran, 1995; Taylor, 1996). A
rapid increase in proline and other amino acids content is an indicator of changes in nitrogen
metabolism. As proline accumulates in high concentration, it has clear role as osmoticum and
27

because of its zwitterionic, highly hydrophilic characteristics, proline acts as a "compatible

solute"(Yancey et al., 1982). Sangwan et al. (1993) reported that the proline accumulation was
highest in C. martinii followed by C. nardus and C. bendulus, and the extent of increase was
lowest in C. winterianus. Sangwan et al. (1994) reported that the proline level was noted to be
3.5 and 2.4 fold higher (over control) in C. nardus under mild and moderate stresses,
respectively.
2.4 EFFECT OF GIBBERELLIC ACID (GA3), ON AROMATIC PLANTS :
2.4.1 Primary Metabolism :
Primary metabolic processes such as photosynthesis, respiration and synthesis of amino acid
influence the growth and development of plants as well as the biogenesis of secondary products
like essential oils (Hess et al., 1975). This is because the initial precursors for secondary plant
products like essential oils are provided by the primary metabolic processes. Efforts have been
made to study the influence of plant growth regulators on primary metabolic processes in
aromatic plants and their bearing on the secondary products.
Application of GA3 on seeds of Phyllanthus emblica increased the leaf and stems protein content,
whereas it was not found to be effective in enhancing the peroxidase specific activity in leaves,
stem and roots; and GA3 was helpful in increasing the total chlorophyll content of the leaves.
(Dhankhar et al.,1997). Effect of PGR on photosynthesis has been studied in Mentha in relation
to triacontanol. Farooqi and Misra (1981) reported that GA3 and IAA application on
Hyoscyamus muticus increased the in-vitro NR activity in the leaves. However, GA 3was more
effective than IAA.
Shukla et al.(1992) studied effect of triacontanol and chloromequat chloride on plant hormones
(GA3 like substances and ABA) and artemisinin in Artemisia annua. Tria application enhanced
the GA like activity, but ABA levels decreased, while chloromequat increased ABA but reduced
GA like substances. Both tria and chloromequat chloride also increased the artemisinin level.
2.4.2 Secondary Metabolism :

28

Attempts have been made to improve the oil Content and yield of aromatic plants by application
of plant growth regulators. Earlier studies exhibited that auxins and GA 3 could not produce any
influence on the oil content in C. flexuosus and C. winterianus(Chatterjee& Ghosh, 1971;
Kokate & Varma, 1971). However, Balyan et al. (1978) demonstrated a significant increase in.
the oil content of C. khasiancisby 24-0 Na salt, whereas in C. citratus, chloromequat increased
the volatile oil and citral content (Ellabben, 1978). Ansari et al., (1988) demonstrated that both
IAA and GA3 enhanced the oil content in C. jwarancusa while IBA decreased the oil yield but
increased piperitone content. IAA and GA application increased oil yield and citral content of C.
citrates (Ansari et al., 1990).
Influence of various growth regulators has been studied on the formation of essential oil in other
aromatic plants. Chloromequat and GA3 are reported to effectively increase the oil yield and the
quality of oil in Ocimum sanctum (Gulati et al., 1974) and in O. basilicum (Eid & Ahmed,
1976). Dey et al., (1981) also reported an enhancement in the contents of eugenol, menthyl
eugenol and caryphyllene contents of the oil in O. sanctum by GA3 and maleic hydrazide. Eid
and Rafaeel (1980b) documented the considerable augmentation in volatile oil yield per plant,
oil content and proportion of citronellal and geraniol in the oil by IAA, alar and chloromequat
application in Pelargonium graveolens. Etheral treatment caused a steady rise in the oil content
in Jasminum grandiflorum, but it was not effective on other species of Jasrninum (Battacharjee
et al., 1984).
In flowers of Rosa damascene however, the oil content was decreased due to ethrel treatment
(Farooqi & Sharma, 1988). Cytokinin such as kinetin, 6-BAP and diphenylamide have also
been found beneficial in augmenting the essential oil content of M. piperita without affecting the
oil composition (Zlatev et al., 1978). Bosela andUdaIova (1977) observed that GA increased
the menthol, neomenthol and isomenthone content in both Mentha piperita and M. crispa.
Similary, Farooqi and Sharma (1988) observed significant increase in the oil content of M
arvensis by chloromequat chloride while ethephon (2 chloroethyl phosphonic acid) at 0.06%
concentration had no significant effect on the oil Content. Among the two oil components
studied, only menthone content was significantly increased by 0.06% ethephon. In Rosa
damascena, ethrel decreased the oil content significantly while kinetin increased the oil yield
significantly (Farooqi & Sharma, 1988). Kinetin application also increased citronellol and
29

geranyl acetate level in the oil (Farooqi et al., 1993). Application of CEPA increased the oil yield
the significant increase being at 0.02% in Rosa damascena (Sharma & Farooqi et al., 1990).

30

METHODOLOGY

3.Plant growth regulators treatment :

The seeding of Cymbopogon martinii were obtained from the Central Institute of Medicinal and
Aromatic plants (CIMAP), Lucknow India and cultivated in pots at Herbal garden of Integral
University, Lucknow. The study was conducted during Feb-May in a randomized block design
with three replications.One seedling was transplanted in each pot. Thre were three pots for each
treatment in one replication.Three foliar sprays of following hormones(GA3,IAA,K and Ethrel
were given at 15 days interval starting from 45 days after transplanting. Tween 80(0.01%) was
used as wetting agent. Inaddition, untreated plants were sprayed with distilled water to serve as
control. The plants were harvested at maximum flowering stage.
The oil content was determined by hydro distillation with Clevenger apparatus and is
expressed on fresh weight basis (Guenther, 1955).

Protein content was estimated according to Lowry et al (1951).

Chlorophyll content was calculated by the method of Arnon (1949).

Proline was estimated calorimetrically by the method of Bates et al (1973).

Peroxidase activity was estimated by Pulter (1974).

Nitrate reductase activity was measured by Hangeman and Hucklesby (1971).

31

3.1 CHLOROPHYLL ESTIMATION :

0.2 g of the tissue was extracted with 25 ml of 80% acetone and absorbance was recorded at
663nm and 645nm in spectrophotometer. From the absorbance values, amount of chlorophyll
was calculated by the method of Arnon (1949).

Where [V/ 1000 W] is constant which is equivalent to 0.125

Where V= volume of Acetone,

W=Weight of the tissue

3.2 ESTIMATION OF NITRATE REDUCTASE (NR) ACTIVITY:

NR (EC 1.6.6.1) activity was measured by in-vivo method according to Hageman and
Hucklesly (1971). The leaves were washed and pressed in filter paper to remove sticking
moisture and cut into small pieces. 0.1g of chopped leaves was taken in 50ml filtering flask
containing reaction mixture (2.5ml KNO3 and 2.5ml phosphate buffer). Each sample was
32

analysed in duplicate. The flasks were kept in 150ml beaker with ice so that the enzyme may not
decompose. The reaction mixture was infiltrated into the tissue by using vacuum pump and
infiltration was done for one minute. The infiltrated material was kept at 33C for 1hr. in dark
(Klepper et al., 1974).
The reaction was stopped by boiling the reaction mixture. The flasks were removed from the hot
plate immediately after the reaction mixture started boiling. The heating also facilitated
movement of nitrite out of the tissue. 0.5ml of reaction mixture was taken in a test tube. 1.0ml of
sulfanilamide (1%) and 1ml of N-(1 Napthyl) ethylene diamine dihydrochloride (0.02%) were
mixed in it. The final volume was made to 10ml. Optical density reading were taken at 540nm
against distilled water on spectrophotometer. The nitrate reductase activity is stated as the
amount of nitrite formed per gram fresh weight per hour.
3.2.1 Standard Curve of Nitrate Reductase :
Sodium nitrite (AR grade) was used for the preparation of standard curve69mg Sodium nitrite
was dissolved in 100ml distilled water in order to obtain solution of 100moles/0.1ml strength.
By successive dilution, solution of 100 mmoles/0.1ml strength was prepared. From this
solution, 0.05ml, 0.1ml, 0.15ml, 0.2ml, and 0.25ml solutions were taken in five different test
tubes. To these test tubes 0.95, 0.9, 0.85, 0.8 and 0.75mldistilled water was added to obtain
solutions of 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 mmoles/ml. Then, 1 ml of
1% sulfanilamide and 1ml of 0.02% N-(1 Napthyl) ethylene diamine dihydrochloride was added
in each test tube. The colour was allowed to develop for 20 minutes. The optical density was read
at 540nm on spectrophotometer. The standard curve is presented in fig. 3.2.

33

Fig. 3.2 Standard Curve for Nitrate Reductase

34

3.3 ESTIMATION OF PROTEIN:

Protein was estimated according to Lowry et al. (1951) in crude enzyme extract. 100l of
supernatant and 100l of 10% trichloroacetic acid (TCA) were mixed and kept for protein
precipitation. After 24 hrs., it was centrifuged for 10 minutes at 10,000 rpm to remove TCA. The
pellet was dissolved in 1ml of 1N NaOH and shaken well on cyclomixer. 0.5 ml from this
solution was taken in the test tube in duplicate and 5ml of alkaline CuSO 4 solution was added to
this and left for 30 minutes, then 0.5 ml of 1/2 strength Follins reagent was added to it. Optical
density was recorded at 660nm on spectrophotometer. Concentration of the protein was
calculated against the standard curve.
3.3.1 Standard Curve of Protein :
For the preparation of standard curve of protein, 5mg of Bovine serum albumin (BSA) was
dissolved in 25ml of 1N NaOH. From this stock solution, different volumes (0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, and 1.0ml) corresponding to concentrations of 20, 40, 60, 80, 100, 120,
140, 160, 180 and 200g respectively were pipetted in test tubes in duplicate. Different volumes
of 0.1 N NaOH were added to corresponding test tubes to make the final volume 0.5ml. Then
5.0ml of alkaline copper solution was added in each test tube and then 0.5 ml of Folins reagent
was added in each test tube and left for 30 minutes. Optical density was measured on
spectrophotometer and standard curve was plotted, which is presented in fig 3.3.

35

36

Fig.3.3 Standard Curve for Protein

3.4 ESTIMATION OF PROLINE:
Proline was estimated calorimetrically by the method of Bates et al., (1973). 1.5 g fresh tissue
was ground in 5ml of 3% sulphosalicylic acid. The homogenate was centrifuged at 10,000 rpm
for 10 minutes. 0.5 ml of supernatant was taken in duplicate. To this 0.5 ml glacial acetic acid
and 0.5 ml of ninhydrin solution was added. Ninhydrin solution was prepared by dissolving 1.25
g ninhydrin in 30ml of glacial acetic acid and 20 ml orthophosphoric acid (6M). The test tubes
were placed in boiling water for 1hour and then 4ml of toluene was added after cooling the tubes.
Optical density was measured at 520nm on spectrophotometer after mixing the contents in
cyclomixer.
3.4.1

Standard Curve of Proline :

For 5.0mg proline was dissolved in 50ml of distilled water. From this stock solution, different
concentrations of proline (20, 40, 60, 80 and 100 ml) were pipetted in test tubes in duplicate. To
this 0.5, 0.4, 0.3, 0.2, 0.1 and 0ml sulphosalicylic acid was added. 0.5ml of acetic acid and 0.5 ml
of nin-hydrin solution was added in each test tube. All the test tubes were placed in boiling water
bath for 1hour and then 4ml of toluene was added after cooling the test tubes. Contents were
mixed thoroughly on cyclomixer. Optical density was measured at 520nm on spectrophotometer
and standard curve was plotted, which is presented in fig 3.4.

37

38

Fig. 3.4 Standard Curve of Proline

39

RESULT AND DISCUSSION

4.1 Statistical analysis

40

The data were statistically analyzed using one way analysis of variance (ANOVA) by GRAPH
PAD Prism 5 software. Mean values were statistically compared by Tukey-Kramer

multiple

comparison test at (P<0.05. Data were presented as Mean SE (n=3).

Table no:1
Analysis of growth and development (plant height, area of the leaves, tiller no and herb
yield) of Palmarosa by foliar spraying method of GA3 (25ppm, 50ppm and 100ppm) in pod

Treatment

Plant Height(cm)

Area of the leaves Tiller no

Herbage

(cm2)

Yield(g)/pod

Untreated

124.556.47

31.911.89

26.002.88

35010.00

GA3 (25ppm)

150.210.76d

40.561.03c

27.003.60a

386.6715.27a

GA3 (50ppm)

165.662.60d

49.983.55d

32.000.57a

45020d

GA3 (100ppm)

191.670.52d

58.092.06d

42.002.00d

52026.45d

The data represented are Mean SE (n=3).

P>0.05-NS=a, P<0.05-b*, P<.001-c**, P<0.001-d***

Table no: 2

41

Effect of GA3 (25ppm, 50ppm and 100ppm) on chlorophyll content, protein content,
enzyme activity like nitrate reductase, proline content and oil content in excised leaves of
Palmarosa .
Treatment

Chlorophyll

Protein (mg/g Nitrate

fr.wt.)
reductase

(mg/g fr.wt.)

Proline

Oil content

(g/g fr.wt.)

(ml/25g.fr. wt.)

(moles
NO2formed/g
fr.wt) hr.
Untreated

3.080.06

2.990.15

7.550.24

206.965.64

0.2800.03

GA3 (25ppm)

2.860.10a

2.690.38a

5.970.28d

282.296.21d

0.260.01a

GA3 (50ppm)

3.570.12c

3.700.16b

8.730.33d

234.525.81d

0.3501b

GA3 (100ppm)

4.430.12d

4.500.20d

12.250.11d

102.818.78c

0.510.01d

Table no:3
Effect of GA3 (25ppm, 50ppm and 100ppm) on oil constituents in Palmarosa by foliar
spraying method in pod.
GA3

% Geraniol

%Geranyl acetate

Untreated

56.

34

GA3 (25ppm)

47

14

GA3 (50ppm)

60

27

GA3 (100ppm)

85

11

Table no: 4
Analysis of growth and development (plant height, area of the leaves, tiller no and herb
yield) of palmarosa in pot by foliar spraying method of IAA (25ppm, 50ppm and 100ppm)
in pod.
42

Treatment

Plant Height(cm)

Area of the leaves Tiller no

Herbage

(cm2)

Yield(g)/pod

Untreated

107.337.33

32.771.59

34.660.57

337.336.42

IAA (25ppm)

100.331.20a

29.471.5a

31.001.00a

31010a

IAA (50ppm)

126.441.38cd

46.611.3d

42.334.61b

421.6718.93d

IAA (100ppm)

110.662.09a

34.500.85a

33.003.00a

340.675.13a

Table no: 5
Effect of IAA (25ppm, 50ppm and 100ppm) on chlorophyll content, protein content,
enzyme activity like nitrate reductase, proline content and oil content and in excised leaves
of Palmarosa .
Treatment

Chlorophyll
(mg/g fr.wt.)

Protein
(mg/g fr.wt.)

Nitrate
reductase

Proline

Oil content

(g/g fr.wt.)

(ml/25g.fr. wt.)

(moles
NO2formed/
g fr.wt) hr.
Untreated

3.020.08

2.770.06

5.930.53

175.466.80

0.260.03

IAA (25ppm)

2.850.11a

2.240.16d

4.810.30b

266.096.13d

0.200.00a

IAA (50ppm)

4.120.20d

3.640.03d

9.850.27d

133.337.63d

0.430.05c

IAA (100ppm)

3.510.10c

2.870.03a

7.950.08d

156.155.14b

0.340.01a

Table no: 6
Effect IAA 25ppm 50ppm 100ppm on oil constituents in Palmarosa by foliar spraying
method in pod.
IAA

% Geraniol

%Geranyl acetate

Untreated

56

36

IAA (25ppm)

58

34
43

IAA (50ppm)

78

14

IAA (100ppm)

47

26

Table no: 7
Analysis of growth and development (plant height, area of the leaves, tiller no and herb
yield) of palmarosa in pot by foliar spraying method of Kinetin (25ppm, 50ppm and
100ppm).
Treatment

Plant Height(cm)

Area of the leaves Tiller no

Herbage

(cm2)

Yield(g)/pod

Untreated

113.002.33

28.261.00

26.332.88

312.332.51

Kinetin (25ppm)

105.992.08b

23.561.02a

23.331.15a

250.336.4d

Kinetin (50ppm)

133.882.91d

41.343.94d

41.331.15d

446.004.00d

Kinetin (100ppm)

118.780.38b

33.442.25a

28.661.15a

327.332.51c

Table no: 8
Effect of Kinetin (25, 50, 100ppm) on chlorophyll content, protein content, enzyme activity
like nitrate reductase, proline content and oil content and in excised leaves of Palmarosa.
Treatment

Chlorophyll
(mg/g fr.wt.)

Protein
(mg/g
fr.wt.)

Nitrate
reductase
(moles
NO2formed/g
44

Proline

Oil yield

(g/g fr.wt.)

(ml/25g.fr
wt.)

fr.wt.)hr.

Untreated

2.940.33

2.640.38

5.260.09

199.092.62

0.210.02

Kinetin (25ppm)

2.450.06a

2.600.19a

4.650.15a

280.805.50d

0.170.02a

Kinetin (50ppm)

4.060.23c

3.830.60b 11.720.52d

125.839.02d

0.370.03d

Kinetin (100ppm)

3.350.20a

3.200.06a

238.095.26d

0.260.01a

7.450.30d

Table no: 9
Effect of Kinetin (25ppm, 50ppm and 100ppm) on oil constituents in Palmarosa by foliar
spaying method in pod.
Kinetin

% Geraniol

%Geranyl acetate

Untreated

63

18

Kinetin (25ppm)

60

15

Kinetin (50ppm)

76

12

Kinetin (100ppm)

70

16

Table: 10
Analysis of growth and development (plant height, area of the leaves, tiller no and herb
yield) of palmarosa in pot by foliar spraying method of Ethrel (100ppm, 250pp, 500ppm) in
pod.
Treatment

Plant Height(cm)

Area of the leaves Tiller no

45

Herbage

(cm2)

Yield(g)/pod

Untreated

1072.64

33.631.62

41.331.15

390.672.36

Ethrel (100ppm)

83.771.17d

26.080.52d

30.661.15d

294.335.77d

Ethrel (250ppm)

92.772.20d

28.920.75c

351.00d

3244.50d

Ethrel (500ppm)

65.992.73d

23.370.58d

250.57d

242.331.52d

Table no: 11
Effect of Ethrel (100ppm, 250ppm and 500ppm) on chlorophyll content, protein content,
enzyme activity like nitrate reductase, proline content and oil content and in excised leaves
of Palmarosa .
Treatment

Chlorophyll

Protein
(mg/g fr.wt.)

(mg/g fr.wt.)

Nitrate
reductase

Proline

Oil content

(g/g fr.wt.)

(ml/25g.fr. wt.)

(moles
NO2formed/
g fr.wt) hr.
Untreated

3.410.10

3.150.05

5.780.14

183.861.16

0.310.01

Ethrel (100ppm)

2.520.10d

2.060.06d

3.720.10d

269.553.71d

0.240.00d

Ethrel (250ppm)

3.080.12b

4.280.15d

6.650.30c

194.367.60a

0.2860.00b

Ethrel (500ppm)

1.770.16d

1.520.08d

2.580.11d

293.532.01d

0.180.10d

Table no: 12
Effect Ethrel (100ppm 250ppm and 500ppm) on oil constituents in Palmarosa by foliar
spraying method in pod.
IAA

% Geraniol

%Geranyl acetate

Untreated

63

18

Ethrel ( 100ppm)

61

19

Ethrel (250ppm)

67

17

Ethrel (500ppm)

52

21
46

SUMMARY AND
CONCLUSION
47

Plant growth regulators have gained wide acceptance for optimizing the yield of plants by
modifying growth and development. Gibbrellic acid (GA3), IAA, K has been reported to play an
important role in growth and development process of aromatic plants (Hassanpouaghdam et al.,
2010), improving the quality and quantity of essential oil. The GA3,IAA and K enhanced
growth of the plants and essential oil yield which might be associated with the regulatory effects
of these plant growth regulators on cell growth and division and gegulatory ezymes used in the
growth and metabolism as well as terpenoid pathway which is associated with the biogenesis of
essential oil. Moreover the enzyme activity like nitrate reductase were significantly increased by
the gradual increment in the applied levels of GA 3 with 100ppm and IAA and K at 50ppm
proving the best foliar application/feeding. Our results are in harmony with the findings of B.
Santosh et al., 1998. A probable reason for the enhancement of NR activity due to GA 3
treatment

might

be

the

de-novo

synthesis

of

NR

enzyme

which

involves

translation/transcription of the associated genes. Application of these PGRs alleviated the

photosynthetic pigment (chlorophyll). Such an increase in the content of this pigment was also
observed by Jhao et al., 1995. Presumably GA3, IAA and K feeded leaves might trap more
sunlight to increase the rate of photosynthesis as compared to control plants. In the present
study it was observed that GA 3 is most potent plant growth regulator which significantly
enhanced the growth and development as well as oil content and quality as compared to IAA, K
and Ethrel a growth retardant. According to Farooqi et al., 2007, GA3 regulates genes
transcription in the terpenoid biosynthetic pathway and their by increase in oil content. As
revealed by the data represented in tables it is clear that GA3 is more potent plant growth
regulator among IAA, Kinetin and Ethrel.GA 3 100ppm IAA and Kinetin at 50ppm brought
about a significant increase in essential oil content, chlorophyll content, nitrate reductase
activity, protein content, while significant decrement of proline content was found and in the
case of Ethrel all the biochemical parameter as well as plant growth was found to be decreased
while the main constituent of palmarosa essential oil i.e. geraniol and geranyl acetate was found
significant at Ethrel 250ppm.
48

REFERENCES
49

Akhila A(2009). Essential Oil-bearing Grasses: The Genus Cymbopogon (Medicinal

and

Aromatic Plants - Industrial Profiles). 1st ed. United States, Florida,

Boca Raton: CRC Press.

A.Misra, N.K. Srivastava, A.K. Srivastava and A.Khan(2009). African Journal of

Pharmacy and Pharmacology Vol. 3(11), pp. 515-520, Available online
http://www.academicjournals.org/ajpp ,ISSN 1996-0816 2009 Academic
Journals.

Ansari SH, Qadry JS, Jain VK(1988). Effect of plant hormones on the growth and
chemical composition of volatile oil of Cymbopogon jwarancusa (Schutt). Indian J
Foresty ;11:143-5.

Azadeh afrigan, Zahra Javdani, Esfandiar Jahantab, Ruhollah Jahanbin and Abdol
Azim Bahari(2013). Scholars research library, Annals of biological research ,
4(6):1-9.

Bakshi IS, Farooqi AHA and Maheshwari SC (1979). Effect of GA 3and IAA on
nitrate reductase activity and floral bud formation in Hyoscyamus muticus.Plant
and Cell Physiol. 20(5), 957.

Brenes, A., Roura, E(2010). Essential oils in poultry nutrition: Main effects and
modes of action. Anim.Feed Sci. Technol., 158, 114.

Chang, J(2000). Medicinal herbs: Drugs or dietary supplements? Biochem.

Pharmacol., 59, 211219.

Cho GH, Kim DI, Pedersen H, Chin CK(1988). Ethephon enhancement of

secondary metabolite synthesis in plant cell cultures, Biotechnol. Prog., 4: 184-188.
50

Duarte MC, Figueira GM, Sartoratto A, Rehder VL, Delarmelina C(2005). AntiCandida activity of Brazilian medicinal plants. J Ethnopharmacol;97(2):305-11.

El-Keltawi NE, Croteau R(1987). Influence of foliar applied cytokinins ongrowth

and

essential

oil

content

of

several

members

of

the

lamiaceae.

Phytochemistry;26(4):891-5.

Farooqi AH, Khan A, Sharma S(2003). Effect of kinetin and chlormequatchloride

on growth, leaf abscission and essential oil yield in Mentha arvensis. Indian
Perfumer 2003;47(4):359-63.

Farooqi AHA and Sharma S(1988). Effect of growth retardants on growth and
essential oil content in Japanese mint, Plant Growth Regulation, 7: 39-45.

Farooqi AHA, Shukla A, Sharma S, Khan A(1996). Effect of plant age and GA3 on
artmisinin and essential oil yield in Artemisia annua L. J Herbs Spices Med Plants;
4:73-80.

Farooqi AHA, Fatima S and Sheema S (2005). Ameliorating effect of chlormequat

chloride and IAA and drought strencultivars of Cymbopogon martinii and
Cymbopogon winterianus. Plant growth regulation.

Farooqi AHA, SharmaS, NaqviAA and KhanA(1993). The effect of kinetin on

flower and oil production in Rosa damascene. Journal of Essential Oil Research5:
305-309.

Fatima S, Farooqi AH, Sharma S(2002). Physiological and metabolic responses of

different genotypes of Cymbopogon martinii and C. winterianus to water stress. J
Plant Growth Regul;37:143-9.
51

Greathead, H(2003). Plants and plant extracts for improving animal productivity.
Proc. Nutr. Soc.,62, 279290.

HagemanRM and HucklesbyDP (1971). Nitrate reductase from higher plants, In:
Methods in Enzymology. 23: 491 (Ed,A.S.Pierto) Academic press.

Haque S, FarooqiAHA, GuptaMM, SangwanRS and Khan A (2007).Effect of

Ethrel, chlormequat chloride and paclobutrazol on growth and pyrethrins
accumulation in Chrysanthemum cinerariaefolium. vis. Plant growth regulator. 51:
263-269.

Hassanpouraghdam M.B., Hajismadi ASL B., Khalighi A(2011). Romanian

biotechnological letters, Copyright 2011 University of Bucharest ,Vol.16, No.14.

Hur MH, Lee MS, Kim C, Ernst E (2012). Aromatherapy for treatment of
hypertension: a systematic review. J Eval Clin Pract 18: 37-41.

Husain A, Singh P and Singh A (1979). Effect of GA 3and IAA on nitrate reductase
activity and floral bud formation in Hyoscyamus muticus.Indian J. Pharm. Sci.
41(1), 46.

Joint meeting of the FAO panel of experts on pesticides residues in food and
environment(1994).UN food and agriculture organization.

Kadner R and DruegeU (2004). Role of ethylene action in ethylene production and
post storage leaf senescence and survival of pelargonium cuttings. Plant growth
Regulation, 43: 187-196.
52

Khanuja SP, Shasany AK, Pawar A, Lal RK, Darokar MP, Naqvi AA(2005).
Essential oil constituents and RAPD markers to establish species relationship in
Cymbopogon Spreng. (Poaceae). Biochem Syst Ecol;33(2):171-86.

Koseva-kovasevaDand StaevD (1978). Effect of some growth regulators and

hydrogen peroxide on the content and quality of peppermint oil. Rest enie din re
Nauki. 15: 21-25.

L.D.Kapoor and B.K.Kaul(2006). Journal of pharmaceutical sciences. Volume 51,

issue 11, pages 1113-1114.

Lee YL, Wu Y, Tsang HW, Leung AY, Cheung WM (2011). A systematic review on
the anxiolytic effects of aromatherapy in people with anxiety symptoms. J Altern
Complement Med 17: 101-108.

Li, T.S.C(2006). The range of medicinal herbs and spices. In Handbook of Herbs
and Spices; Peter, K.V., Ed.; Woodhead Publishing Limited:Cambridge, UK, 2006;
Volume3, pp.113-125.

Linden JC, Haigh JR, Mirjalili N and Phisaphalong M(2001). Gas Concentration
Effects on Secondary Metabolite Production by Plant Cell Cultures, Advances in
Biochemical

Engineering/Biotechnology,Vol.

72,

Springer-Verlag

Berlin

Heidelberg, 27-62.

LowryOH, RusenNJ, FarrAL and RandallRJ (1951). Protein measurement with the
folin reagent. Journal Biochem: 193: 265-275.

53

MishraHA, MishraA and SatputeGK (1998). Effect of ethephon on growth and oil
yield of Palmrosa (Cymbopogon martinii).Wats. Journal of Herbs, Spices and
Med.Plants:1-8.

Mpho Edwin Maudu, Fhatuwani Nixwell Mudau and Irvine Kwaramba

Mariga(2011).School of Agricultural and Environmental Sciences, University of
Limpopo, P Bag X 1106, Sovenga 0727, South Africa.

Negi, P.S(2012). Plant extracts for the control of bacterial growth: Efficacy, stability
and safety issues for food application. Int. J. Food Microbiol., 156, 717.

Piccaglia, R.; Marotti, M.; Giovanelli, E.; Deans, S.G.; Eaglesham, E(1993).
Antibacterial and antioxidant properties of mediterranean aromatic plants. Ind.
Crops Prod., 2, 4750.

Poyh JA, Ono EO (2006). Rendimento do leo essencial de Salvia officinalis L. sob
acao de reguladores vegetais. Acta Sci Biol Sci 28(3): 189-193.

Rabie KA. (1996). Studies on the interaction between gibberellin and

benzyladenine in regulating growth, yield and phytohormone content in wheat
plants. Annals Agric. Sci. Ain Shams Univ. Cairo 41: 99-110.

Rajeswara RB, Rajput DK, Patel RP(2009). Essential oil profiles of different parts
of

palmarosa (Cymbopogon martinii (Roxb.) Wats. var. motia Burk.). J Essent

Oil Res;21:519-21.

RademacherW (2000). Growth Retardants: Effects of Gibberellin Biosynthesis and

other Metabolic Pathways. In Annual. Review of plant physiology and plant
Molecular Biology by Jones. Bohnert and delmer.5 1:501-531.
54

SangwanNS, FarooqiAHA, FatimaS,and Sangwan RS(2001). Regulations of

essential oil production in plants. Plants growth regulation, 34: 3-21.

Setzer WN (2009) Essential oils and anxiolytic aromatherapy. Nat Prod Commun 4:
1305-1316.

Singh K, Chowdhury A, Singh DV(1996). Effect of diuron and nitrogen weed

growth, oil yield and Nutilization in citronella Java (Cymbopogon winterianus).
Indian Perfumes;40:47-52.

Smith CA, Collins CT, Crowther CA (2011). Aromatherapy for pain management in
labour. Cochrane Database Syst Rev 6 : CD009215.

Soenarko, S.91977).The genus Cymbopogon Sprengel 9(3):225-375.

Subir K Bose, Ritesh Kumar Yadav, Smrati Mishra, Rajender S Sangwan, A K

Singh, B Mishra, A K Srivastava, Neelam S Sangwan(2013). Plant Physiol
Biochem 2013 May 27;66:150-8. Epub.

Tillett J, Ames D (2010). The uses of aromatherapy in womens health. J Perinat

Neonatal Nurs 24: 238-245.

Yim VW, Ng AK, Tsang HW, Leung AY (2009) A review on the effects of
aromatherapy for

patients with depressive symptoms. J Altern Complement Med

15: 187-195.

55

Yogita Rohamare , T. D. Nikam and K. N. Dhumal(2013). International J. Seed

Spices 3(2), 34-41.

Zakaria Hazzoumi, Youssef Moustakime and Khalid Amrani Joutei(2014).

SpringerPlus , 3:321. http://www.springerplus.com/content/3/1/321.

56