Essential Oil
Bearing Grasses
The genus Cymbopogon
Edited by
Anand Akhila
Medicinal and Aromatic Plants - Industrial Profiles
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Essential oil-bearing grasses' the genus Cymbopogon / editor: Anand Akhila.
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ISBN 978-0-8493-7857-7 (hardcover, alk. paper)
1. Cymbopogon. 2. Cymbopogon--Industrial applications. 3. Cymbopogon--Therapeutic use. I.
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1 The Genus Cymbopogon
Botany, Including Anatomy,
Physiology, Biochemistry,
and Molecular Biology
Cinzia M. Bertea and Massimo E. Maffei
CONTENTS
1.1 Introduction
1.2 Anatomy
1.2.1 General Considerations
1.2.1.1 Leaf Anatomy
1.3 Biochemistry
1.3.1 Characterization of the Photosynthetic Variant..
1.3.2 NADP+Inhibition of NADP-MDH
l.3.3 pH and Temperature Dependence of NADPH-MDH and NADP-ME
1.3.4 CO 2 Assimilation and Stomatal Conductance
1.4 Molecular Biology
1.4.1 Randomly Amplified Polymorphic DNA (RAPD) Markers
1.4.2 Simple Sequence Repeat Markers (SSRs)
1.5 Physiology and Ecophysiology
1.5.1 Cymbopogon martinii
1.5.2 Cymbopogon fiexuosus
1.5.3 Cymbopogon winterianus
1.5.4 Other Cymbopogon Species
References
1.1
I
4
4
5
7
8
9
9
10
II
II
13
14
14
16
17
18
20
INTRODUCTION
Among monocots forming the family of Gramineae some grasses produce essential oils that are
a valuable source for the flavor industry. Two grasses are known for their industrial potential for
essential oil production: Vetiveria zizanioides Stapf, which has been the subject of a monograph in
the series Medicinal and Aromatic Plants-Industrial Profiles (Maffei 2002) and Cymbopogon,
which is the subject of this book and which updates the monograph edited by Kumar et al. (2000).
Cymbopogon is a genus comprising about 180 species, subspecies, varieties, and subvarieties. It
is native to warm temperate and tropical regions of the Old World and Oceania. Table 1.1 lists the
several species, subspecies, varieties, and subvarieties as reported by the International Plant Names
Index (2004), published on the Internet http://www.ipni.org (accessed September 10, 2007).
The name Cymbopogon was introduced by Sprengel in 1815 (Sprengel 1815) and at that time
the genus consisted of a few species, which were then moved to the genus Andropogon. In fact,
both Cymbopogon and Andropogon belong to the tribe Andropogoneae, a monophyletic tribe that
2
Essential Oil-Bearing Grasses: The Genus Cymbopogon
TABLE 1.1
List of Cymbopogon Species, Subspecies, Varieties, and Subvarieties as Reported
in The International Plant Names Index (2004)
Cvmbopogon acutispathaceus De Wild
e. afronardus Stapf
e. ambiguus A. Camus.
e. andongensis Rendle
e. angustispica Nakai
e. annamensis A. Camus.
e. arabicus Nees ex Steud.
e. arriani Aitch.
e. arundinaceus Schult.
e. baginnicus Stapf
e. hassacensis A. Camus.
e. bequaertii De Wild
e. bhutanicus Noltie
e. bombycinus A. Camus.
e. bombvcinus var. bombvcinus (RBr) Domin.
e. bombvcinus var. townsvillensis Domin.
e. bombvcinus var. typicus Domin.
e. bracteatus Hitchcock
e. caesius (Hook and Arn.) Stapf
e. caesius subsp. giganteus (Chiov) Sales
e. calcicola C.E. Hubb.
e. calciphilus Bor
e. cambodgiensis E.G. Camus and A. Camus
e. chevalieri A. Camus
e. chrvsargyreus Stapf
e. circinnatus Hochst. ex Hookf.
e. citratus Stapf
e. citriodorus Link
e. claessensii Robyns
e. clandestinus Stapf
e. coloratus Stapf
e. commutatus Stapf
e. commutatus var.jammuensis (Gupta) H.B. Naithani
e. condensatus Spreng.
e. confertiftorus Stapf
e. connatus Chiov.
e. cyanescens Stapf
e. cvmbarius Rendle.
e. densiflorus Stapf
e. dependens B.K. Simon
e. dieterlenii Stapf ex Phillips
e. diplandrus De Wild.
C. distans (Nees ex Steud.) Will Watson.
e. divaricatus Stapf
e. eherhardtii A. Camus.
e. effusus A. Camus.
e. elegans Spreng.
e. exaltatus A. Camus.
e. exaltatus var. ambiguus Domin.
e. exaltatus var. exaltatus (RBr) Domin.
e. exaltatus var. genuinus Domin.
e. exaltatus var. gracilior Domin.
e. exaltatus var. lanatus (RBr) Domin.
e. exarmatus Stapf
e. excavatus Stapf
Cifamiliaris De Wild
e.jigarianus Chiov.
e. filipendulus Rendle
e. finuimus Rendle
e.fiexuosus Stapf
e. flexuosus var. assamensis S.c. Nath and K.K. Sarma
e.floccosus Stapf
e. foliosus Roem and Schu It.
e. gazensis Rendle
e. gidarba [Buch-Harn. ex Steud.] Haines
e. giganteus Chiov.
e. glundulosus Spreng.
e. glaucus Schult.
e. globosus Henrard.
e. goeringii A. Camus
e. goeringii var. hongkongensis S. Soenarko
e. graws Domin.
e. hamatulus A Camus
e. hirtus Stapf ex Burtt Davy
e. hirtus subsp. villosum (Pignatti) Pignatti
e. hispidus Griff.
e. hookeri (Munro ex Hackel) Stapf ex Bor
e. humboldtii Spreng.
e. iwarancusa Schult.
e. jinshaensis R. Zhang and C.H. Li
e. jwarancusa subsp. olivieri (Boiss.) S. Soenarko
e. kapandensis De Wild
e. khasianus (Hackel) Stapf ex Bor
e. ladakhensis B.K. Gupta
e. lanatus Roberty
e. laniger Duthie
e. lecomtei RendJe
e. lepidus (Nees) Chiov.
e. liangshanensis S.M. Phillips and S.L. Chen
e. lividus (Thwaires) Willis
e. luembensis De Wild
e. mandalaiaensis Soenarko
e. marginatus Stapf ex Burtt Davy
e. martinii Stapf
he Genus Cymbopogon
The Genus Cymbopogon
as Reported
TABLE 1.1 (continued)
list of Cymbopogon Species, Subspecies, Varieties, and Subvarieties as Reported
in The International Plant Names Index (2004)
e. martinianus Schult.
e. mekongensis A. Camus
C. melanocarpus Spreng.
e. micratherus Pilg.
e. microstachys (Hookf) S. Soenarko
e. microthecus A. Camus
e. minor B.S. Sun and R. Zhang ex S.M. Phillips and S.L. Chen
e. minutiflorus S. Dransf
e. modicus De Wild
e. motia B.K. Gupta
e. munroi (C.B. Clarke) Noltie
e. nardus (L.) Rendle
e. nardus subvar. bombvcinus (R.Br.) Roberty
e. nardus vat. confertiftorus (Steud.) Stapf ex Bar
e. nardus subvar. exaltatus (R.Br.) Roberty
C. nardus subvar. grandis Roberty.
e. nardus subvar. lanatus (R.Br.) Roberty
e. nardus var. luridus (Hookf.) Gavade and M.R. Almeida
e. nardus subvar. procerus (R.Br.) Roberty
e. nardus subvar. refractus (R.Br.) Roberty
e. nardus subvar. schultzii Roberty
e. nervatus A. Camus
e. nyassae Pilg.
e. obtectus S.T. Blake
e. olivieri (Boiss.) Bor
e. osmastonii R. Parker
e. pachnodes (Trin.) Will Watson
e. papillipes (Hochst. ex A. Rich) Chiov.
e. parkeri Stapf
e. pendulus Stapf
e. phoenix Rendle
e. pilosovaginatus De Wild
e. pleiarthron Stapf
C. plicatus Stapf
e. plurinodis Stapf ex Burtt Davy
e. polyneuros Stapf
e. pospischilii (K. Schum) C.E. Hubb
e. princeps Stapf
e. procerus A. Camus
e. procerus var. genuinus Domin.
e. procerus var. procerus (R.Br.) Domin.
e. procerus var. schultzii Domin.
e. prolixus (Stapt) Phillips
e. prostratus Sweet
e. proximus Stapf
e. proximus var. sennarensis (Hochst.) Tackholm
3
C. pruinosus Chiov.
e. pubescens (vis) Fritsch.
e. queenslandicus S.T. Blake
e. quinhonensis (A. Camus) S.M.
Phillips and S.L. Chen
[transferred to Andropogon (Phillips and Hua 2(05)J
e. ramnagarensis B.K. Gupta
e. rectus A. Camus
e. reflexus Roem and Schult.
e. refractus A. Camus
e. rufus Rendle
e. ruprechtii Rendle
e. scabrimarginatus De Wild
e. schimperi Rendle
e. schoenanthus Spreng.
e. schoenanthus subsp. velutinus Cope
e. schultzii Roberty
e. sennaarensis Chiov.
e. setifer Pilg.
e. siamensis Bor
e. solutus Stapf
e. stipulatus Chiov.
e. stolzii Pilg.
e. stracheyi (Hookf.) Raizada and Jain
e. strictus Bojer.
e. stypticus Fritsch.
e. suaveolens Pilger
e. subcordatifolius De Wild
e. tamba Rendle
e. tenuis Gilli
e. thwaitesii (Hookf) Willis
e. tibeticus Bor
e. tortilis (Presl.) A. Camus
e. tortilis subsp. goeringii (Steud.) TKoyama
e. traninhensis (A. Camus) SSoenarko
e. transvaalensis Stapf ex Burtt Davy
e. travancorensis Bar
e. tungmaiensis L. Liu
e. umbrosus Pilg.
e. validus Stapf ex Burtt Davy
e. vandervstii De Wild
e. versicolor (Nees ex Steud.) Will Watson
e. virgarus Stapf ex Rhind.
e. virgatus Stapf ex Bor
C. welwitschii Rendle
e. winterianus Jowitt
e. xichangensis R. Zhang and B.S. Sun
Source: Published on the Internet hnp://www.ipni.org [accessed September 10.2007].
4
Essential Oil-Bearing Grasses: The Genus Cymbopogon
includes 85 genera. Mathews and coworkers (2002) found strong support for a core Andropogoneae
that includes, among others, Andropogon and Cymbopogon, and support for its relationship with an
expanded Saccharinae that includes Microstegium. The limited difference in the plant traits between
Andropogon and Cymbopogon, has argued the possibility that species belonging to Cymbopogon
might be a subgenus of Andropogon. Most of Andropogoneae have pairs of spikelets in the inflores
cence, one sessile and one on a pedicel, although in some species one or the other of these spikelets
appear to be suppressed. The inflorescences form is also highly variable (Mathews et al. 2002).
Morphologically, the main difference in the genus Cymbopogon is the presence of some pair of
spikelets, for each spike, with unisexual male flowers, whereas in the Andropogon spikelets are
usually sessile and often sterile. Cymbopogon plants are tall (up to and above I m) perennial plants,
with narrow and long leaves that are mostly characterized by the presence of silica thorns aligned
on the leaf edges. Leaves bear glandular hairs, usually each with a basal cell that is wider than the
distal cell (see Section 1.2). Representative of the Andropogoneae exhibit C 4 photosynthesis, with
NADP-ME as the primary decarboxylating enzyme (Mathews et al. 2002), they usually have a chro
mosome number of five, with ploidy levels ranging from tetrapJoids to 24-pJoid. Polyploidy, either
as alloploidy or segmental alloploidy, is frequent. Representative specimens of various species of
the genus Cymbopogon have been cytogenetically studied by Spies and coworkers (Spies et al.
1994). The monophyly of Cymbopogon has also been clearly demonstrated, and the genus is sister
to Heteropogon (Mathews et al. 2002).
Among the several aromatic species belonging to the genus Cymbopogon the most important
in terms of essential oil production are C. martinii, also known as palmarosa; C. citratus, better
known as lernongrass; and the so-called East Indian lemongrass, Cymbopogon flexuosus, native to
India, Sri Lanka, Burma, and Thailand; whereas, for the related West Indian C. citratus, a Malesian
origin is generally assumed. C. nardus and C. winterianus produce the famous citronella from Sri
Lanka and lava, respectively. Also known to produce essential oils are C. schoenanthus, or camel
grass; C. caesius, or inchi/kachi grass; C. afronardus, C. clandestinus; C. coloratus; C. exaltatus;
C. goeringii; C. giganteus; C. jwarancusa; C. polyneuros; C. procerus; C. proximus; C. rectus;
C. sennaarensis; C. stipulatus; and C. virgatus (Guenther 1950b). The main constituents of
Cymbopogon essential oils will be described in other chapters of the book.
Many laboratories in several countries are deeply involved in studying various aspects of cyrn
bopogons, using variously derived genetic resources. The work already done covers a wide array of
topics, including botanical identification, plant description, cytogenetics, and cell, tissue, and organ
in vitro cultures. Physiology and biochemistry of stress tolerance and essential oil biosynthesis,
genetics and biotechnology, and agrotechnology involved in crop production and disease and pest
control chemistry of terpenes, biological activities of essential oil terpenoids and trade and market
ing aspects (reviewed by Kumar et al. 2000).
The major objective of this monograph is to update the literature and give references on the afore
mentioned topics of cymbopogons, with particular attention to industrial aspects. In the following
sections of this introductory chapter, we will explore the anatomy and biochemistry of the photosyn
thetic apparatus, also considering the most recent advances in molecular biology of Cymbopogon.
We will conclude the chapter with some physiological and ecophysiological considerations.
1.2
1.2.1
ANATOMY
GENERAL CONSIDERATIONS
Higher plants can be divided into two groups, C, and C 4 , based on the mechanism utilized for pho
tosynthetic carbon assimilation related to anatomical and ultrastructural features. A cross section
of a typical C 1 leaf reveals essentially one type of photosynthetic, chloroplast-containing cell, the
mesophyll, and in these plants atmospheric CO 2 is fixed directly by the primary carbon-fixation
enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), In contrast, a typical C 41eaf
1.2.1.1
First desc .
and C. refi
followed by
conducted by
1.2.1.1.1
The Genus Cymbopogon
core Andropogoneae
relationship with an
planttraits between
ing to Cymbopogon
ikelets in the inflores
her of these spikelets
Mathews et al. 2002).
nee of some pair of
.ropogon spikelets are
1m) perennial plants,
f silica thorns aligned
· 1that is wider than the
4 photosynthesis, with
y usually have a chro
oid. Polyploidy, either
· s of various species of
'COworkers (Spies et al.
and the genus is sister
on the most important
sa; C. citratus, better
onflexuosus, native to
. C. citratus, a Malesian
us citronella from Sri
"schoenanthus, or camel
• coloratus; C. exauatus;
Co proximus; C. rectus;
main constituents of
various aspects of cym
covers a wide array of
cell, tissue, and organ
sential oil biosynthesis,
.on and disease and pest
ids and trade and markete references on the afore
aspects. In the following
mistryof the photosyn
( biology of Cymbopogon.
I considerations.
hanism utilized for pho
features. A cross section
plast-containing cell, the
primary carbon-fixation
contrast, a typical C 4 leaf
5
has two distinct chloroplast-containing cell types, the mesophyll and the bundle sheath (or Kranz)
cells, and they differ in photosynthetic activities (Hatch 1992; Maurino et al. 1997). The operation
of the C 4 photosynthetic mechanism requires the cooperative effort of both cell types, connected by
an extensive network of plasmodesmata that provides a pathway for the flow of metabolites between
the cells.
The C 4 pathway is a complex adaptation of the C 3 pathway that overcomes the limitation of photo
respiration and is found in a diverse collection of species, many of which grow in hot climates. It
was first discovered in tropical grasses (e.g., sugarcane and maize) and is now known to occur in
16plant families. It occurs in both monocotyledonous and dicotyledonous plants, and is particularly
prominent in species of the Gramineae, Chenopodiaceae, and Cyperaceae (Edwards and Walker
1983).About half of the species of the Poaceae are included among the C 4 plants (Smith and Brown
1973). The key feature of C 4 photosynthesis is the compartmentalization of activities into two spe
cialized cell and chloroplast types. Rubisco and C 3 photosynthetic carbon reduction (PCR) cycle
are found in the inner ring of bundle sheath cells. These cells are separated from the mesophyll
and from the air in the intercellular spaces by a lamella that is highly resistant to the diffusion of
CO 2 (Hatch 1988). Thus, by virtue of this two-stage CO 2 fixation pathway, the rnesophyll-located
C4 cycle acts as a biochemical pump to increase the concentration of CO 2 in the bundle sheath an
estimated lO-fold over atmospheric concentrations. The net result is that the oxygenase activity of
Rubisco is effectively suppressed, and the PCR cycle operates more efficiently.
C4 plants have two chloroplast types, each found in a specialized cell type. Leaves of C 4 plants
show extensive vascularization, with a ring of bundle sheath (BS) cells surrounding each vein and
an outer ring of mesophyll (M) cells surrounding the bundle sheath. CO 2 fixation in these plants is
a two-step process.
There are three variants on the basic C 4 pathway, and the biochemical distinctions are correlated
with the ultrastructural differences of Kranz cells (Gutierrez et al. 1974; Hatch et al. 2007; Hatch
et al. 1975). The three C 4 variants can be distinguished ultrastructurally by using combinations
of two characters of bundle sheath cell chloroplasts, by the degree of granal stacking, and by the
chloroplasts position (Gutierrez et al. 1974).
For this reason, comparative grass leaf anatomy has become the object of intensive investigation
in relation to photosynthesis along with biochemical studies.
1.2.1.1 Leaf Anatomy
First descriptions of the genus Cymbopogon were given by Breakwell (1914) on C. bombycinus
and C. refractus under the name of Andropogon bombycinus R. Br. and A. refractus, respectively,
followed by a leaf structure description done by Vickery (1935) and Prat (1937). Further studies were
conducted by Metcalfe (1960). The descriptions given by these authors are very similar and still valid.
1.2.1.1.1
Generic Characters
Both adaxial and abaxial epidermises of Cymbopogon species contain short-cells, over the veins,
solitary, paired or in short or long rows, the proportion of each type varying with the species. Silica
bodies are located over the veins, mostly crossed to dumbell shaped. Microhairs are present usually
each with the basal cell wider than the distal cell, the latter frequently tapering to a pointed apex,
or hemispherical. Stomata with subsidiary cells range from low or tall dome-shaped to triangular,
the proportions of each type varying in different species and sometimes in separate preparations
from a single species. The vascular bundles are small, mostly angular, but less conspicuous in some
species than in others. The mesophyll presents a distinctly radial chlorenchyma. Bundle sheaths are
single (Metcalfe 1960).
Figure I.lA shows an electron scanning micrograph of a C. citratus leaf blade. It is possible to
observe developed prickle hairs with rather elongated bases over the veins. The abaxial epidermis
also reveals several stomata, with narrow guard cells associated with subsidiary cells, typical of
grasses (Figure UB).
6
Essential Oil-Bearing Grasses: The Genus Cymbopogon
~\.
~
.'
,:&~, '
,
_':«.
:..r~ ...?,
,.,.'- ..........4~~.·
~'.;.:"~"""
r~ A\·',~,
iii
.J;.(.. .
l
FIGURE 1.1 (A) Electron scanning micrograph of C. citratus leaf blade. Prickle hairs are evident on the
veins (white arrows) (80x). (B) Electron scanning micrograph of a stoma seen from the surface. Guard cells
are narrow in the middle and enlarged at the end, while subsidiary cells are triangular (2000x). (C) Semi-thin
cross section of C. citratus leaf stained with toluidine blue. The vascular bundle in a minor vein is surrounded
by a layer of sheath cells, with chloroplasts arranged in a centrifugal position. (D) Bundle sheath chloroplast
without grana and with few starch grains. (E) Mesophyll chloroplast with most of the thylakoid stacked in
grana and without starch grains. (F) Bundle sheath chloroplast showing immunolabeling against Rubisco.
Colloidal gold particles (white arrows in the high magnification) strongly label the stroma. (G) Enlargement
of plasmodesmata connecting mesophyll and bundle sheath cells, providing metabolite flow between the two
photosynthetic tissues.
1.3
Genus Cymbopogon
The Genus Cymbopogon
7
1.2.1.7.7.7 Leaf Ultrastructure of Cymbopogon citratus The structure of parenchymatic bundle
sheath (BS) cells is particularly important in distinguishing C 3 and C 4 species. A commonly men
tioned anatomical feature of C 4 plants is the orderly arrangement of mesophyll cells with reference
to the BS, the two together forming concentric layers around the vascular bundle. The anatomi
cal differences between plants exhibiting a C 4 photosynthetic carbon assimilation pathway can be
disclosed by electron microscopical observations of the BS (Chapman and Hatch 1983; Edwards
and Walker 1983; Gutierrez et al. 1974; Hatch 1988; Hatch et al. 1975; Jenkins et al. 1989). In the
NADP-ME type, chloroplasts are peripherally arranged and grana are deficient or absent in bundle
sheath cells. Two other distinctive features are the presence or absence of a mestome sheath, a layer of
cells intervening between metaxylem vessel elements and laterally adjacent BS Kranz cells, and the
presence or absence of a cell wall suberized lamella (SL). The mestome sheath occurs in NAD-ME
and PCK species, while the SL is present in bundle sheath cell walls of several NADP-ME and PCK
species (Eastman et al. 1988; Hattersley and Watson 1976). The number of mitochondria in the
NADP-ME subtype is lower than in the NAD-ME one because, in the latter, the enzymes involved
in the transformation of aspartate to CO 2 and pyruvate are present in these organelles (Hatch et al.
1975). Anatomical studies conducted on C. citratus leaves indicated the presence of the C 4 Kranz
anatomy in this plant along with several ultrastructural features typical of NADP-ME species.
In C. citratus cross sections of minor veins, the vascular bundle appears surrounded by one layer
of sheath cells, in which the chloroplasts are located in a centrifugal position (Figure 1.lC). No
mestome sheath between metaxylem vessel elements and laterally adjacent Kranz cells is observed.
In the bundle sheath, ultrastructural analyses show a suberized cell wall lamella, and the presence
of agranal chloroplasts, containing numerous starch grains (Figure I.lD). Figure I.L E shows meso
phyll chloroplasts with most of the thylakoids stacked in grana. The two photosynthetic tissues are
connected by a certain number of plasmodesmata that provide a pathway for the flow of metabolites
between the mesophyll and bundle sheath cells (Figure I.LG).
These observations are in accordance with previous anatomical descriptions related to the genus
Cymbopogon reported by Rajendrudu and Das (198 l). These authors reported on the leaf anatomy
and photosynthetic carbon assimilation in five species of Cymbopogon (C.fiexuosus, C. martinii var.
motia, C. nardus, C. pendulus, and C. winterianus) a Kranz-type leaf anatomy with a centrifugal
position of starch-containing chloroplasts in the bundle sheath cells. Starch was exclusively local
ized in the bundle sheath cells that were typically elongated parallel to the veins and nearly twice
as long as wide in the species of Cymbopogon. A narrow leaf interveinal distance was a common
feature among the five Cymbopogon species. A xylem-mestome sheath of cells between metaxylem
vessels and laterally adjacent bundle sheath cells of primary vascular bundles was totally absent in
the five Cymbopogon species (Rajendrudu and Das 1981).
7.2.7.7.2
hairs are evident on the
m the surface. Guard cells
ular(2000x). (C) Semi-thin
oR a minor vein is surrounded
. ~ Bundle sheath chloroplast
of the thylakoid stacked in
., labeling against Rubisco.
:·tIle stroma. (G) Enlargement
ite flow between the two
Rubisco Immunolocalization
High-resolution immunolocalization of Rubisco by electron microscopy showed that labeling
occurred only in the bundle sheath chloroplasts of C. citratus. For these experiments, purified rabbit
polyclonal antibodies raised against Rubisco were employed. Bound antibodies were then visu
alized by linking conjugated gold-labeled goat antirabbit polyclonal antibodies. Gold particles
appeared to be uniformly distributed throughout the stroma (Figure I.L F). These studies conducted
on C. citratus leaves provide evidence for the localization of Rubisco in the stroma of bundle sheath
chloroplasts, as expected for a C 4 plant (Bertea et al. 2003).
1.3
BIOCHEMISTRY
In a preliminary physiological study conducted by (Maffei et al. 1988) on C. citratus grown in
humid temperate climates, some of the PEP-carboxylase kinetic characteristics, and Rubisco and
glycolate oxidase activities were found to be comparable to those of C 4 plants.
8
Essential Oil-Bearing Grasses: The Genus Cymbopogon
The C, mechanism was also confirmed by the 13C/llC stable isotope ratio analyses (Sl3C =
-l3.0). These results are in accordance with sl3e values measured on other species or Cvmbopogon
(Rajendrudu and Das 1981) in which Sl3C value of -ll.O for C. flexuosus, -9.7 for C. martinii, -l1.6
for C. nardus, -lO.3 for C. pendulus. and -11.3 for C. winterianus, respectively, were recorded.
From a biochemical point of view, the three types of the basic Co pathway differ mainly in the
C 4 acid transported into the bundle sheath cells (malate and aspartate) and in the way in which
it is decarboxylated; they are named (based on the enzymes that catalyse their decarboxylation)
NADP-dependent malic enzyme (NADP-ME) found in the chloroplasts, NAD-dependent malic
enzyme (NAD-ME) found in mitochondria, and phosphoenolpyruvate (PEP) carboxykinase (PCK),
found the cytosol of the bundle sheath cells (Edwards and Walker 1983; Ghannoum et al. 2001;
Hatch et al. 1975; Jenkins et al. 1989; Huang et al. 2001). Furthermore, a characteristic leaf anatomy,
biochemistry, and physiology are associated with each of the C4 types (Dengler and Nelson 1999;
Hattersley and Watson 1976). A clear indication of the C 4 photosynthetic pathway of C. citratus and
the variant to which it helongs was obtained by estimating the activities of NADP-ME (EC 1.1.1.40),
NADP-MDH (EC 1.1.1.82), PPDK (EC 2.7.9.1), NAD-ME (EC 1.1.1.39), and PCK (EC 4.1.1.49) as
well as some kinetic characteristics of NADP-ME and NADP-MDH. Adaptation to a particular
environment is a complex process involving a number of physiological, morphological, and ecologi
cal factors (Ghannoum et al. 2001; Huang et al. 2001).
Therefore, enzyme activities were recorded at the low and high temperatures typical of humid
temperate climates, in order to evaluate the adaptability of C. citratus. In order to estimate increases
or decreases in the reaction rate due to changes in the protonation state, groups involved in the catal
ysis and/or binding of substrates as a consequence of pH fluctuations, activities were also recorded
at different pH values.
1.3.1
1.3.2
CHARACTERIZATION OF THE PHOTOSYNTHETIC VARIANT
Further studies dealing with the characterization of the C 4 variant indicated an NADP-depenqent
malic enzyme photosynthetic pathway in C. citratus.
The biochemical subtype was established through the estimation of the highest activities of
NADP-dependent malic enzyme (NADP-ME, EC 1.1.1.40), NADP-dependent malate dehydroge
nase (NADP-MDH, E.C 1.1.1.82), pyruvate, orthophosphate dikinase (PPDK, E.C 2.7.9.1), NAD
dependent malic enzyme (NAD-ME, E.C 1.1.1.39),and phosphoenolpyruvate carboxykinase (PCK,
E.C 4.1.1.49) and some kinetic, along with some chemical-physical parameters of NADP-ME and
NADP-MDH.
Extraction and partial purification sequentially involved precipitation with crystalline ammo
nium sulfate, dialysis, and anion exchange (DEAE-Sephacell). Both, extraction and assays were
conducted according to Ashton (1990). The low activity values of PPDK (90.28 nKat mg:' prot),
PCK «1 nKat rng" prot), and NAD-ME (52.51 nKat mg:' prot) in C. citratus leaf extracts did not
allow to determine the kinetic characteristics, such as Km and Vmax, and/or other chemical-physical
parameters of these enzymes. NADP-MDH and NADP-ME presented relatively high activity values
(15.93 mKat mg:' and 12.56 mKat mg" prot, respectively). NADP-ME activity was 239-fold greater
than NAD-ME activity. The kinetics of NADP-MDH and NADP-ME were therefore measured
to gain a clearer picture of the photosynthetic pathway. The low activity of PPDK found in plant
extracts of C. citratus agrees with the literature data for C 4 plants (Ashton 1990) and with data on
species of the same photosynthetic subtype (Ashton 1990; Bertea et al. 2001). The low levels of
NAD-ME and PCK activities found in our extracts clearly indicated the absence of a C4 variant
utilizing these two pathways. The relatively high activities found for NADP-MDH allowed us to
determine some of its kinetic characteristics (Km and Vmax), which were comparable to those of
plants belonging to the NADP-ME photosynthetic variant (Ashton 1990; Bertea et al. 2003).
1.3.3
The Genus Cymbopogon
ratio analyses (b13C =
speciesof Cymbopogon
9.7 for C. martinii, -11.6
'vely, were recorded.
'way differ mainly in the
:and in the way in which
their decarboxylation)
, ' NAO-dependent malic
) carboxykinase (PCK),
; Ghannoum et al. 2001;
cteristicleaf anatomy,
gler and Nelson 1999;
thway of C. citratus and
NAOP-ME (EC 1.1.1.40),
"1lIId PCK (EC 4.1.1.49) as
~aptation to a particular
rphological, and ecologi
tures typical of humid
er toestimate increases
ps involved in the catal
ivities were also recorded
the highest activities of
ndent malate dehydroge
OK, E.C. 2.7.9,1), NAD
, vatecarboxykinase (PCK,
eters of NADP-ME and
with crystalline ammo
'extraction and assays were
. 'K (90.28 nKat mg:' prot),
ratus leaf extracts did not
r otherchemical-physical
, tively high activity values
tivity was 239-fold greater
were therefore measured
" y of PPOK found in plant
ton 1990) and with data on
'ial. 2001), The low levels of
the absence of a C4 variant
NAOP-MDH allowed us to
re comparable to those of
; Berteaet al. 2003).
9
Km values obtained for OAA and NADPH (NADP-MDH) were 29.0 (±0.014) mM and 31,67
(±0.09) mM, respectively. With regard to NADP-ME, the apparent Km values for NADP" and malate
were 19.40 (±0.08) and 242.0 (±0.008) mM, respectively. In the case of NADP-MDH, Vmax values
for OAA and NADPH were 12.52 (±0.021) and 14.97 (±0.012) mKat mg- I prot, respectively.
NADP-ME Vmax values for malate and NADP" were 8.63 (±0.507) and 18.60 (±0.007) mKat
mg" prot. respectively. In general. relatively high activities of NADP-MDH and NADP-ME allowed
a partial characterization of these enzymes and provided evidence for an NADP-ME subtype for
C. citratus. The apparent kinetic properties of both enzymes were comparable to those of plants
belonging to this subtype (Ashton 1990; Hatch et al. 2.007; Hatch et al. 1975), and were consistent
with a high photosynthetic activity. even when the plant was cultivated in a temperate climate.
1.3.2
NADP+ INHIBITION
OF
NADP-MDH
Inhibition studies were carried out by measuring the NADP-MDH-catalyzed reaction at varying
concentrations of NADP" and constant concentrations of OAA (1.0 mM) and NADPH (0.2 mM).
NADP-MDH activity was increasingly inhibited by increasing NADP" concentrations. The activ
ity value recorded in the presence of 0.25 mM NADP" was only 38% of the activity measured in
absence of the oxidized coenzyme.
In Zea mays, activation of NADP-MDH is regulated by oxidation and reduction of cysteine
residues (thioredoxin-rnediated system) (Lunn et al. 1995), and interconversion of the reduced and
oxidized forms is influenced by the NADPH/NADP" ratio (Trevanion et at. 1997). A high NADPHI
NADP" ratio leads to a more active enzyme; thus, high rates of OAA reduction only occur in reduced
conditions. The percentage of inhibition caused increasing NADP" concentration in our DEAE
preparations was in accordance with the observations reported earlier. The relatively high activities
of NADP-ME detected enable us to characterize the enzyme in C. citratus. A low Km value was
calculated for free NADf>+. These results confirm the high affinity of NADP" for its binding site in
all isoforms of this enzyme (Rothermel and Nelson 1989). A higher Km value was calculated for
malate in accordance with literature data.
1.3.3
pH
AND TEMPERATURE DEPENDENCE OF
NADPH-MDH
AND
NADP-ME
pH studies were carried out by using a buffer system that contained an equimolar mixture of buffers
adjusted to different pH values with KOH (Bertea et al. 2001).
Assays were performed at different pH values, 6.0 to 10.5 for NADP-MDH, and 6.0 to 10 for
NADP-ME, using the DEAE-preparation. Maximal activity of NADP-MDH enzyme was observed
at pH 8.3, in agreement with the published data (Ashton 1990). An increase in activity was
observed starting from the lowest pH value (6.0) up to pH 8.3. At pH 7.0-7.5, the activity was com
parable to that at pH values ranging between 9.5 and 10.0. At pH 10.5 the activity was comparable
to that recorded at pH 6.0. Temperature changes also affected the reaction rate. The influence of
temperature on enzyme activities was determined for both NADP-MDH and NADP-ME by adding
the substrates to standard assay mixtures equilibrated at the appropriate temperatures. The assays
were performed by using the DEAE-preparation. Apparent activation energy was calculated from
Arrhenius plots.
When enzyme activity was measured using the standard assay system at temperatures ranging
from 20°C to 49°C, maximal activity was detected at 35°C, while at 49°C activity was lower, but
still much higher than that at 20°C, in accordance with the typical behavior of C 4 photosynthetic
enzymes. From a linear Arrhenius plot of the data, in a temperature range from 20°C to 38°C, the
activation energy of the reaction was calculated to be 6970.8 cal mol:'. The highest NADP-ME
activity was recorded at pH 8.3, in accordance with the enzyme characteristics (Edwards and
f:'
I
~'.
10
Essential Oil-Bearing Grasses: The Genus Cymbopogon
Andreo 1992), whereas at pH 10.0 the activity was higher than the activity recorded at pH 6.0 and
6.5. With regard to temperature, maximal activity was measured at 45°C, the lowest at 20°e. Also
in this case, activity response to temperature changes was typical of C 4 plants (Edwards and Andreo
1992). The activation energy of the reaction, calculated in a temperature range from 20°C to 45°C,
was 7605.2 cal mol:".
Climatic conditions exert an evident effect on the physiological status of photosynthetic enzymes,
and variations in light, temperature, moisture, etc., may influence the cytosolic and stromal pH
(Ashton 1990). In C 4 plants, NADP-MDH has subunits of 42 kDa, and the native enzyme apparently
occurs as either a tetramer or a dimer. The tetramer is the more active form; it is stable at alkaline
pH values and at high temperatures (Ashton 1990). NADP-ME is a tetramer with a molecular weight
of about 280 kDa, and it is more stable at pH values above 8.0 (Edwards and Andreo 1992).
This enzyme exists as a dimer and a monomer, both of which are active. Differences in pH can
dramatically alter the activities of these photosynthetic enzymes. Temperature is another critical
parameter. When it is low, photosynthetic rates of C 4 plants may fall below those of C3 ones. The
response of enzyme activities to such changes depends on the photosynthetic pathway adopted,
resulting in a different optimum range of temperatures over which the highest growth rate can be
maintained (Fitter and Hay 1987).
Because they originated in tropical and subtropical areas, the optimum temperature for photo
synthesis in C 4 plants is 30°C-40°C, which is approximately LOoC higher than in C, plants (Leegood
1993). However, C4 photosynthesis is usually sensitive to low temperature; the minimum tempera
ture for photosynthesis in several C4 tropical grasses is 5°C_10°C (Casati et al. 1997). Activities at
different temperatures and pH values of C. citratus NADP-MDH and NADP-ME indicated that this
species is a C4 NADP-ME plant, which is able to retain its photosynthetic mechanism even when
cultivated in temperate climates.
1.3.4
CO2 ASSIMILATION AND STOMATAL CONDUCTANCE
A very low compensation point (between 8 and 15 ppm CO 2 ) was calculated for C. citratus. This
result is typical for a C 4 plant. Stomatal opening increased in response to CO 2 concentration up to
157 ppm. However, at higher CO 2 values a decrease was recorded, thus indicating a clear effect
of the CO 2-concentrating mechanism present in C4 plants (data not shown). In order to evaluate
changes in photosynthesis as a function of leaf age, CO 2 assimilation and stomatal conductance
were also measured at different developmental stages of C. citratus leaves. A general increase for
both parameters was observed, starting from primordial up to mature leaves, while a decrease in
CO 2 assimilation and stomatal conductance was recorded in old leaves. Thus, primordial leaves
presented the lowest CO 2 assimilation value (9.01 mmol CO 2 drrr? S-I), while the highest values
were recorded in young and mature leaves, without appreciable differences (22.71 and 23.94 mmol
CO 2 drrr? S-I, respectively). With regard co stomatal conductance, the lowest value was measured in
old leaves (55.00 mM H 20 dm? S-I), while the highest occurred in mature ones (165.27 mM H20
drrr? S-I).
From a physiological point of view, the remarkable differences between the photosynthetic
responses of C 1 and C4 plants to CO 2 concentration become apparent when calculating the CO 2
compensation point. In plants with CO 2-concentrating mechanisms, including C 4 plants, CO 2
concentrations at the carboxylation sites are often saturating. Plants with C 4 metabolism have
a CO 2 compensation point of or close to zero, reflecting their very low levels of photorespiration.
The results obtained in C. citratus are in accordance with the values previously recorded on other
species of Cymbopogon (Raiendrudu and Das 1981). In addition, the C 4 mechanism allows the plant
to maintain high photosynthetic rates at lower partial CO 2 pressures in the intercellular spaces of
the leaf, which require lower rates of stomatal conductance for a given ratt: of photosynthesis. For
these reasons, measuring the CO 2 compensation point and stomatal conductance can be useful to
distinguish between C 3 and C4 pathways.
1.4
1.4.1
The Genus Cymbopogon
ded at pH 6.0 and
:lowest at 20°e. Also
wards and Andreo
from 20°C to 45°C,
syntheticenzymes,
lie and stromal pH
enzyme apparently
·itis stable at alkaline
. a molecular weight
Andreo 1992).
.Differences in pH can
,. re is another critical
those of C3 ones. The
ic pathway adopted,
st growth rate can be
temperature for photo
in C3 plants (Leegood
the minimum tempera
al. 1997). Activities at
-ME indicated that this
mechanism even when
d for C. citratus. This
CO2 concentration up to
indicating a clear effect
n). In order to evaluate
stomatal conductance
. A general increase for
Yes, while a decrease in
Thus, primordial leaves
while the highest values
(22.71 and 23.94 mmol
st value was measured in
ure ones (165.27 mM u.o
. tween the photosynthetic
when calculating the CO 2
including C 4 plants, CO 2
with C4 metabolism have
levelsof photorespiration.
viously recorded on other
mechanism allows the plant
m the intercellular spaces of
, rate of photosynthesis. For
Conductance can be useful to
1.4
11
MOLECULAR BIOLOGY
The morphological variation and oil characteristics of various species and varieties of Cymbopogon
have been reported, but such information is not sufficient to precisely define the relatedness among
the morphotypes and chemotypes. For instance, C. martinii var. sofia and C. martinii val'. motia are
morphologically almost indistinguishable, but show distinct chernotypic characteristics in terms of
oil constituents (Guenther 1950a). Conversely, phenotypically and taxonomically well distinguish
able species produce oils of almost identical chemical compositions, such as lemongrass oils from
C. citraius and C. fiexuosus (Khanuja et al. 2005). Such phenotypic traits, whether morphological
or chemotypic, are basically the phenotypic expression of the genotype, while DNA markers are
independent of environment, age, and tissue, and expected to reveal the genetic variation more
conclusively in assessing such variations. Introgression of various traits, intermittent mutations,
and selection through human intervention may lead to variation in chemotypic characters across
geographical distributions (Kuriakose (995). While natural hybridization may lead to the formation
of morphological or chemotypic intermediates, defining taxa purely on this basis may not be appro
priate. Molecular markers provide extensive polymorphism at DNA level used for differentiating
closely related genotypes (Pecchioni et al. 1996) and also to find out the extent of genetic diversity
(Jain et al. 2003).
.
Different types of molecular markers have been developed and used in various plant species
including grasses in the recent past years.
1.4.1
RANDOMLY AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS
DNA-based markers such as randomly amplified polymorphic DNA (RAPD) (Welsh and
McClelland 1990) have been employed not only for cultivar identification but also for phylogenetic
and pedigree studies in a number of food, forage, and fiber crops (Chalmers et al. 1992; Kresovich
et al. 1994). RAPDs have provided rare biotype specific markers in medicinal and aromatic plants
such as vetiver (Adams and Daffern 1997) and Artemisia annua (Sangwan et al. 1999).
Randomly primed polymerase chain reaction provides a simple and fast approach to detecting
DNA polymorphism, with allelic RAPD marker variations being detected as a plus or minus allele
(Welsh and McClelland 1990). In particular, the approach provides multi locus profiling of DNA
sequence differences of genotypes when genetic knowledge is lacking. Several studies have been
carried out on Cymbopogon species by employing the RAPD approach.
A study using RAPD markers was carried out by Shasany and coworkers (2000) to trace the
ancestors of cultivar Java II within C. winterianus. The species C. winterianus Jowitt is believed to
have originated from the well-known species C. nardus, type Maha Pengiri, referred to as Ceylonese
(Sri Lankan) commercial citronella. It was introduced into Indonesia and became commercially
known as the Javanese citronella. The Javanese type C. winterianus material was introduced into
India for the commercial cultivation of this crop during 1959. Varieties of this species have been
developed later by the use of breeding procedures from the same introduced material. The authors
carried out a comparative analysis of the morphological characters, chemical traits (oil percent
age and constituents), and RAPD profiles to assess the diversity and relationships among the Java
citronella cultivated forms, which were systematically developed for their suitability to different
climatic regions, and also their differences and similarities to the believed parent species C. nardus
(Purseglove 1975). All these accessions were analyzed at the molecular level for the similarity and
genetic distances through RAPD profiling, using 20 random primers. More than 50% divergence
was observed for all the C. winterianus accessions in relation to C. nardus accession CN2. The clus
tering based on the similarity matrices showed a major cluster of six accessions, consisting of two
subclusters. The accession C. nardus CN2 got carved out along with two C. winterianus accessions,
CW2 and CW6. On the other hand, the accessions CW2 and CW6 demonstrated distinct identities
compared to CN2 at the DNA level (Shasany et al. 2000).
12
Essential Oil-Bearing Grasses: The Genus Cymbopogon
The same approach was used by Sangwan et al. (2001) on eleven elite and popular Indian cul
tivars of Cymbopogon aromatic grasses of essential oil trade types-citronella, palmarosa, and
lemongrass. They were characterized by means of RAPOs to discern the extent of diversity at the
DNA level between and within the oil biotypes. Primary allelic variability and the genetic bases of
the cultivated germplasm were computed through parameters of gene diversity, expected heterozy
gosity, allele number per locus, SENA, and Shannon's information indices. The allelic diversity
was found to be in this order: lemongrass > palmarosa > citronella. Lemongrasses displayed higher
(1.89) allelic variability per locus than palmarosa (1.63) and citronella (1040). Also, RAPOs of diag
nostic and curatorial importance were discerned as "stand-along" molecular descriptors. Principal
component analysis (PCA) resolved the cultivars into four clusters: one each of citronella and pal
rnarosa, and two of lemongrasses (one of C. flexuosus and another of C. pendulus and its hybrid
with C. khasianus). Proximity of the two species-groups of lemongrasses was also revealed as they
shared the same dimension in the three-dimensional PCA (Sang wan et al. 2001).
The same authors analyzed the elite and popular cultivars of C. martinii for genomic and
expressed molecular diversity using RAPO, enzyme, and SOS-PAGE protein polymorphisms. The
allelic score at each locus of the enzymes, as well as presence and absence profiling in RAPOs,
and overall occurrence of band types were subjected to computation of gene diversity, expected
heterozygosity, allele number per locus, and similarity matrix. These, in turn, provide inputs to
derive primary account of allelic variability, genetic bases of the cultivated germplasm, putative
need for gene/trait introgression from the wild or geographically diverse habitat in elite selections.
'PRel' possessed the highest number of unique bands based on RAPD polymorphism. In variety
'IW31245E,' diaphorase and glutamate oxaloacetate transaminase isozymes generated two unique
bands as dia-III2 and got-II4. 'RRL(B)77' exhibited three unique bands; one produced by esterase
as allele est-III and two by malic enzyme (me-IIIt3). Only one unique band was generated by
malic enzyme in variety 'Trishna.' But sofia had three unique bands, two contributed by diaphorase
(dia-II3 and dia-II4) and one by glutamate oxaloacetate transaminase (got-II2). SDS-PAGE analysis
revealed the presence of unique polypeptide fragments (97.7 to 31.6 kOa) in varieties 'IW31245E,'
'RRL(B)77,' 'Tripta,' 'Trishna,' 'PRCl,' and sofia, generated as a diagnostic marker. In general,
molecular distinctions associated with varieties. motia and sofia were clearly noticed in C. martinii
(Sangwan et al. 2003).
Khanuja et al. (2005) analyzed 19 Cymbopogon taxa belonging to 11 species, 2 varieties, I
hybrid taxon, and 4 unidentified species for their essential oil constituents and RAPO profiles to
determine the extent of genetic similarity and thereby the phylogenetic relationships among them.
Remarkable variation was observed in the essential oil yield ranging from 0.3% in Cymbopogon
travancorensis Bor to 1.2% in Cymbopogon martinii (Roxb.) Wats. var. motia. Citral, a major essen
tial oil constituent, was employed as the base marker for chemotypic clustering. Based on genetic
analysis, elevation of Cymbopogon fiexuosus var. microstachys (Hook. E) Soenarko to species sta
tus and separate species status for C. travancorensis Bor, which has been merged under C.fiexuosus
(Steud.) Wats., were suggested toward resolving some of the taxonomic complexes in Cymbopogon.
The separate species status for the earlier proposed varieties of C. martinii tmotia and sofia) is fur
ther substantiated by these analyses. The unidentified species of Cymbopogon have been observed
as intermediate forms in the development of new taxa (Khanuja et al. 2005).
Somaclonal variants that arise through the tissue culture have been reported in a large number of
species. The significance of somaclonal variation in crop improvement depends upon establishing a
genetic basis for variation (Larkin and Scowcroft 1981). The use of the molecular marker is becom
ing widespread for the identification of somaclonal variant. In particular, RAPD markers have
proved useful for this purpose owing to its ability to analyze DNA variation at many loci using small
amounts of tissue (Munthali et al. 1996; Wallner et al. 1996). Screening of somaclonal variants with
improved oil yield and quality have been reported in two species of Cymbopogon, C. winterianus
and C. martinii (Mathur et al. 1988; Patnaik et al. 1999), but RAPDs were not used to establish
The Genus Cymbopogon
popular Indian cul
ella, palmarosa, and
tent of diversity at the
d the genetic bases of
ity, expectedheterozy
. The allelic diversity
ssesdisplayed higher
. Also, RAPOs of diag
. r descriptors. Principal
. h of citronella and pal
pendulus and its hybrid
also revealed as they
2001).
rtinii for genomic and
in polymorphisms. The
e profiling in RAPOs,
'gene diversity, expected
turn, provide inputs to
ed germplasm, putative
bitat in elite selections.
lymorphism. In variety
sgenerated two unique
one produced by esterase
band was generated by
eontributed by diaphorase
, -(12). S-OS-PAGE analysis
. ~in varieties 'IW31245E,'
stic marker. In general,
y noticedin C martinii
II species, 2 varieties, I
ts and RAPD profiles to
'selationships among them.
0.3% in Cymbopogon
tia. Citral,a major essen
, ustering. Based on genetic
'F.) Soenarko to species sta
"merged under Cflexuosus
eomplexes in Cymbopogon.
, ii (motia and sofia) is fur
pagan have been observed
5).
rted in a large number of
depends upon establishing a
molecularmarker is becom
lar, RAPD markers have
'on at manyloci using small
of somaclonalvariants with
. ymbopogon, C winterianus
were not used to establish
13
the genetic basis of this somaclonal variation. The paper of Nayak and coworkers (2003) reports the
quantitative and qualitative analysis of selected somaclones of jamrosa (a hybrid Cymbopogon) in
the field, screening and selection of agronomically useful somaclonal variants with high oil yield
and desirable quality, and detection of gross genetic changes through RAPD analysis.
In this study. the high oil yield somaclonal variants SCI and SC2 were subjected to RAPD analy
sis and the result was compared with RAPD profile of the control. A total of 22 arbitrary primers
were utilized for initial screening for their amplifying ability. Of these, 12 primers successfully
amplified jamrosa DNA with reproducible banding pattern. In general, 2-11 amplified fragments
were scored, depending upon primers, ranging in molecular sizes from 266 bp to 1.9 Kb. The test
samples SCI, SC2. and the control could be suitably distinguished by the presence of specific mark
ers or by their absence. Out of the two somaclones analyzed, relatively less distinctness in the ampli
fied DNA of SC2 was detected using the primers tested. Banding pattern of this somaclone SC2
and the control plant was similar whereas in other variants, somaclone (SCI) DNA polymorphism
was observed by having distinct banding pattern. As indicated by RAPDs gross genetic changes
have occurred in somaclone (SCl). The results obtained by Nayak and coworkers are in agreement
with detection of somaclonal variants by RAPD analysis in Populus deltoides (Rani et al. 1995),
garlic (AI-Zahim et al. 1999) and in rice (Yang et al. 1999). Taylor et al. (1995) also reported that
RAPD analysis proved suitable for detecting gross genetic changes occurring in sugarcane tissues
subjected to prolonged in vitro culture. This work has demonstrated the scope of selecting improved
clones of jamrosa with high oil yield and quality through somaclonal variation and suitability of
RAPDs for detecting gross genetic changes in somaclonal variants at DNA level.
1.4.2
SIMPLE SEQUENCE REPEAT MARKERS (SSRs)
Kumar and coworkers (2007) developed a set of simple sequence repeat markers from a genomic
library of Cymbopogon jwarancusa to help in the precise identification of the species (includ
ing accessions) of Cymbopogon. For this purpose, they isolated 16 simple sequence repeats
containing genomic deoxyribonucleic acid clones of C. jwarancusa, which contained a total of
32 simple sequence repeats with a range of I to 3 simple sequence repeats per clone, The major
ity (68.8%) of the 32 simple sequence repeats comprised dinucleotide repeat motifs followed
by simple sequence repeats with trinucleotide (21.8%) and other higher-order repeat motifs.
Eighteen (81.8%) of the 22 designed primers for the above simple sequence repeats amplified
products of expected sizes, when tried with genomic DNA of C. jwarancusa. Thirteen (72.2%)
of the 18 functional primers detected polymorphism among the three species of Cymbopogon
(C jlexuosus, C. pendulus, and C. jwarancusai and amplified a total of 95 alleles (range 1-18
alleles) with a PIC value of 0.44 to 0.96 per simple sequence repeat. Thus, the higher allelic
range and high level of polymorphism demonstrated by the developed simple sequence repeat
markers are likely to have many applications such as in improvement of essential oil quality by
authentication of Cymbopogon species and varieties, and mapping or tagging the genes control
ling agronomically important traits of essential oils, which can further be utilized in marker
assisted breeding (Kumar et al. 2007). Considering the high reproducibility and polymorphic
nature of the SSRs, the SSRs developed by Kumar and coworkers (2007) may be utilized for
identification/authentication of superior accessions/species of the genus Cymbopogon with cor
rectness and certainty to ensure production of high-quality oil. The SSR markers developed
during this kind of study might also be used to resolve the taxonomic disputes, study the genetic
diversity, and for genetic mapping and QTL (quantitative trait loci) analysis. The SSRs due to
their codominant nature may be specifically useful for the identification of interspecific hybrids,
which have been shown to be superior in terms of both their yielding high quantity and better
quality of essential oils.
14
1.5
Essential Oil-Bearing Grasses: The Genus Cymbopogon
PHYSIOLOGY AND ECOPHYSIOLOGY
As discussed, Cymbopogon has a photosynthetic machinery that allows the plant to perform high
rates of carbon assimilation and, at the same time, save water. In species that produce essential oil,
the biogenesis of terpenoids relies on photosynthetic carbon dioxide reduction on the one hand and
availability of water and nutrients, on the other. For this reason several studies have been conducted
in order to assess which nutrients and at what conditions were required for an optimal production
of both biomass and essential oils. In this section, we will discuss the most important Cymhopogon
species in terms of yield of biomass and essential oil production as related to nutrition. Furthermore,
when available, references to biotechnological applications will be also reported.
1.5.1
CYMBOPOGON MART/Nil
Water requirement, productivity, and water use efficiency of palmarosa (c. martiniis were studied
under different levels of irrigation (0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, and 1.5 IW:CPE ratio). Growth,
herb, and essential oil yield increased significantly up to 0.5 IW:CPE ratio. At 0.5 IW:CPE ratio
palmarosa produced 47.3 t ha" yr' of fresh herb and 227.3 kg ha' yr' of essential oil. Further
increase in irrigation levels caused an adverse effect on growth and yield of palmarosa. Irrigation
levels did not affect the quality of oil in terms of its geraniol and geranyl acetate contents. Water
requirement of palmarosa was worked out to be 89.1 cm. The highest water use efficiency of 2.97 kg
ha- l crrr ' oil was recorded at 0.1 IW:CPE ratio, at 0.5 IW:CPE ratio (optimum) it was 2.55 kg ha"
crrr" oil. Irrigation scheduled at 0.5 IW:CPE ratio gave the highest net return of Rs 51 963 ha'
yr" (Singh et al. 1997). In C. martinii the application of 160 kg N/ha per year produced the high
est amount of biomass and essential oil, and increased the net profit and NPK uptake by the crop
(Rao et al. 1988); furthermore, dressing of 40 kg K/ha enhanced the yield of biomass by 13.6% and
6.5% and that of oil by 12.9% and 6.1%, compared with 20 and 80 kg Kzha, respectively (Singh
et al. 1992). In the same species, harvesting the crop at early seeding (l12-l 15 days after planting)
gave 25% more herbage and 5l% more oil yield over harvesting vegetative stage, while the oil so
produced had higher content (90.1%) geraniol (Maheshwari et al. 1992). Highest dry-matter yield,
essential oil yield, and maximum net return of palmarosa were recorded by applying Azotobacter
at 2 kg/ha together with 20 kg N + 20 kg P/ha under rainfed condition in a shallow black soil
(Maheshwari et al. 1998). Intercropping of blackgram-blackgram or sorghum fodder-ratoon with
palmarosa gave additional yields of 660 kg/ha seed and 16.6 t/ha fodder, respectively, compared
with the sole crop of palmarosa (Rao et al. 1994). Moreover, sowing of pigeon pea in alternate rows
parallel to palmarosa proved most efficient and economic, as it provided higher economic returns,
bonus income, and monetary advantage, and the oil content and quality in terms of total geraniols
of palmarosa were not adversely affected by adoption of intercropping (Maheshwari et al. 1995).
However, in palmarosa-pigeon pea intercropping systems, competition exists mainly for light rather
than for nutrients and moisture, possibly because the two crop components acquire their nutrients
and moisture from different soil layers (Singh et al. 1998). Concerning essential oil production of
palmarosa, changes in fresh weight, dry weight, chlorophyll, and essential oil content and its major
constituents, such as geraniol and geranyl acetate, were examined for both racemes and spathe at
various stages of spikelet development (Dubey et al. 2000). The essential oil content was maximal
at the unopened spikelets stage and decreased significantly thereafter. At unopened spikelets stage,
the proportion of geranyl acetate (58.6%) in the raceme oil was relatively greater compared with
geraniol (37.2%), whereas the spathe oil contained more geraniol (61.9%) compared with geranyl
acetate (33.4%). The relative percentage of geranyl acetate in both the oils, however, decreased sig
nificantly with development, and this is accompanied by a corresponding increase in the percentage
of geraniol. Analysis of the volatile constituents from racemes and spathes (from mature spikelets)
and seeds by capillary GC indicated 28 minor constituents besides the major constituent geraniol.
(E)-Nerolidol was detected for the first time in an essential oil from this species. The geraniol
~
The Genus Cymbopogon
'the plant to perform high
'that produce essential oil,
tion on the one hand and
ies have been conducted
for an optimal production
important Cymbopogon
to nutrition. Furthermore,
rted.
(c. martiniit were studied
1.5 IW:CPE ratio). Growth,
ratio. At 0.5 IW:CPE ratio
I of essential oil. Further
d of palmarosa. Irrigation
'yl acetate contents. Water
r use efficiency of 2.97 kg
. imum) it was 2.55 kg na'
return of Rs 51 963 ha'
r year produced the high
NPK uptake by the crop
ld of biomass by 13.6% and
K/ha, respectively (Singh
12-115 days after planting)
ive stage, while the oil so
), Highest dry-matter yield,
by applying Azotobacter
'on in a shallow black soil
rghum fodder-ratoon with
r, respectively, compared
pigeon pea in alternate rows
higher economic returns,
y in terms of total geraniols
(Maheshwari et al. 1995).
exists mainly for light rather
ents acquire their nutrients
essential oil production of
tial oil content and its major
both racemes and spathe at
ial oil content was maximal
. ,At unopened spikelets stage,
ively greater compared with
,9%) compared with geranyl
oils, however, decreased sig
ing increase in the percentage
thes (from mature spikelets)
major constituent geraniol.
this species. The geraniol
15
content predominated in the seed oil, whereas the geranyl acetate content was higher in the raceme
oil (Dubey et al. 2000).
Biotechnology is a powerful and consolidated technique for understanding plant growth and
development as well as for improving biomass and yield of crops. Callus could be induced from
nodal explant of mature tillering plant of C. martinii in different basal media supplemented with
2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (Kin). Shoot bud was regenerated from such
calli in MS and B5 basal media modified with various combinations of phytohormones, vitamins,
and amino acids. Root formation was induced either in white basal medium or half-strength MS or
B5media containing naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA). High survival
percentage of regenerated plants in soil was obtained after acclimatization in normal environment
(Baruah and Bordoloi 1991). A detailed characterization of chromosomal status was carried out in
callus, somatic embryos, and regenerants derived from in vitro cultured nodal and inflorescence
explants of C. martinii (2n = 20). Both the callus lines revealed considerable ploidy variations (tet
raploids to octoploids and hyperoctoploids), and the degree of polyploidization increased with the
culture age.Prequencies of various polyploid cells were significantly higher in nodal callus lines
(3.6% to 46.3%) than the inflorescence callus lines (1.9% to 23.6%) when analyzed over 520 days
of culture. Somatic embryos derived from both the callus lines retained a predominantly diploid
chromosome status throughout (99.0% to 93.1%). Root tip analysis of about 70 regenerants ran
domly taken from cultures of various ages (days 20 to 520) revealed only diploid chromosome
numbers (2n = 20) implying a strong relative stability of diploidy among the regenerants (Patnaik
et al. 1996). Chromosome counts of cells in suspensions, calli, and somatic embryos derived from
cultures of different ages revealed the presence of diploids, tetraploids, and octaploids (Parnaik
et al. 1997). Sodium chloride tolerant callus lines of C. martinii were obtained by exposing the cal
lus to increasing concentrations of NaCI (0-350 mM) in the MS medium. The tolerant lines grew
better than the sensitive wild-type lines in all concentrations of NaCl tested up to 300 mM. Callus
survival and growth were completely inhibited, resulting in tissue browning and subsequent death
at 350 mM NaCl. The selected lines retained their salt tolerance after 3-4 subcultures on salt-free
medium, indicating the stability of the induced salt tolerance. The growth behavior, the Na", K+,
and proline contents of the selected callus lines were characterized and compared with those of
the NaCl-sensitive lines. The Na" levels increased sharply, while the K+ level declined continu
ously with the corresponding increase in external NaCI concentrations in both lines, but the NaCl
tolerant callus lines always maintained higher Na" and K+ levels than that of the sensitive lines.
The NaCI-selected callus line accumulated high levels of proline under salt stress. The degree of
NaCl tolerance of the selected lines was in negative correlation with the K+/Na+ ratio and in positive
correlation with proline accumulation (Patnaik and Debata 1997a). The embryogenic potential of
NaCl-tolerant callus selected even at 300 mM could be improved significantly by the incorporation
of gibberellic acid (GA(3)) and abscisic acid (ABA), in the medium where, with 2 mg/L of GAOl
and I mg/L of ABA, the highest rates of embryogenesis (44.5%, 28.8%, and 18.6%) were achieved
against 17.5%,8.2%, and 1.8% on medium devoid of GAiJ), and ABA at 50%, 150%, and 250 mM
of NaCl, respectively (Patnaik and Debata 1997b). Finally, plants regenerated from cell suspension
cultures of palmarosa were analyzed for somaclonal variation in five clonal generations. A wide
range of variation in important quantitative traits, for example, plant yield, height, tiller number,
oil content and qualitative changes in essential oil constituents geraniol, geranyl acetate, geranyl
formate, and linalool, were observed among the 120 somaclones screened. Eight somaclones were
selected on the basis of high herb and oil yield over the donor line and high geraniol content in the
oil. Based on performance in the field trials, three superior lines were selected, and maintained for
five clonal generations. The superior lines exhibited a reasonable degree of stability in the traits
selected (Patnaik et al. 1999).
Palmarosa was also found to be associated with a vesicular-arbuscular mycorrhizal (VAM)
fungus, Glomus aggregatum. Glasshouse experiments showed that inoculation of palmarosa with
G. aggregatum caused a twofold and threefold biomass production as compared to nonmycorrhizal
Essential Oil-Bearing Grasses: The Genus Cymbopogon
16
plants. These findings indicate the potential use of YAM-fungi for improving the production of this
essential oil-bearing plant (Gupta and Janardhanan 1991). Furthermore, when the interactive effects
of phosphate solubilizing bacteria, N-2 or fixing bacteria, and arbuscular mycorrhizal fungi (AMF)
were studied in a low phosphate alkaline soil amended with a tricalcium insoluble source of inor
ganic phosphate on the growth of C. martinii, The rhizobacteria behaved as a "mycorrhiza helper"
and enhanced root colonization by G. aggregatum in presence of tricalcium phosphate at the rate of
200 mg kg:' soil (PI level) (Ratti et a1. 20m).
A dramatic increase in PEP carboxylase activity and oil biosynthesis was observed under
drought conditions in C. martinii (Sangwan et al. 1993). The physiological and biochemical
basis of drought tolerance in C. martinii has been elucidated on the basis of growth and metabolic
responses (Fatima et a1. 2002).
1.5.2
CYMBOPOGON FLEXUOSUS
Cymbopogon fiexuosus (also known as 1emongrass) is a perennial, multicut aromatic grass that
yields an essential oil used in perfumery and pharmaceutical industries and vitamin A. It has a
long initial lag phase. The growth and herbage and oil production of C. jiexuosus in response to
different levels of irrigation water (IW) 0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, and 1.5 times cumulative pan
evaporation CPE evaluated on deep sandy soils showed that an increment in the level of irriga
tion increased the plant height up to 0.7 IW:CPE ratio. However, the response of irrigation levels
on tiller production of lemongrass differed with the season of harvest. Oil content had an inverse
relationship with the levels of irrigation, whereas significantly higher herb and essential oil yields
were recorded at 0.7 IW:CPE ratio, irrespective of season of harvest (Singh et al. 2000). Application
of nitrogen (0, 50, 100, and 150 kg N ha- l yr ') and phosphorus to C. fiexuosus crops maintained
the fertility of the soil, while potassium depletion was noticed (Singh 2001). When the effects of
phosphorus (at 0, 17.75, and 35.50 kg ha- l yell, potassium (at 0,33.2,66.4, and 99.6 kg ha- l yr') and
nitrogen (at 100 and 200 kg ha- l yell and potassium (at 0, 33.2 and 66.4 kg ha- l yr") were studied
on herbage and oil yield of Ci fiexuosus. it was found that plants produced significantly higher herb
age and oil yields compared with controls (Singh et al. 2005; Singh and Shivaraj 1999). Spraying of
iron-complexed additives on C. flexuosus increased iron translocation and the dry-matter produc
tion. Application of iron chelates and salts increased the vegetative herb yield, and oil and citral
content. While maximum geraniol and less citral were obtained in the chlorotic plants, Fe recov
ered plants possessed more citral and less geraniol. The maximum recovery of total chlorophyll
and nitrate reductase activity were recorded in the crop when Fe-EDTA chelates were sprayed at
22.4 ppm (Misra and Khan 1992). In Ci jiexuosus. a closer plant spacing of 45 x 45 ern resulted in
higher herb and oil yields compared to wider spacing of60 x 60 em. Application of 150 kg N ha- l
yr- l resulted in higher herb and oil yields. Higher nitrogen applications also increased the plant
height and number of tillers per clump. The oil content and quality were not influenced by spacing
and nitrogen levels (Singh et al. 1996b).
As for C. martinii, intercropping of C. flexuosus with the food legumes such as blackgram
(Vigna mungo (L) Hepper), cowpea (Vigna unguiculata (L) Walp), or soybean (Glycine max (L)
Merr.) prompted extra yields over and above that of pure cultures, without affecting the oil yield
(Singh and Shivaraj 1998).
The influence of different foliar applications of the triacontanol (Tria.)-based plant growth regu
lator Miraculan on growth, CO 2 exchange, and essential oil accumulation in C. fiexuosus showed
increased rates in plant height, tillers per plant, biomass yield, accumulation of essential oil, net
CO 2, and exchange and transpiration compared to the untreated control, but the number of leaves per
tiller remained unaffected. Application of Miraculan also increased micronutrient uptake and total
chlorophyll and citral content but decreased chlorophyll alb ratio and stomatal resistance. Increase
in shoot biomass, photosynthesis, and chlorophyll were significantly correlated with essential oil
content (Misra and Srivastava 1991). Only young and rapidly expanding C. fiexuosus leaves were
1.5.3
. The Genus Cymbopogon
ing the production of this
whenthe interactive effects
'mycorrhizal fungi (AMF)
insoluble source of inor
as a "mycorrhiza helper"
m phosphate at the rate of
f
r
sis was observed under
ogical and biochemical
's of growth and metabolic
lticut aromatic grass that
and vitamin A. [t has a
C. flexuosus in response to
1.5 times cumulative pan
nt in the level of irriga
ponse of irrigation levels
Oil content had an inverse
herb and essential oil yields
, h et aL 2000). Application
"jtexuosus crops maintained
2001). When the effects of
.A. and99.6kg ha' yr ') and
4 kg ha' yr') were studied
significantly higher herbShivaraj 1999). Spraying of
and the dry-matter produc
b yield, and oil and citral
chlorotic plants, Fe recov
overy of total chlorophyll
A chelates were sprayed at
:, g of 45 x 45 em resulted in
;Application of 150 kg N ha'
ns also increased the plant
not influenced by spacing
legumes such as blackgram
.,. soybean (Glycine max (L)
ithout affecting the oil yield
f
·a.)-based plant growth regu
'on in C. flexuosus showed
ulation of essential oil, net
butthe number of leaves per
. 'icronutrient uptake and total
stomatal resistance. Increase
correlated with essential oil
ing C. flexuosus leaves were
The Genus Cymbopogon
17
found to have the capacity to synthesize and accumulate essential oil and citral. The pattern of the
ratio of the label incorporated in citral to that in geraniol, during leaf ontogeny, evinced parallelism
with the geraniol dehydrogenase activity. The elevated levels of glucose-6-phosphate dehydrogenase,
6-phosphogluconate dehydrogenase, NAOP"-malic enzyme, and NAOP"-isocitrate dehydrogenase
coincided with the period of active essential oil biogenesis accompanying early leaf growth (Singh
et al. 1990). Thus, there is an active involvement of oxidative pathways in essential oil biosynthesis.
The time-course (12 h light followed by 12 h dark) monitoring of the C-14 radioactivity in starch and
essential oil, after exposure of the immature (\5 days after emergence) leaf to (CO)-C-14, revealed
a progressive loss of label from starch and a parallel increase in radioactivity in essential oil. Thus,
there was indication of a possible degradation of transitory starch serving as the source of carbon
precursor for essential oil (monoterpene) biogenesis in the tissue (Singh et al. 1991).
Biotechnological applications revealed that C. fiexuosus plants derived from somatic embryoids
were more uniform in all the characteristics examined when compared with the field performance
of plants raised through slips by standard propagation procedures (Nayak et al. 1996).
A fungal endophyte, Balansia sclerotica (Pat.) Hohn., has been found to establish a perennial
association with the commercially grown East Indian C. fiexuosus cv. Kerala local (syn. = 00-19).
Endophyte-infected plants produced 195% more shoot biomass and 185% more essential oil than
the endophyte-free control plants when grown experimentally under glasshouse conditions. The
essential oil extracted from the endophyte-infected plants is qualitatively identical with that of
endophyte-free plants and is free of toxic ergot alkaloids. Thus, B. sclerotica-infected East Indian
C. flexuosus has potential for agricultural exploitation (Ahmad et al. 2001).
1.5.3
CYMBOPOGON WINTERIANUS
Java citronella (c. winterianus) is a perennial, multiharvest aromatic grass, the shoot biomass of
which, on steam distillation, yields an essential oil extensively used in fragrance and flavor industries.
Fresh C. winterianus (Java citronella) herbage and essential oil yields were significantly influenced
by application of N up to 200 kg ha:' yr', while tissue N concentration and N uptake increased only
to ISO kg N ha", The oil yields with neem cake-coated urea (urea granules coated with neem cake)
and urea super granules were 22 and 9% higher over that with prilled urea, and urea supergranules
were significantly increased up to 200 kg N ha' while with neem cake-coated urea, response was
observed only to 150 kg N ha'! Estimated recovery of N during two years from neem cake-coated
urea, urea supergranules, and prilled urea were 38%. 31%, and 21%, respectively (Singh and Singh
1992).The interaction between N doses and nitrification inhibitors was also significant. Nitrification
inhibitors performed better at the highest N dose (450 kg N ha- ' yr"), and the increase in the essen
tial oil yields was to an extent of 27.3% to 34.6% when compared with "N alone" treatment. The
nitrification inhibitors also increased the apparent N recoveries by citronella considerably. The oil
content in the herb and its quality were not affected by the treatments. The nitrification inhibitors
increased citronella yields and improved N economy (Puttanna et al. 2001). In Java citronella sig
nificant positive correlations were observed between fresh matter, citronellol content, dry and fresh
matter yields, and total essential oil content (Omisra and Srivastava 1994). When the effect of depth
(25,37.5, and 50 mm) and methods (ridge and furrow, and broad bed and furrow method) of irriga
tion acid nitrogen levels (0, 200, and 400 kg N ha' yr') were studied on herb and oil yields of Java
citronella, highest herb and oil yields were achieved with the application of 400 kg N, maintaining
25 mm depth of irrigation, while the content and quality of oil were not affected either by irrigation
or nitrogen (Singh et al. 1996a).
Among food legumes, greengram (Vigna radiata (L.) Wilez.), and among vegetables, c1usterbean
(Cyamopsis psoraloides O. c., syn. Cyamopsis tetragonoloba (L.) Taub.), tomato (Lycopersicon
esculentum Mill.) and lady's finger (Abelmoschus esculentus Moench.) as intercrops of C. win
terianus did not decrease its biomass and essential oil yield and produced bonus yields of these
crops over and above that of Java citronella. Maximum monetary returns were recorded by Java
Essential Oil-Bearing Grasses: The Genus Cymbopogon
18
citronella intercropped with tomato or greengram. However, Java citronella intercropped with red
gram iCajanus cajan (L.) Millsp.), horsegram (Macrotyloma uniflorum (Lam.) Verd, syn. Dolichos
biflorus Roxb.), and brinjal (Solanum melongena L.) suffered significant biomass and essential oil
yield reductions. Horsegram proved to be the most competitive iruercrop, producing least yields and
minimum monetary returns (Rao 2000).
Changes in the utilization pattern of primary substrate, viz. [U-C-14] acetate. (C0 2)-C-14
and [U-C-14] saccharose, and the contents of C-14 fixation products in photosynthetic metabo
lites (sugars. amino acids, and organic acids) were determined in Fe-deficient Java citronella in
relation to the essential oil accumulation. An overall decrease in photosynthetic efficiency of the
Fe-deficient plants as evidenced by lower levels of incorporation into the sugar fraction and essen
tial oil after (C0 2)-C-14 had been supplied was observed. When acetate and saccharose were fed
to the Fe-deficient plants, despite a higher incorporation of label into sugars, amino acids, and
organic acids, there was a lower incorporation of these metabolites into essential oils than in control
plants. Thus, the availability of precursors and the translocation to a site of synthesis/accumulation,
severely affected by Fe deficiency, is equally important for the essential oil biosynthesis in citro
nella (Srivastava et al. 1998). Lal and coworkers (2001) observed that improvement of oil quality
with high citronellal content and low elemol content in Java citronella is believed to be achievable.
although some compromise will have to be made in oil yield.
Nutrient acquisition and growth of Java citronella was also studied in a P-deficient sandy soil
to determine the effects of mycorrhizal symbiosis and soil compaction. When a pasteurized sandy
loam soil was inoculated either with rhizosphere microorganisms excluding VAM fungi (nonmycor
rhizal) or with the VAM fungus, Glomus intraradices Schenck and Smith (mycorrhizal) and sup
plied with 0, 50, or 100 mg P kg' soil, G. intraradices was found to substantially increase root and
shoot biomass, root length, nutrient (P, Zn, and Cu) uptake per unit root length, and nutrient concen
trations in the plant, compared to inoculation with rhizosphere microorganisms when the soil was at
the low bulk density and not amended with P. Little or no plant response to the VAM fungus was
observed when the soil was supplied with 50 or 100 mg P kg:" soil and/or compacted to the highest
bulk density. At higher soil compaction and P supply, the VAM fungus significantly reduced root
length. Nonmycorrhizal plants at higher soil compaction produced relatively thinner roots and had
higher concentrations and uptake of P, Zn, and Cu than at lower soil compaction, particularly under
conditions of P deficiency (Kothari and Singh 1996).
Pythium aphanidermatum was the predominant fungus recovered from the roots of Java citro
nella showing lethal yellowing in the northern part of India. Roots of infected plants showed marked
discoloration, and the cortical region was completely disintegrated and sloughed from the vascular
tissue. Diseased plants were chlorotic and stunted. Rotting was often found to spread from roots to
stem, leading to severe chlorosis and death of the infected plants. The pathogenicity of the fungus
was established. The disease is a potential constraint to citronella cultivation in nonarid climates
where the crop is irrigated extensively (Alam et al. 1992). Another disease affecting commercial
plantations of Java citronella is a collar rot and wilt disease. The causal organism was identified as
Fusarium moniliforme, anamorph of Gibberellafujikuroi. Isolates of the pathogen differed in their
pathogenicity on the host plant under glasshouse conditions. Differences were also observed in
growth rates, pigment production. and sporulation between isolates (Alam et al. 1994).
1.5.4
OTHER CYMBOPOGON SPECIES
Application of graded levels of lime up to 10 t/ha on acid soil (pH 4.2) raised the pH up to 6.7. It
increased the dry herbage of C. khasianus linearly. Increase of soil pH decreased N, P, K, Fe, and
Zn contents in dry herbage significantly but increased the Ca and Mg contents. Liming showed a
positive effect on the uptake of N, P, K, and Ca. However, Fe and Mg declined beyond lime levels
of 23 and 5.0 t, respectively. Uptake of Zn was found fluctuating. Oil content (2.00%-2.07%; DWB)
and geraniol (80.2%-81.0%) in the oil were unaffected by the lime treatments (Choudhury and
•The Genus Cymbopogon
lla intercropped with red
.(Lam.) Verd, syn. Dolichos
biomass and essential oil
producing least yields and
"
. -14] acetate, (C0 2)-C-14
in photosynthetic metabo
ficient Java citronella in
nthetic efficiency of the
sugar fraction and essen
and saccharose were fed
sugars, amino acids, and
ntialoils than in control
of synthesis/accumulation,
oil biosynthesis in citro
improvement of oil quality
believed to be achievable,
'in a P-deficient sandy soi I
,When a pasteurized sandy
ing YAM fungi (nonmycor
ith (mycorrhizal) and sup
. tantially increase root and
length, and nutrient concen
isms when the soil was at
to the YAM fungus was
compacted to the highest
significantly reduced root
ively thinner roots and had
action, particularly under
m the roots of Java citro
ted plants showed marked
sloughedfrom the vascular
nd to spread from roots to
pathogenicity of the fungus
ivation in nonarid climates
isease affecting commercial
organism was identified as
pathogendiffered in their
es were also observed in
et al. 1994).
2) raised the pH up to 6.7. It
decreased N, P, K, Fe, and
contents. Liming showed a
declined beyond lime levels
ntent(2.00%-2.07%; OWB)
.treatments (Choudhury and
The Genus Cymbopogon
19
Bordoloi 1992). Experiments were also conducted to measure the rate of C. caesius litter decompo
sition and to identify fungal flora associated with the litter during different stages of decomposition
in a tropical grassland. Rate of litter decomposition was several times higher than in temperate
grasslands. Buried litter decayed more rapidly, and this rate was not influenced by climatic condi
tions. In contrast, surface litter recorded a lower decomposition rate, which was dependent on tem
poral (seasonal) fluctuations. Total nitrogen, available phosphorus, and potassium contents of the
stem litter decreased during the initial stages of incubation.
Thirty-five species of fungi were isolated from the litter during the different stages of litter
degradation. Most belonged to Hyphornycetes, which are active decomposers (Senthilkumar et al.
1992). C. nardus var. confertiflorus and C. pendulus were grown under mild and moderate water
stress for 45 and 90 d to investigate the impact of in situ drought stress on plants in terms of relative
water content, psi, concentration of proline, activities of PEP carboxylase and geraniol dehydroge
nase, and geraniol and citral biogenesis. The results revealed that the species exhibited differential
responses under mild and moderate stress treatments. In general, plant growth was reduced consid
erably, while the level of essential oils was maintained or enhanced.
Significant induction in catalytic activity of PEP carboxylase under water stress was one of the
consistent metabolic responses of the aromatic grasses. The major oil constituents, geraniol and
citral, increased substantially in both the species. Activity of geraniol dehydrogenase was also
modulated under moisture stress. The responses varied depending upon the level and duration of
moisture stress. The observations have been analyzed in terms of possible relevance of some of
these responses to their drought stress adaptability/tolerance (Singhsangwan et al. 1994). In vitro
plants of C. citratus were established, starting from shoot apices derived from plants cultivated
under field conditions. The effect of the immersion frequency (two, four, and six immersions per
day) on the production of biomass in temporary immersion systems (TIS) of I L capacity was stud
ied. The highest multiplication coefficient (12.3) was obtained when six immersions per day were
used. The maximum values of fresh weight (FW; 62.2 and 66.2 g) were obtained with a frequency
of four and six immersions per day, respectively. However, the values for dry weight (OW; 6.4 g)
and height (8.97 ern) were greater in the treatment with four immersions per day. The TIS used in
this work for the production of lemongrass biomass may offer the possibility of manipulating the
culture parameters, which can influence the production of biomass and the accumulation of sec
ondary metabolites. We describe for the first time the in vitro production of Cymbopogon citratus
biomass in TIS. In vitro regeneration of C. polyneuros was obtained through callus culture using
leaf base, node, and root as explants. Callus was induced from different explants with 2-5 mg/L
alpha-naphthalene acetic acid (NAA) and 1-2 mg/L kinetin in Murashige and Skoog's (MS) basal
medium. High frequency shoots were noticed from leaf-base callus supplemented with 3.5 mg/L
6-benzylaminopurine (BA), I-arginine, adenine, and a low level of NAA (0.2 mg/L). About 80-85
shoot buds were obtained from ca. 200 mg of callus per culture. The individual shoots produced
root in the presence of 0.5-3 mg/L indole 3-butyric acid or its potassium salt.
Regenerated plants were cytologically and phenotypically stable. Regenerants were transplanted
into soil and subsequently transferred to the field (Das 1999). C. nardus could be propagated via
tissue culture using axillary buds as explants. The aseptic bud explants obtained using double steril
ization methods produced stunted abnormal multiple shoots when they were cultured on Murashige
and Skoog (MS) medium supplemented with 1.0 mg L-I or 2.0 mg L-I benzyladenine (BA). Stunted
shoots that cultured on MS + 1.0 mg L -I RA + 1.0 mg L-I N-6-isopentenyl-adenine (2iP) could
induce elongation of shoots from about 60% of the stunted shoots. Normal multiple shoots could
be induced at the highest (19.7 shoots per bud) from the bud explants within 6 weeks when cul
tured on proliferation medium consisted of MS supplemented with 0.3 mg L -I BA and 0.1 mg L-I
indole-3-butyric acid (IBA). The separated individual shoot produced roots when transferred to
basic MS solid medium. The essential oils that were contained in the mature plants namely citro
nellal, geraniol, and citronellol, were also found in the in vitro C. nardus plantlets. Citronellal was
the main essential oil component in the matured plants, while geraniol was the main component in
20
Essential Oil-Bearing Grasses: The Genus Cymbopogon
the in vitro plant lets (Chan et al. 2005). The occurrence, mode of infection, and the extent of dam
age caused by Psilocybe kashmeriensis sp. nov. Abraham on oil grass C. jawarancusa in Kashmir
valley is discussed herein. A brief description of the new agaric species is also offered (Abraham
1995). Dormant vegetative slips of jamrosa (c. nardus var. confertifiorus x C. jwarancusa) were
subjected to various doses of gamma rays. Plants raised from them were screened with a view to iso
late improved clones of the crop. Five mutant clones isolated exhibited variation in quality/quantity
of essential oil. These changes in oil characters were attributed to microlevel mutations induced by
gamma rays (Kak et al. 2000).
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