Diss. ETH No.

14182

Vetiveria zizanioides:

an

approach

to obtain essential

oil variants via tissue culture

A dissertation submitted to the

SWISS FEDERAL INSTITUTE OF TECHNOLOGY

for the

ZRICH

degree

of

Doctor of Natural Sciences

Presented

by

RUTH ELISABETH LEUPIN

Dipl.

Natw. ETH

Born October 19, 1967

Citizen of Ksnacht

(ZH)

and Muttenz

(BL)

Accepted

of the recommendation of

Prof. Dr. B. Witholt, examiner Prof. Dr. N. Amrhein, co-examiner Prof. Dr. K. H. Erismann, co-examiner
Dr. C. Ehret, co-examiner

Zrich 2001

ii

Ill

Acknowledgements
After

finishing

my thesis, it is

now

time to leave the Institute of

Biotechnology.
a

It

will not be the

building

that I will miss, but all the

people

who made it such

nice

place

to work.

Therefore, I would like

to say thanks to all of you.

First of all, I would like to express my the

gratitude

to Prof.

Witholt, who gave

me

opportunity
his

to carry out this

project

at the IBT under his

supervision.

apreciated period.

continuing support

and encouragement

throughout

the entire

My

thanks also to Prof. Erismann for his advise

on

tissue cultures and for

being

my co-examiner. He initiated this

interesting project together

with Givaudan.

am

grateful

to Dr. Ehret

(Givaudan)

for his

help regarding

vtiver oil and for

agreeing

to be co-examiner.

appreciated
so

Prof. Amrhein's

agreeing

to be

co-examiner, especially since he

showed Davos.

much interest in my poster at the

Biology Symposium

1998 in

My

thanks to Helena, for all the administration, the

organization

of

pleasant
there if

events such as river

rafting,

Christmas

parties,

etc and for

always being

I had

some

language problems.

Thanks to all the present and former IBT-members for their useful discussions and advise, for the

pleasant

time

during

basketball games, coffee breaks,

a

lunches, barbecue,... in short, for creating such

nice

working atmosphere.
Jin

Particularly

I would like to thank Nicolas,

Guy, Qun, Marjan,

time

Byung

and all

the others in my lab. I

A

really

had

good

working together nearly

with them.

special

thanks to Theo, who in the last years

on

became my

supervisor. reading

Our discussions
of my

experiments
and

and results, his advise and his critical

were

manuscripts

chapters

of great

help

to me.

IV

I also would like to

me

acknowledge

all the

Phytotech

Labor members who

helped

during

my time in Bern.

Special
and

thanks to my
to my

family members,

who

always

were me

there to support me,

were

especially

sister, Marianne, who helped

if there

too many

hungry plantlets

to transfer to new medium.

This work

was

supported by by

Givaudan-Roure

Forschung

AG Dbendorf,

Switzerland and

the Swiss Federal Office for Economic

Policy, project
as

no.

2561.1 of the Commission for

Technology

and Innovation,

well

as

by

the

Institute of

Biotechnology,

ETHZ.

Table of contents
page

Summary
Zusammenfassung
Abbreviations

vi

viii

Chapter Chapter

Introduction

Compact

callus induction from in vitro from Java

plantlets

of vtiver
27

(Vetiveria zizanioides) Chapter

3

Compact

Callus Induction and Plant

Vtiver

Regeneration

of

non-flowering

(Vetiveria zizanioides)

from Java

43

Chapter

Liquid

Culture Induction and Plantlet

Regeneration

of
61

Vtiver

( Vetiveria zizanioides)
methods for

Chapter

Comparing analysis
and

detecting quantitative composition

71

qualitative changes

in the vtiver oil

Chapter

Comparison

of Vtiver oil extraction

by

water distillation

and solvent extraction

95

Chapter

Optimization

of small scale extraction,

purification

and

concentration methods for from vtiver roots

analysis

of the essential oil

117

Chapter

Initial

study

on

induction of vtiver oil

production

and
145

accumulation in tissue culture: material

preparation

Chapter

Conclusions and outlook

155

References

166

Curriculum vitae

185

VI

Summary
Vtiver oil, isolated from the roots of the is
an

tropical

grass Vetiveria zizanioides, Since it is

a

important

raw

material for the

perfume industry.
now

very

complex
it

essential oil, vtiver oil could up to

not be

produced artificially. Therefore,

a

would be of interest to obtain vtiver variants with

different odor

(oil

compo

sition)

or a

higher

oil

yield.
not be

Such variants

can

produced

via traditional

breeding,

since the used

or

vtiver variant is not culture

flowering.
crown or

Therefore

plant regeneration

via callus

liquid

starting

from

leaf slices

was

chosen to obtain somaclonal

was

variants. A successful

regeneration

within 18 weeks

reached via callus

on

up to 55 % of the

crown

slices and up to 60 % of the leaf slices with up to 100 concentrations

plantlets phenoxy

per slice

by changing growth regulator

(2,4-dichloro-

acetic acid,

6-benzylaminopurine), (light, dark).

Other

sucrose

concentrations and described in

cultivation conditions

regeneration methods,

literature, did

not work with the used

non-flowering
more

vtiver from Java.

are

Since, starting from in vitro plantlets,

until the
in
an

than 15-22 months

a

needed

plant

contains the would be

complete

vtiver oil,

pre-screening

of the

plantlets

early stage

advantageous. Unfortunately,
are

the genes involved in

the

biosynthesis
for

of vtiver oil

not known. As a result it is necessary to

screen

phenotypical changes
assess

and

especially

for the

production

of

more or

altered oils. To

quantitative
material

and

qualitative changes

in the vtiver oil

composition
and

of the

plant

already

in tissue cultures, methods to extract

analyze

the vtiver oil from small

on

samples

had to be

optimized

and

compared.
and

Methods based thin

olfactive detection, inhibition of microbial and gas

was

growth

analysis by
therefore

layer chromatography (TLC)

The olfactive detection and not accurate
was

chromatography (GC) pre-screening,

were

compared.
was

useful for

but the
more

analysis

subjective

enough.
for
a

GC

analysis provided
of

detailed information, while TLC

preferred

preliminary analysis

many

samples.
were

To extract the oil, water distillation and solvent extraction

optimized

and

compared.
buffer at

The distillation times could be reduced

by using

0.5 M

phosphate

pH

8 instead of water. Furthermore, this substitution resulted in the

distillation of less acidic

compounds. By combining

water distillation with solid

VII

phase extraction,

an

approach
The

was

developed

to distill several small

samples

(about

100

mg)

in

parallel.

procedure

for solvent extraction at

room

temperature could easily be miniaturized for extracting 100 mg vtiver

1.5 ml hexane.

roots in

Unfortunately

additional non-volatile and increased noise

were

compounds during
GC

were

extracted,

which caused base line Neither TLC

nor

shifting

analysis.

column

chromatography samples.

able to

remove

the non-volatile

compounds

from small scale

The choice of hexane extraction is

favorable since it is less labor intensive than water distillation combined with solid

phase

extraction.
we were

In this

study,

able to regenerate

plantlets

via in vitro culture and

optimized
in

methods to extract and This

means

analyze large

numbers of very small

samples

parallel.

that

we now

have all tools to

in such

develop

somaclonal

variants in vitro and test the oil this work will be lation in the

produced
the

plantlets.

The next stage in and

accumu

dependent
or

on

ability
an

to induce oil

production

plantlets

in vitro tissue in
an

early stage.

Future work should include

development

of such

induction method and the

application

and further

development

of the extraction and

analysis

methods.

VIM

Zusammenfassung
Vetiverl, welches
aus

den Wurzeln des

tropischen

Grases Vetiveria

zizanioides isoliert wird, ist ein

es

wichtiges

Rohmaterial der Parfmindustrie. Da

es

ein sehr

komplexes

therisches

l ist, konnte

bis

jetzt

noch nicht
an

knstlich

hergestellt

werden. Es besteht deshalb ein grosses Interesse

Vetiver-Varianten mit unterschiedlichem Geruch d.h. hherem

Olzusammensetzung

oder

lgehalt.
aus

Bei der verwendeten Vetiver-Kultivar

Java versagen die traditionellen

Zchtungsmethoden, Weg

da die Pflanze nicht blht. Aus diesem Grunde wurde der

ber die Produktion somaklonaler Varianten via Kallus- oder wobei Rhizom- und Blattscheiben als

Flssigkultur
benutzt der

gewhlt,

Ausgangsmaterial

wurden. Eine

erfolgreiche Regeneration

via Kalli wurde durch

Optimieren
6-

Wachstumregulator-Konzentrationen (2,4-Dichlorophenoxyessigsure,

Benzylaminopurin)
der

und des

Zuckergehaltes

im Medium sowie durch

ndern

Kulturbedingungen (Kultur

im Licht oder im

Dunkeln)

erreicht. Mit der

zu

optimierten

Methode konnten innerhalb 18 Wochen auf bis

zu

55 % Rhizom-

scheiben und auf bis bis

zu

60 % Blattscheiben Pflanzen

regeneriert werden,

mit

100 Pflanzen pro Scheibe. Andere in der Literatur beschriebene

Regenerationsmethoden
Java
zu

fhrten bei dem nicht-blhenden Vetiver-Kultivar Resultaten.

in vitro Pflanzen dauert
es

aus

keinen

positiven
den

Ausgehend
oder

von

regenerierten

15-22 Monate

lnger,

bis die Wurzeln das

vollstndige

Vetiverl enthalten. Fr die

Zchtung

neuer

wertvoller Vetiver-Varianten wre deshalb eine Vor-Selektion in

einem frhen Stadium vorteilhaft. Leider sind die fr die Vetiverl verantwortlichen Gene nicht bekannt. Deshalb
muss

Biosyntese

nach

phnotypischen

Vernderungen
werden. Zur

insbesondere und

des

lertrags

und des

lqualitt gesucht
der Vetiverl-

qualitativen

quantitativen Bestimmung

Zusammensetzung
methoden und

wurden fr kleine Probenvolumen die entwickelt. Zur

ntigen

Extraktions

Analytik

Analyse

wurden Methoden basierend auf Dnnschicht Die

von

olfaktorischer Detektion, bakterieller

Wachstumshemmung,

chromatographie (DC)

und

Gaschromatographie (GC) getestet.

zum

olfaktorische Detektion wurde

Vor-Screenen auf das Vorhandensein

IX

Vetiverl genutzt. fr

GC-Analysen ergaben
bevorzugt

detaillierte Informationen, whrend DC wurde.

Voranalysen

vieler Proben

Zur Extraktion des Extraktion

les wurden Wasserdestillation und Lsungsmittel-

optimiert

und

verglichen.

Die Destillationsdauer konnte reduziert

ersetzt wurde.
von

werden, indem das Wasser durch 0.5 M Phosphatpuffer (pH 8)

Gleichzeitig

wurden dadurch

weniger

Suren destilliert. Die Kombination

die

Wasserdestillation und

Festphasen-Extraktion ermglicht

gleichzeitige
bei

Destillation vieler kleiner Proben

(ca.

100

mg).

Die

Lsungsmittel-Extraktion
werden

Raumtemperatur

konnte leicht auf kleine

Mengen bertragen

(100

mg

Vetiver-Wurzeln in 1.5 ml

Hexan). Gleichzeitig

werden dabei auch nicht

flchtige

Substanzen extrahiert, welche die GC

Analytik
den

erschweren und weder Proben

durch DC noch durch

Sulenchromatographie

von

kleinvolumigen

getrennt werden knnen. Gegenber der mit Festphasen-Extraktion

kombinierten Destillation hat die Hexan-Extraktion den Vorteil, dass sie arbeitsintensiv ist.
In dieser Arbeit
ren

weniger

gelang
zu

es, Pflanzen via in vitro Kulturen effizient

zu

regenerie
sehr

und Methoden

entwickeln fr eine

parallele

Extraktion und
zur

Analyse

kleiner

Probemengen.

Somit sind
zur

nun

alle

Werkzeuge

Produktion

soma-

klonaler Varianten und

Analyse

der

le dieser regenerierten Pflanzen

ob die

vorhanden. Der nchste Schritt ist Akkumukation

abhngig davon,

lsynthese

und

in einem frhen Stadium der Pflanze induziert werden kann.

Zuknftige

Arbeiten sollten neben der

Entwicklung

einer solchen Induktions der Extraktions- und

methode auch die

Anwendung
beinhalten.

und

Weiterentwicklung

Analyse-Methoden

Abbreviations

AA medium AFLP BA

Mller and Grfe medium

(Mller

and Grfe

1978)

amplified fragment length polymorphism 6-benzylaminopurine

cleaved column

CAPS CC
cfu

amplified polymorphic chromatography

units

sequence

colony forming
chloroform

Chi CoA

Co-enzyme

2,4-D
DOXP
DMAPP

2,4-dichlorophenoxy
1

acetic acid

-deoxy-D-xylulose-5-phosphate
IPP isomer

dimethylallyl diphosphate, dimethyl sulphoxide dry weight

flame ionization detector

DMSO
dw
FID FPP

farnesyl diphosphate
gas

GC GGPP GPP
Hex HexN

chromatography

geranylgeranyl diphosphate geranyl diphosphate

hexane hexane extract of vtiver roots

evaporated
on a

with

nitrogen

HexSil HMG-CoA HMGR HPLC

IAA IPP

hexane extract of vtiver roots loaded

silica column

(S)-3-hydroxy-3-methylglutaryl-CoA 3-hydroxy-3-methylglutaryl-CoA
high
pressure
reductase

liquid chromatography

indole acetic acid

isopentenyl diphosphate
internal standard kinetin Luria-Bertani medium

IS
Kin LB medium

(Sambrook

et al.

1989)
von

M65 medium

Streptomyces

medium

(Deutsche Sammlung

Mikroorganismen

und Zellkulturen GmbH,

Braunschweig,

Germany)

XI

material X

estimated amount of oil in segment X based

mass

on

GC

analysis

MS MS medium

spectroscopy
and

Murashige

Skoog

medium

(Murashige

and

Skoog

1962)
MTBE MVA

methyl ferf-butyl
mevalonate

ether

N6 medium
NAA

medium described

by

Chu et al.

(1975)

a-naphthalene phosphate

acetic acid

P-buffer

buffer chain reaction

a

PCR

polymerase

Px/pHy
RAPD RFLP

distillation with random

phosphate

buffer

(x M),

at a

pH

amplified polymorphic

DNA

restriction

fragment length polymorphism

rpm TLC
UV
v/v

rotations per minute thin

layer chromatography light

ultra violet

volume per volume

xii

Chapter

Introduction

1. Vetiveria zizanioides

1.1. Plant

synonyms: Phalaris zizanioides L.

(1771), Andropogon
Guzman and

muricatus Retzius

(1783),

A. zizanioides

(L.)

Urban

(1903) (de

Oyen 1999)

Vetiveria zizanioides

also known

as

vtiver

(grass),

khus

or

khus-khus, is

perennial tropical
grass to the

grass. It

belongs

like maize,

sorghum,

sugarcane and lemonname

family

of Poaceae

(Gramineae).

The

generic

Vetiveria

comes name

from the Tamil word "vtiver" zizanioides

was

meaning

"root that is

dug up".

The

specific

first

given by

the Swedish taxonomist Carolus Linnaeus in

1771, meaning "by the riverside" (Vietmeyer and Ruskin 1993).

Vtiver is native to India. The exact location of is not

origin
nor

precisely known;

some

say that it is native to

thern India, others say that it is native around

Bombay
name

(Vietmeyer

and Ruskin

1993).

As the

specific

suggests, vtiver grows particularly

in rich
not

on

river-banks and it
can

marshy
stand

soil

(Anonymous 1976). However,

of inundation, it
can

only

long periods drought,

also

withstand extreme

survives temperatures

between -9C and 45C, is fire-resistant and is able to

grow in any type of soil

regardless

of

fertility, salinity

or

Figure

1.1: Vetiveria
a

pH (pH

4.0

9.6) (Anonymous 1976; de Guzman and

Sreenath et al.

zizanioides in

Oyen 1999;
large, densely

1994).
a

green-house
Vtiver grows in tufted

clumps high.

from

stout, compact rhizome

(crown)
the

with erect culms up to 3 meters

The roots bind the soil beneath

plant, reaching depths

of up to 4 meters and Ruskin

(de

Guzman and

Oyen 1999;

Sreenath et al. 1994;

Vietmeyer

1993).

The wild type from North and sets fertile seeds.

India flowers under suitable It is

a

marshy

conditions become

regularly
a

"colonizer" and therefore

might

weed

(de

Guzman and

Oyen
non-

1999).

The "domesticated" type from South India consists of vtiver

flowering

and
no

flowering

plants (Anonymous 1976).

If

they

flower

they produce

viable seeds. It mination

are

might

be that the seeds

are

sterile

or

the conditions for ger These vtiver

seldom met

(Vietmeyer

and Ruskin

1993).

plants

replicate by vegetative propagation

via side shoots.

1.2. Use of vtiver

Vtiver has been cultivated in India for centuries and is

out the

now

found

through

tropics

and in many

subtropical
are

areas

(de

Guzman and

Oyen 1999).

Besides many others, there used

as

two main reasons

why

vtiver is cultivated: it is
an

protection against

erosion and the roots contain

essential oil.

1.2.1. Protection

against

erosion

Due to its

densely packed,

stiff and

tough

grass stems and the

deeply
are

penetrating
used
as

root

system that works

as an

anchor, non-seeding vtiver plants

living

dams to slow down run-off water, to trap nutrient-rich top soil and
to combat erosion and increase moisture conservation

generally therefore,

(Adams

et ai.

1998; de Guzman and Oyen 1999; Vietmeyer and Ruskin 1993).

are

Since 90 % of the roots

found within

radius of 20

cm

from the vtiver

plant

(de

Guzman and
cultivated

Oyen 1999;
nearby
and

Salam et ai.

1993),

vtiver does not interfere with

plants

can

be used in natural

hedges

beside crops.

Erosion is combated with vtiver

et ai.

hedges

in

more

than 160 countries

(Adams

1998).
or

Since

lack of

genetic diversity
et ai.

increases

susceptibility by plants
from around the

diseases world

insects, Adams

(1998)

tested vtiver
DNA

by

random

amplified polymorphic genetic

(RAPD)

for additional nonfertile

germplasm

to broaden the

base for erosion-control

projects. They
a

found

that the vtiver cultivated outside of South Asia have been derived from

single

genotype. More nonfertile vtiver lines will be analyzed

to assure the

genetic

diversity

of cultivated vtiver

(Adams

et ai.

1998).

1.2.2. Roots contain

an

essential oil

Vtiver contains

an

essential oil, called vtiver oil. The oil

occurs

primarily

in

the roots, but traces of it in the

foliage

may nevertheless account for the

plant's
The

inherent resistance to pest and diseases

aroma

(Vietmeyer

and Ruskin

1993).

of the essential oil is and

basically

of

heavy, woody, earthy character,

pleasant

extremely persistent (de

are

Guzman and

Oyen 1999;

Sreenath et al.

1994).

These aromatic roots

either used

directly

to weave them into mats,

fans, sachets and

Guzman and

ornaments or

put among clothes

and Ruskin

to

keep
or

insects away
are

(de
by

Oyen 1999; Vietmeyer

yield

1993),

they

extracted deodo

steam distillation to

the essential oil, which is used in

perfumes,

rants, soap and other toilet articles

of interest to the cosmetic and also due to its

(de

Guzman and

Oyen 1999).
only

Vtiver oil is

perfumery industry,
more

not

due to its scent, but

ability

as

fixative, keeping

volatile oils from

evaporating

(Anonymous 1976).

1.2.3.

Phytoremediation

of polluted

areas

Since vtiver is

highly

tolerant to arsenic, cadmium, chromium, copper, lead,

nickel and zinc in the soil, it is suitable for the rehabilitation of lands contami nated with these elements. The vtiver thus retain the

plants

do not

only prevent

erosion and

polluted

soil at these sites, but when harvested and removed of

from the sites, and the soil

can

disposed

safely elsewhere,

the level of
et al.

heavy

metals in

be

gradually

lowered with time

(Chen

2000; Truong 1999;

Truong

and

Claridge 1996).

1.2.4. Medicinal

applications

In medicine, both the

plant

and its essential oil

are

used. A paste made of the

roots is claimed to have an almost

magic effect,

when

applied

to

bruises, swell

ings

or

burns. A stimulant drink is made from fresh rhizomes in India, and in Pradesh

Madya

(India)

the

plants

are

used

as an

anthelmintic

(de

Guzman and

Oyen 1999;

Sreenath et al.

1994).

1.2.5. Other

uses

Young

leaves of vtiver

can

be used

as

fodder for cattle and goats, whereas

or

the dried grass is used for

can

making

brooms and

for

thatching

of huts. The grass

also be used for

-

making writing
a

printing

paper. Since the

pulp

is short-

fibered, 30
added

40 % of

long-fibered pulp (e.g. Eulaliopsis binata)

de Guzman and

has to be

(Anonymous 1976;

Oyen 1999).

2. Variants of

plants

2.1. Production of variants

2.1.1. Traditional

breeding

and selection

Traits of different variants

can

be combined

by

traditional

breeding

and the

resulting plants

can

then be screened for the desired trait. However, the desired

a

traits have to be present in

sexually compatible plant.

2.1.2. Tissue culture: somaclonal variation and mutation

Plant cells fraction of the

propagated

in culture

can

undergo

variations.

Although only

variability present
due to
some

in the cell cultures is

actually

recovered in the

regenerated plants,
and Vasil variants. The variations

degree

of selection

during regeneration (Vasil

another way to obtain

1986), regeneration

via tissue culture

provides

are

divided in

genetic

and

epigenetic.

Genetic variation is

used to describe heritable variation that is

sexually

transmitted to the progeny of

regenerated plant.

This

phenomenon

is called somaclonal variation. which is at the

Epigenetic
same

variation is used to describe

non-hereditary variation,
a

time

reversible and is often the result of The

changed
on

gene

expression (Pierik 1987).

source

frequency

of variations

depends

factors like cultivar,

of the

tissue, the culture method (e.g. callus culture, suspension culture, regeneration
via

organogenesis

or

somatic

embryogenesis),

duration of the

disorganized

phase

and

growth regulators

used
in

(Karp 1994;

Maddock 1985; Pierik

1987).
and

In

somaclonal variants,

changes

ploidy level,

chromosome

breakage

rearrangements, gene amplification, single-gene mutation, variation in

quantitative traits,

and activation of

transposable
there is

elements have been

no

reported

(Maheshwari
character of

et al.

1995). Nevertheless,

guarantee that any

even

specific

interest will be among somaclones, since

in

cases

where somaclonal variation has occurred at been observed in

a

high frequencies
some

and

changes

have

whole range of characters,

characters do not

change

(Karp 1994).
Since many of the variations observed in somaclones
obtained variation
are

similar to those

by

traditional novel

breeding (Karp 1994;

of

Vasil and Vasil

1986),

somaclonal
a con

as a

source

variability

for

plant improvement

remains

troversial issue. However, in

asexually propagated species

where alternative

breeding approaches

are

limited, the prospects of somaclonal variation remain

promising (Karp 1994).

2.1.3. Genetic

engineering

A third

possibility

to obtain

plants

with

desired trait is

by genetic engineer
genes,

ing. By introducing
useful

and

expressing by

or

inactivating specific

plants
be

with

phenotypes

unachievable

conventional

plant breeding

can

generated.
Essential

requirements

for the

production

of

transgenic plants

are

(Birch

1997; Maheshwari etal. 1995):

a) availability
ration, which
is often and
are

of

target tissue including cells competent for plant regene

also accessible to the gene transfer treatment. Tissue culture

to achieve a workable

employed

efficiency

of gene transfer, selection,

regeneration

of transformants. To avoid somaclonal variation, the tissue

in the

culture

phase, especially

unorganized state,

should be minimized

or even

eliminated, by transferring genes into intact tissue expiants and regenerating

these without substantial in vitro culture.

b)

method to introduce DNA which cells.

can

be

expressed

in

plants,

into the

regenerable

Depending

on

the

plant material,

methods like

Agrobacterium-me\a\e transformation, microprojectiles, protoplasts,

of
a are

bombardment with DNA-coated

treatment of

and

electroporation

or

polyethylene glycol plant

used to introduce DNA into the

cells. Efficient

expression
genes

foreign

gene in
an

plants requires

the

adaptation
as

of the DNA to

plant

and selection of since the

appropriate promoter

well

as

polyadenylation

sequences,

plant transcription machinery particularly promoter plants

does not

recognize foreign

gene control

sequences,

sequences of many bacterial genes, while the

codon usage of

and bacteria differs

significantly.

The introduction of
a

introns has been shown to enhance number of systems.

expression

of genes several-fold in

c)

procedure

to select for transformed cells and to

regenerate plantlets in

satisfactory frequency conferring

has to be available. To
a

recover

transformed cells, genes

resistance to
or

selective chemical agent, genes

or even

conferring
to

pheno-

type allowing visual

physical screening,
can

PCR

screening

identify

plants containing

transferred genes

be used.

2.2. Detection of variants

2.2.1. Detection of variants

by comparing phenotypes

Depending

on

the trait of interest, different methods have to be chosen to

detect variants. Selection for

morphological changes
area

like

herbage yield,

tiller

number, diameter of the bush, length and

of the flowers
or

of the

longest leaf, morphology

leaves, fresh and dry weight ratio and harvest of crops, is the
can

easiest, since these changes

be monitored
stress

during

normal

growth. However,
chemicals

to select for traits like resistance

or

against
or

(temperatures, water,
rate or

fungi,

bacteria

or

insect

attack)
are

for

changed production
or

spectrum of

secondary
conditions

metabolites which
or

only produced

accumulated under

specific

in

specific developmental stage,

conditions. The results
can

the

plantlets

can

only

be tested

under these

specific

be influenced
to

by developmental
between
soma-

and environmental factors and it is not clonal and

possible

distinguish

epigenetic

variants.

2.2.2. Detection of variants at the molecular level

Somaclonal variations
the

might

be detected at the molecular level.

are

By screening by

plants

for

changes

within the DNA, the results

not influenced

developmental techniques

and environmental factors. There

are

several molecular
on

available for

checking

the genome, based restriction

analysis

of random

amplified polymorphic

DNA

(RAPD),

fragment length polymorphism

microsatellite repeat

(RFLP), amplified fragment length polymorphism (AFLP),

polymorphism (also polymorphic
known
as

simple

sequence
et al.

repeat)

and cleaved

amplified

sequences

(CAPS) (Jones

1997; Rafalski and Tingey 1993;

a

Ridout and Donini

1999). However,

after detection of

change

in the DNA, it is
a

not known what kind of effect it has on the

plant

or even

whether there is

detectable effect.

3. Essential oil

Essential oils
a

are a

mixture of
an

fragrant, highly plant

and of
a

volatile substances isolated botanic

by

physical

process from

odiferous

single

species

or

variety

(Encyclopaedia

Britannica 1986,

Oyen

Dung 1999).

The essential oil is

named after the aromatic stored in canals

or

plant

from which it has been derived.

They

are

often

specialized storage
oil ducts and

and secretory sites like

glandular hairs,

resin

schizogenous glands (Banthorpe 1988;

a

Charlwood and

Charlwood

1991).

Essential oils may be found in any part of

plant

and

can

be

extracted from roots, wood, bark, leaves, flowers, fruit and seed for commercial

purposes

(Oyen

and

Dung 1999).
uses

Essential oils have many

for

man.

Most obvious is their role in fra

grance materials, but

they

are

also used

as

flavoring

materials and in medicine

(Oyen

and

Dung 1999).

3.1. Extraction methods

Depending

on

the

plant material,

distinct processes like distillation, solvent

are

extraction, enfleurage and expression

and

used to

produce

essential oil

(Oyen

Dung 1999;

Roth and Kornmann 1997; Weiss

1997b).

3.1.1. Water

or

steam distillation

Essential oils distillation.

in in
a a

are

most often obtained from

plant plant

materials material

by

water or steam

During

water

distillation,

water and steam

are

boiled

together

pot by direct fire, whereas during separate

steam

distillation, the

steam is

produced

generator. As

result, the plant material does

not come in

contact with the hot wall of the

pot and therefore charring and decomposing of

and

compounds
Weiss

can

be avoided

(Oyen

Dung 1999;

Roth and Kornmann 1997;

1997b).

3.1.2. Solvent extraction

Certain oils
non-volatile

are

obtained

by

solvent extraction,

process which also extracts

a

compounds, yielding

after removal of the solvent

substance called
a

concrete. Solvent extracted oils are

generally

considered to reflect

plant's

natural odor

more

accurate than distilled oils.

Repeated washing produces

an

of the concrete absolute. If

with alcohol to

remove waxes

and other inert matter

ethanol is used to extract the resinoid.

plant material,
or

the

resulting

extract is called

By using

carbon dioxide

supercritical

fluid extraction the

problems

associated with solvent removal

can

be avoided, since carbon dioxide is odor

less, tasteless and inexpensive (Oyen and Dung 1999; Roth and Kornmann
1997; Weiss 1997b).

3.1.3. En fleu rage

Enfleurage
oils

is

method to capture the odor of delicate, heat sensitive flower

on wax or

by absorption

fat. The

enfleurage

process is

mainly

advanta for

geous for flowers, like

several

jasmine,

that continue to

produce

aroma

compounds
is called

days

after

they

have been

picked.

The

resulting product

pomade (Oyen

and

Dung 1999;

Roth and Kornmann 1997; Weiss

1997b).

10

3.1.4.

Expression

Citrus
are

peel oils,

like those of
In

bergamot, grapefruit, lemon, expression

methods the fruit

lime and orange,

extracted

by expression.
or

peel

is

com

pressed,

lacerated

abraded to rupture the oil cells

(Oyen

and

Dung 1999;

Roth and Kornmann 1997; Weiss

1997b).

For small scale extraction of essential oils, methods like simultaneous distil lation
-

solvent extraction

(Bicchi

et al.

1983; Godefroot et al. 1981), headspace

solid-phase
et al.

micro extraction and

(Field

et al.

1996),

microwave extraction and Ciola 1991 ;

on

(Craveiro

1989)

supercritical

fluid extraction

(Blatt

Sugiyama

and Saito

1988)

have been described.

Depending

the extraction method

to a

used, the composition and yield of essential oils may vary

large

extent

(Boutekedjiret
For

etal. 1997; Pino etal. 1996; Scheffer 1996; Simndi etal.

1999).

example,
a

the

supercritical

carbon dioxide extraction of than steam distillation and lower

ground

fennel seeds
same

resulted in

higher yield (10 %)

(3 %),

almost the

yield

as

hexane extraction

(10.6 %),

yield

than alcohol extraction

(15.4 %). Sensory

and the distilled oil hexane extracts

evaluation showed that the carbon dioxide extraction

were more

product

intense in odor and taste than the alcohol and

(Simndi

etal.

1999).
are

These differences

can

be

explained by

the fact that not all components

extracted

equally

well

by

each process and the process

because individual components may

undergo changes during

(Oyen

and

Dung 1999).

3.2.

Analysis

methods

Until

few decades ago, the human nose,

a

supported by

the measurement of
were

physical
means

characteristics and

few

simple

chemical

analyses

the

only

of

verifying

the

density, purity

and the naturalness of essential oils. The used to characterize essential oils with aqueous
are

physical
relative

characteristics most

commonly

density,
rotation

refractive index, and

miscibility (usually

alcohol)
on

and

optical

(Oyen

Dung 1999). However,

was

the information

the

chemical

composition

of essential oils

greatly improved by

the

development

11

of

analyses

methods

involving chromatographic separation

can now

and pure natural and

essential oil and modified oils

be

more

easily distinguished (Oyen

Dung 1999).

Different methods like gas column

chromatography (GC), high chromatography analysis

and thin

pressure
chroma

liquid chromatography (HPLC), tography (TLC)

layer

have been used for the

of essential oils

(Banthorpe

1991; Croteau and Ronald 1983; Harborne 1984b; Kubeczka 1985).

3.2.1. Gas

chromatography (GC)

GC has been the classical tool for

analysis

and isolation of volatile data

com

pounds, providing qualitative

and

quantitative

(Banthorpe
a

1991 ; Harborne

1984b; Kubeczka 1985). To detect the compounds,

flame ionization detector

(FID)
can

is

commonly

used. However the human

more

nose or mass

spectroscopy (MS)
or

also be used to obtain

information about the odor

the structure of
nature of
even

the

compounds (Gardner

and Bartelett
a

1999).

Since the very

complex

many essential oils does not allow

on a

complete chromatographic separation

gas

high

resolution

capillary column,

chromatography-tandem

mass

spectrometry and comprehensive gas chromatography (GCxGC) have been

used

(Cazaussus

etal. 1988; Marriott etal. 2000; Sellier et al.

mass

1991). By using

gas

chromatography-tandem
were

spectrometry, Cazaussus etal. 1988 and

each component of
a

Sellier etal. 1991

able to

analyze

overlapping peaks
resolution of vtiver

separately.

Marriott etal.

(2000)

obtained

much

superior
than

essential oil with Another

comprehensive

gas

chromatography

by

normal GC.
a

option

to obtain a total delineation of the

components is by

prior

fractionation of the essential oil, distillation

or

by

chemical class

separation,

fractional

liquid chromatography (Kubeczka 1985).

3.2.2. Thin

layer chromatography (TLC)

Due to its

simplicity

and

rapidity,

TLC is still

one

of the most

important
TLC is

methods for the

analysis

of essential oils

(Kubeczka 1985). Furthermore,

a

also useful in combination with GC to obtain

better

separation (Harborne
of

1984b)

or as a

pilot technique

for column

chromatography separation
or

essential oils

(Kubeczka 1985).

To detect the TLC spots, UV

different

12

staining

methods

can

be used

(Cosicia 1984; 1970).

Croteau and Ronald 1983;

Gibbons and

Gray 1998;

Merck

3.2.3.

High

pressure

liquid chromatography (HPLC)

HPLC is not often used for the

analysis
obtained

of volatile

compounds,

since

relatively good separations

HPLC is useful for

were

by

GC. However, in contrast to GC,

separation

of temperature labile and of non-volatile

compounds (Kubeczka 1985). Depending phase,

on

the mix, different columns based

on

normal

phase,

reversed

size exclusion,

partition

and

affinity

materials

can

be chosen

(Banthorpe

1991)

and

by changing

from isocraticto
et al.

gradient elution,

better resolution has

been obtained

(Schwanbeck

1982).

For the detection of the eluates, UV

absorption,

refraction index

changes

or mass

spectrometry

can

be used

(Banthorpe 1991). However,

chromophoric

since most of the essential oil components lack

groups, low UV

monitoring

from 200 to 220

to HPLC

nm

is necessary, with

which limits the few solvents

application

of such

analysis

separations

only

(Kubeczka 1985).

3.2.4.

Sensory analysis

The human

nose

is

useful tool to

analyze qualitative changes

more

in essential

oil, since the human

nose

is still about 1000 times

sensitive than modern

analyzers (Oyen
onset of

and

Dung 1999). However,

the measurement is limited

by

the

olfactory fatigue

after around 30 minutes and the

sensitivity

to odorants

varies

widely

both with the nature of the odorant and from person to person. In
was

addition, the sensitivity of individuals

and their

found to vary with their state of health and Bartelett

endocrinological

condition

(Gardner

1999).
and Bartelett 1999;

Therefore, the development of electronic

Weiss
tors

noses

(Gardner

1997b)

or a

detector made of cell cultures with

specific olfactory interesting.

recep

(Metzger

2001 ;

Monastyrskaia

et al.

1999)

would be

13

3.3. Chemical

components

Essential oils

are

complex

mixtures of sometimes hundreds of chemical

can

compounds.

Most of these

compounds

be

grouped

into 4

major

groups:

aliphatic compounds (acyclic organic compounds), terpenes

and terpene

derivatives, benzene derivatives and miscellaneous compounds (Oyen and

Dung 1999).

3.4.

Terpenoids

3.4.1. General

In

primary metabolism, terpenoids

are

involved in

photosynthesis (caro-

tenoids, chlorophyll, phylloquinone, plastoquinone), in respiration (ubiquinone,

cytochrome a), lipid development

and

membrane structure

(triterpenoids, sterols), regulation

of

growth (gibberellins, cycle

control

abscisic acid, brassinosteroids,

some

cytokinins)

and in cell

(prenylated proteins). Terpenoids,

which

are

not essential for

viability (e.g. monoterpenoids, sesquiterpenoids

are

and ditermetabolites.

penoids
Some

as

present in essential oils)

for the and

classified

as

secondary

are

important

plant

to

adapt

to environmental conditions e. g.

plant-plant, plant-insect
most of the

plant-microorganism

interactions. However, for

et al.

terpenoids

the function is unknown

(Cane 1999a; Champenoy

1999; Kribii et al. 1999; Newman and Chappell 1999).

Terpenoids building block, isoprenoids. variety

more

share the

a common

characteristic:

they

all derive from the

same

isoprene

unit

(C5). They
acyclic,

are

therefore also called

are

Some

terpenoids

are

but most For

cyclic,

with

wide

of carbon skeletons

(Kleinig 1989).

sesquiterpenoids (C15), already

than 300

sesquiterpene

carbon skeletons have been identified and oxidized

or

thousands of

naturally occurring

otherwise modified derivatives have and microbial

sources

been isolated from marine, terrestrial

plant,

(Cane

1999b).

14

3.4.2.

Terpenoid biosynthesis

3.4.2.1.

Biosynthesis

of the

isoprene

unit

Two

biosynthesis pathways

have been described for the the mevalonate

production

of the from

precursor

isopentenyl diphosphate (IPP):

via mevalonate

pathway starting

acetyl-CoA
or

(MVA) (Figure 1.2)

and the non-mevalonic via

pathway 1-deoxy-

DOXP

pathway

from pyruvate and

glyceraldehyde-3-phosphate
a

D-xylulose-5-phosphate (DOXP) (Figure 1.3). Only

use

minority

of the bacteria The DOXP with

some

the MVA

pathway,

while the DOXP

pathway

is

more common.

pathway

is found either alone

(e.g.

Bacillus subtilis, Escherichia

coli),

genes of the MVA

pathway

or

in addition to the

complete

MVA

pathway
the MVA
in

(Boucher

and Doolittle

2000). Non-photosynthetic eukaryotesuse

plants
and

pathway exclusively.
unicellular green

In most

algae

both

pathways exist, except

algae (e.g.

Scenedesmus
which
seem

obliquus, Chlamydomonas
to have lost the MVA

reinhardtii and Chlorella

fusca)

pathway

after

acquisition

of the DOXP

pathway (Boucher
was

and Doolittle

2000).

Within the

plant

cells, the terpenoid biosynthesis

observed in the

cytoplasm,

mitoplasm

(the

mitochondrial
In the

matrix),

and the

plastoplasm (the plastid stroma) (Kleinig

via the MVA For the

1989).
DOXP

cytoplasm,

IPP is

produced plastids.

pathway,

whereas the

pathway

is located in the

biosynthesis
on

of the

prenyl
IPP

side-chain of the formation

ubiquinones,

the mitochondria

depend

cytosolic pools
other

(Lichtenthaler 1999).
and the

Whether the two cellular IPP and

of the

cytoplasm

plastids cooperate
as

exchange

IPP

or

prenyl-

diphosphates,
or

such

geranyl diphosphate (GPP), farnesyl diphosphate (FPP)

is not known at present. Several also Lichtenthaler

geranylgeranyl diphosphate (GGPP),

some

observations suggest at least

exchanges (see

1999).

3.4.2.1.1. MVA

pathway (Figure 1.2)

In the first two reactions of the MVA

pathway,

three

acetyl-CoA

condense

resulting

in

(S)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA).
two

In animals and

yeasts these

steps

are

catalyzed by

two

separate enzymes, acetyl-CoA

acetyltransferase (EC 2.3.1.9)

and HMG-CoA

synthase (EC 4.1.3.5) (McGarvey

15

and Croteau reaction is

1995),

whereas in radish
a

seedlings

this double condensation and Bach

catalyzed by

single

monomeric

protein (Weber

1994).

Acetyl-CoA
SCoA
Acetyl-CoA
CoA-SH

c
o

Acetoacetyl-CoA
Acetyle-CoA+
CoA-SH

H20

ho,

HO

JL3CJk.
C
9
HO
OH
2 NADPH 2 NADP+
+

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA)
H+
CoA-SH

Mevalonate

HO

ATP

ADP

9
HO

HO.

Mevalonate-5-P
OP
ATP ADP

c
9
HO HO

Mevalonate-5-PP
OPP
ATPr

ChiADP +P,

OPP

Isopentenyl-PP (IPP)

Figure 1.2. Mevalonate (MVA) pathway 1 : Acetoacetyl-CoA synthase

3: HMG-CoA reductase 5:

2: HMG-CoA

synthase

4: Mevalonate kinase

Phosphomevalonate

kinase

6:

Phosphomevalonate decarboxylase

16

The next step from HMG-CoAto

to

(/^-mevalonate

is the first reaction

unique
HMG-

terpenoid biosynthesis

and is irreversible. This reaction is

catalyzed by

CoA reductase

(HMGR,

EC

1.1.1.34),

membrane associated enzyme. In

animals, fungi and perhaps insects, HMGR is the key regulatory step controlling

isoprenoid

metabolism

(Newman
case

and

Chappell 1999).

In

plants,

it is

tightly

regulated, specially
and Croteau mevalonate

in the

of sterol and much

phytoalexin synthesis (McCaskill

diverse

1998).

Plants

produce

more

products

from the

pathway

in various tissues and

organelles. They only

two in

contain

multiple

genes for HMGR, that vary in number from

Arabidopsis
In all
are

to over 12 in

potato (McCaskill and Croteau 1998; Stermer

et al.

1994).

plants

investi

gated,

HMGR is encoded

by

small

family

of genes which

expressed including

differentially during development

and in response to external stimuli

light, plant growth regulators, wounding, pathogen

attack and exogenous sterols

(Lange

et al.

1998; McGarvey and Croteau 1995; Stermer etal. 1994;

Weissenborn etal.

1995).
use

There is also evidence that the of alternate

use

of different

promoters allows the

gene, with the

transcription

start sites within a

single variably

subsequent expression

of different forms of HMGR with

extended N-terminal

regions (Lumbreras

etal. 1995; McCaskill and Croteau

1998).
The

phosphorylation

of mevalonate to mevalonate-PP is

catalyzed by

two

separate soluble kinases, mevalonate kinase (EC 2.7.1.36) and phosphomevalonate kinase

(EC 2.7.4.2).

The

following

reaction is

catalyzed by
in the

diphosphomevalonate decarboxylase (EC 4.1.1.33), resulting building

block IPP

terpenoid

(McGarvey

and Croteau

1995).

3.4.2.1.2. DOXP

pathway (Figure 1.3)

In the first

step of the DOXP pathway,

C2-unit derived from pyruvate is DOXP. This transketolase-

transferred to

glyceraldehyde 3-phosphate, forming

type condensation reaction is thiamine-dependent and is catalyzed by the

enzyme

DOXP-synthase (Lichtenthaler 1999;

intermediate of IPP

Lois etal.

2000).

DOXP is not

only

an

synthesis

but is also involved in the of the

biosynthesis

of
in

thiamine and

pyridoxol

in

plastids. Analysis
and

DOXP-synthase expression
of mRNA

tomato indicated

developmental

organ-specific regulation

17

accumulation and showed fruit

strong correlation with carotenoid synthesis during

development (Lois

et al.

2000). 2-C-methyl-D-erythritol
requiring
In
4-

The next transformation step from DOXP to

phosphate

is

catalyzed by

DOXP reductoisomerase,
and Croteau
to the

NADPH and

manganese

(or magnesium) (Lange

reactions

1999).

plants,

the

remaining enzymatic
been

leading

synthesis

of IPP have not yet

or

fully

elucidated

(Lois

et al.

2000)

and whether IPP

as

its isomer

dimethylallyl diphosphate (DMAPP)

unresolved

is formed

the first

isoprenoid

C5-unit is

(Lichtenthaler 1999).

o
o
+

Pyruvate
OH

D-Glyceraldehyde-3-P
OP OH
-
TPP TPP

^-*OH o

COoj2

1TPP

OP OH

-Deoxy-D-xylulose-5-P

C ^-

,_

NADPH

H+

NADP+

.0P

2-C-Methyl-D-erythritol-4-P

Isopentenyl-PP (IPP)

OPP

OPP

Dimethylallyl-PP (DMAPP)

Figure
1: 2:

TPP: thiamine

1-deoxy-D-xylulose-5-phosphate (DOXP) pathway diphosphate 1-Deoxy-D-xylulose-5-phosphate synthase 1-Deoxy-D-xylulose-phosphate reductoisomerase

1.3.

18

3.4.2.2.

Elongation

IPP is not reactive

enough

to initiate the condensation to

higher terpenoids.
which

It

first needs

an

activation step

catalyzed by

IPP isomerase

(EC 5.3.3.2),

converts IPP to its

allylic

DMAPP

(Figure 1.4) (Koyama

and

Ogura 1999;
equi

McGarvey

and Croteau

1995).

The isomerization is reversible, but the

librium is towards DMAPP formation transferases

(Ramos-Valdivia

etal.

1997). Prenylwith DMAPP the

catalyze

the

multistep elongation

reaction
on

beginning

and IPP to form linear

terpenoids. Depending

the

prenyltransferase,
may
serve as

product

may be released from the enzyme surface,

or

the is

substrate for additional condensations. The controlled

isoprenoid

chain

length

tightly

by

the relevant enzyme

(Cane 1999a).

Different types of

prenylterpenoid

transferases have been

recognized,

with two types involved in pure

reactions, i.e. catalyzing either head-to-tail (1'-4 prenyltransferase: e.g. geranyl

diphosphate synthase (GPP synthase

or

dimethylallylfranstransferase,
or

EC

2.5.1.1), farnesyl diphosphate synthase (FPP synthase

geranylfrans-

transferase, EC 2.5.1.10) and geranylgeranyl diphosphate synthase (GGPP

synthase

or

farnesylfranstransferase,

EC

2.5.1.29)),

or

head-to-head

condensation of

prenyl diphosphates (1'-1 prenyltransferase:

EC

e.g.

squalene
and

synthase (or farnesyl-diphosphate farnesyltransferase,

2.5.1.21)

phytoene synthase (or geranylgeranyl-diphosphate geranylgeranyltransferase,

EC

2.5.1.32)). Furthermore,
of

other types of

prenyltransferase
an

are

involved in the

prenylation pathway

proteins
an

and in the reaction of

intermediate of the

terpenoid

with

intermediate from another

biosynthetic pathway

to form

mesoterpenoids (e.g. ubiquinones, plastoquinones, chlorophyll) (Koyama

and

Ogura 1999;
In

Kribii etal. 1999; Ramos-Valdivia etal.

FPP and GGPP

1997).
by multigene families,
etal.

plants,

synthases

are

encoded

e.g.

A. thaliana contains at least two genes for FPP

synthase (Cunillera

1997)

and 5 genes for GGPP

in
a

synthase (Okada

etal.

2000).

Each gene is
et al.

expressed

different tissue

during plant development (Cunillera

1997; Okada et al.

2000).

19

PPO

OPP

X~

OPP

X
Geranyl-PP (GPP) Linalyl-PP

Isopentenyl-PP (IPP)

Dimethylallyl-PP (DMAPP)

OPP

^OPP
Nerolidyl-PP Squalene

Farnesyl-PP (FPP)

OPP

Geranylgeranyl-PP (GGPP)

Phytoene

Figure 1.4. Names and terpenoid pathway (C5

structures of the
-

parent compounds of the central

C20)

3.4.2.3.

Cyclization

The next metabolic branch

point

of the

terpenoid biosynthesis
some a

is the

cyclization

of the

prenyldiphosphates.

Due to

geometric

barrier of GPP

and FPP to form

cyclohexanoid compounds,
ester

preliminary
was

isomerization to the for such

cases

corresponding tertiary allylic

before the

(Figure 1.4)
,

proposed

ionization-dependent cyclization

rearrangements including methyl

et al.

migration

and proton elimination take

are

place (Bohlmann

1998; Cane

1999b).

These transformations

catalyzed by terpenoid synthases acyclic

and

(cyclases), leading
each class

to a broad range of et al.

cyclic parent

skeletons of

(Bohlmann

1998).

20

The monoterpene

synthases
GPP
or

and

diterpene synthases

show strict substrate

specificity

in

accepting

GGPP, respectively, whereas several sesqui

as an

terpene synthases accept GPP, although inefficiently,

alternate to FPP

(Bohlmann

et al.

1998).
of

Many terpenoid synthases multiple-gene copies,

diversification. In that

secondary

metabolism and then

are

represented by
the basis for

arose

by duplication
seven

provided

grand

fir at least

very closed related genes encode

monoterpene synthases with distinct product patterns.

Since genes of the

secondary

metabolism
are

are

not essential for to

growth

and

development,

functional mutations

tolerated, leading

product diversity,
et al.

which may be beneficial in interactions with other

organisms (Bohlmann

1998).
The

expression
in

of

terpenoid synthases
cells

is often tissue trichomes in

or

cell-specific.
or

It is up-

regulated
certain

specialized

(e.g. glandular
or

mint),

restricted to

developmental stages
et al.

short

periods

of transient defense reactions

(Bohlmann

1998).

3.4.2.4. Transformations

Subsequent

transformations of the basic parent skeletons involve various

modifications and isomerisations to form many thousands of different

terpenoids,
even

e.g. alcohols,

epoxides, aldehydes, ketones, nitrogen-containing

acids and lactones and

halogen-, sulfur,

and

forms have been described.

more or

Modifications of the carbon skeleton itself that exhibit

atoms than the

fewer carbon

original

structure have also been observed.

Finally, composite

structures such as esters and

glycosides

are

also known
are

(Kleinig 1989). Many

P-

of the

hydroxylations

or

epoxidations

involved
et al.

performed by cytochrome

450 mixed function oxidases

(Bohlmann

1998; McGarvey and Croteau

1995).

21

3.5. Vtiver oil

Vtiver oil is

an

essential oil extracted from the roots of Vetiveria zizanioides.

It is of interest to the cosmetic and but also due to its

perfumery industry,

not

only

due to its scent,

ability

to fix some more volatile essential oils

(Anonymous

1976).

Vtiver oil is used

unprocessed

as a

component for perfumes, deodo

raw

rants, soap and other toilet articles, and it is

material for vetiverol alcohols

(bi-

and

tricyclic primary, secondary,

and

tertiary sesquiterpene
than 100 GC/MS

(Anonymous
et al.

1976))

and

vetiveryl

acetate

(more

peaks (Demole

1995)), resulting
and

from vetiverol after

acetylation (Weiss 1997a).

Since vetiverol

vetiveryl

acetate have milder odors than the basic oil and

are

equally good

fixative

properties, they

used in

more

expensive products (Anonymous

1976;Sreenath etal. 1994).

For its essential oil, Vtiver is

mainly

cultivated in Haiti, West Java, India,

two vtiver

Runion, China and Brazil. The essential oils of the

vary in

types of India

quality

and

quantity.

The oil from wild vtiver

(mainly

the North Indian whereas

type),

called khus oil, is poor in

more

yield

but

superior

in oil

quality,

cultivated vtiver contains inferior

oil, but the oil, called vtiver oil, suffers from

on

quality (Lavania 1988). Depending quality oil,

the

origin,

the

quality
one

of vtiver oil

varies. The best

and therefore the most

expensive

(Table 1.1), Oyen 1999).

originates

from Runion and is called Bourbon oil

(de

Guzman and

Table 1.1: Prices of vtiver oil

in the 1990's1 2000
2

[US$ kg"1]
vtiver oil, bourbon vtiver oil, Haiti vtiver oil, Java
35-155 90-100 54-62

[US$ kg"1]
137 93

origin origin origin

vtiver oil, Indonesia

71

vetiveryl

acetate

160

160.50

1: Data from de Guzman and Oyen 1999 2: Data from Chemical Market Reporter (2000) Vol. 258, No 5

22

The

occurrence

and distribution of the vtiver oil in the roots

In young roots
no

was

studied

by

microscopical analyses.

oil

was

detected and

only

in about six in the

months old roots, the volatile oil first cortical

appeared

in the form of oil

drops mainly

layer

outside the endodermis. In older roots, cells in the cortical

parenchyma
essential oil,

were

lysed forming lysigenic lacunae,

in oil ducts.

which

are

filled with

resulting

Specialized

oil cells could not be observed

(Kartusch

and Kartusch 1978; Viano etal. 1991a; Viano etal.

-

1991b). (Anonymous
of 15 and

The roots should be harvested at the age of 15

18 months

1976),

since it is uneconomical to harvest

a

prior

to a minimum

maturity

months, while

period

of 21

24 months is also uneconomical

(Virmani

Datta 1975; Weiss Vtiver oil is distillation.

1997a).
extracted from fresh
or

mainly

air-dried vtiver roots

by

steam
a

Occasionally,

roots are also extracted with

benzene, yielding
Weiss

vtiver concrete The

(Anonymous 1976; (up

to 3 %

de Guzman and

Oyen 1999;

1997a).
not

yield

of oil

(dw) (de

Guzman and
on

Oyen 1999)) depends

only

on

the vtiver type cultivated, but also of the grass, the

the climate, the soil,

frequent

cutting

harvesting time,
soil with lower

methods of distillation, time of distil

a

lation. For

example,

on

clay content,
the oil

marked

improvement

in

quality

is observed but

simultaneously
so

yield

decreases. On

sandy pond

and river banks the decrease is ical

extreme that distillation becomes uneconom

(Virmani

and Datta

1975).

3.5.1.

Composition

The vtiver oil is It consists

extremely complex, containing bicyclic

and

more

than 300 components.

mainly

of

tricyclic sesquiterpenoids (hydrocarbons,

alcohols, ketones, aldehydes, acids) (Cazaussus etal. 1988; de Guzman and

Oyen 1999; Vietmeyerand 1992)

and

Ruskin

1993),

but

monoterpenoids (Nikiforov

etal.

phenols (Garnero

1971 ; Shibamoto and Nishimura

1982)

have also

been detected.

Many

of the vtiver oil components have been isolated, identified and

in

even

synthesized (references
al. 2000a ;

Smadja 1994; Weyerstahl

Weyerstahl

etal. 1999;

Weyerstahl

et

Weyerstahl

etal. 2000b;

etal. 2000c; Wolf

1996).

In the

typical

vtiver oils

(Haiti, Runion, Java,

Brazil and South

India) sesquiterpen-

23

oids with valencane, zizaane

eremophilane, eudesmane, epieudesmane, vetispirane,

and

(or tricyclovetivane), prezizaane, guaiane

were

cyclocopacaphane Weyerstahl
et al.

skeletons

found

(Anonymous 1976;

Garnero 1971 ;

2000a; Weyerstahl et al. 2000b). However, the major representatives of the

vtiver oil, considered

(+)-a-vetivone, (-)--vetivone
as

and khusimol

(Figure 1.5),

can

be

the

"fingerprint"

of vtiver oil

(de

Guzman and

Oyen 1999;

Demole etal.

1995).

(+)-u-vetivone

(-)--vetivone

khusimol
or

khusilal

Figure

1.5. Characteristic

components of typical vtiver oils

khus oil

The khus oil from Northern India differs characterized

markedly

from the

typical

vtiver oil. It is

by

the presence of

antipodal

cadinane and eudesmane

sesqui-

terpenoids (e.g. khusol, khusinol, laevojunenol, epikhusinol

and isokhusinola

oxide)

and contains

large

amount of khusilal

(Figure 1.5),

laevorotatory

C14-

terpenoid.

The khus oil has

sources

laevorotatory characteristics,

while the vtiver oil

from all other

is

dextrorotatory (Table 1.2) (Andersen 1970; Anonymous

1976; de Guzman and Oyen 1999; Garnero 1971; Kalsi etal. 1964; Kalsi etal.

1985).

Table 1.2:

Physical properties
wild North Indian

of Vtiver oil
cultivated

South Indian

Bourbon

23

Java23
0.980-1.022 1.521-1.530 +17 to+46
1 :22

Haiti4
0.986-0.998 1.521-1.526 +22 to +38
1 :2
3

vtiver1
relative

vtiver1
0.992-1.015 1.516-1.530 +10 to+25 0.986-1.015 1.521-1.530 +14 to+32
1:1-2

density
rotation
in

0.990-1.032 1.512-1.523 -50 to-130

refraction index

optical

Miscibility

80% ethanol

insoluble

(vol)
1: Data from Anonymous 1976 2: Data from de Guzman and Oyen 1999 3: Data from Masada 1976 4: Data from Bauer
et al 1988

24

The odor of vtiver oil is very

complex.

It is unclear, which

compounds

are

responsible

for the

typical

vtiver scent

(de

Guzman and

Oyen 1999;
depend

Demole et
on a

al. 1995; Kraft et al.

2000).

It appears that the scent does not but rather

on a

single

group of

compounds

combination of skeletons and

functional groups

contributing

to the total sensory

impression (Nikiforov

et al.

1992; Weyerstahl et al. 2000c; Wolf 1996). Therefore and due to the problem to

chemically synthesize economically possible

up to
now

pure

sesquiterpenoids,
vtiver odorant

it has not been

et al.

to

produce

synthetic

(Kraft

2000;

Spreitzerefa/. 1999).

4.

Scope
Since

of this work

synthetic

vtiver oil cannot yet be manufactured

(Kraft

etal. 2000;

Spreitzer

etal. 1999;
in

Weyerstahl

etal.

2000c),

there is oil

an

interest in the
or

perfume industry

new

vtiver variants with

higher

yields
a

different odor oil

tonalities. New vtiver variants have been obtained with odor with
a

higher

yield

or an

different note

by

traditional

breeding

and selection

(Gupta

etal.

1983; Lai etal. 1997; Lai etal. 1998; Sethi 1982; Sethi and Gupta 1980).

However, traditional breeding with non-flowering South Indian type vtiver is

not

possible.

Therefore

regeneration
was

via in vitro cultures the

was

chosen to obtain

variants. This decision

supported by

finding

of Shreenath and

Jagadishchandra (1991)

that rgnrants of

commercially important

Cymbopogon species (C. flexuosus,

motia and C. and

C. nardus, C. winterianus, C. martini! var.

variations in oil content

jwarancusa )
1991

showed

significant

(Sreenath

Jagadishchandra major

and of Mathur et al.

(1988)

who recorded variations

for six

constituents of the essential oil of the aromatic grass C.

winterianus and

(citronellal, citronellol, geraniol, citronellylacetate, geranylacetate,

etal.

elemol) (Mathur

1988).

The aim of this variants with

a

study

is to extend this work to vtiver, and to obtain vtiver

higher

vtiver oil

yield

or a

different oil

composition

via tissue

culture. To reach this

goal,

the

project

was

divided in two parts: first to

develop

25

the necessary tools and variants.

In the first

technologies

and second to obtain

plantlets

and

identify

part,

we

optimized

methods to regenerate vtiver

plantlets
the

via in

vitro cultures. To obtain compact calli for influence of different factors like culture conditions
on

subsequent regeneration,
medium

starting material,
were

composition

and

callus induction

tested
on

(Chapter 2).

The influence of

growth

conditions and callus induction media

were

the

subsequent plantlet
The

regeneration optimized
vtiver

examined to achieve
were

an

optimal plantlet regeneration.

with earlier

media and conditions

compared

procedures

for

regeneration
via

described in the literature studies

on

(Chapter 3).

To regenerate

plantlets
media

liquid cultures,
on

the influence of callus and

liquid

culture

composition
were

the

liquid

culture

production

and the

subsequent plantlet

regeneration
We also the and

performed (Chapter 4).

different methods to the oil

compared

identify

oil variants. To pre-screen

an

regenerated plants, plant

synthesis

has to be induced in

earlier stage There of in vitro

material has to be
to extract and
a

analyzed

for oil content and

composition. samples
per

fore, methods
tissue
are

analyze large

numbers of small

needed. To find

balance between the time

required

analysis, analysis

the accuracy of the methods

were were

analysis

and the amount of oil necessary, different

compared (Chapter 5).

and

Water distillation and solvent extraction

first

optimized

compared

on a

larger

scale

(=

5 g vtiver

roots) (Chapter 7).

(Chapter 6)

before small scale extraction

experiments

were

started
on

To induce the oil

ness

production
as

in tissue cultures, initial studies in vitro

the useful

of different tissue such

to

plantlets, analyze
on

root cultures and callus and the

were

feasibility

produce enough

tissue to

the oil content

done

(Chapter 8).Finally, general

conclusions

the results

presented

in this thesis

and considerations of how the oil could be induced and accumulated in tissue cultures
are

written in

chapter

9.

26

27

Chapter
plantlets

2:

Compact

callus induction from in vitro from Java

of vtiver

(Vetiveria zizanioides)

Ruth E. Witholt

Leupin,

Marianne

Leupin,

Charles Ehret, Karl H. Erismann and Bernard

Partially published
115-123

in Plant

Cell, Tissue and Organ Culture, 2000, 62(2):

28

ABSTRACT

Crown and leaf slices of in vitro

zizanioides from Java concentrations of
sucrose were

plantlets
on

of

non-flowering

Vetiveria various

cultured

agar medium
acetic acid

containing
0.1
-

2,4-dichlorophenoxy

(2,4-D,
0
-

10 mg

I"1),

(10

100 g

I"1), 6-benzylaminopurine (BA,

(NAA,
0
or

2 mg

I"1)

and After 4
-

a-naphthalene

acetic acid

0.1 mg

I"1)

in the dark

(23C).

weeks in culture, compact calli tration

were

observed. A

relatively

low 2,4-D

concen

(0.5

mg

I"1)

was

best for compact callus induction. The induction of

compact calli could be increased by adding 0.5 mg I"1 BA and increasing the
sucrose

concentration up to 75 g I"1. The auxin NAA did not increase the

compact callus induction significantly. Combining the above results, the optimal
medium for the of
a

generation

of compact calli from Vetiveria zizanioides consisted

modified

Murashige

and

Skoog

medium

supplemented

with 0.5 mg I"1 2,4-D,

0.5 mg I"1 BA and 75 g I"1

sucrose.

INTRODUCTION

Vetiveria zizanioides is

tropical

grass. It

belongs

to the

subfamily

of

Panicoideae, which includes maize, sorghum, sugarcane and lemongrass

(Vietmeyer
plants.
The

and Ruskin

1993).

There

are

flowering

and

non-flowering

vtiver of the

wild-growing variety commonly

found in North India is

are

mostly

former type, whereas in South India both types As not all vtiver

found

(Anonymous 1976).
low,

plants

flower and the

germination
shoots

rate of the seeds is

vtiver propagates itself There

are

through axillary why

(Vietmeyer

and Ruskin

1993).

two reasons

vtiver is of interest. A traditional and well

on

established the

use

of vtiver is based

its

deep

and fibrous roots, which allow

plant

to be used for

protection against
and Ruskin

soil erosion and loss of moisture Another

an reason

(Erskine 1992; Vietmeyer

cultivated in the of
more

1993).

why

vtiver is

tropics,

is that the roots contain

et ai.

essential oil which consists This oil is used

as a

than 150

sesquiterpenoids (Akhila

1981).

component for perfumes, scenting soaps and

as a

fixative to prevent the

evaporation

of

more

volatile oils

(Vietmeyer

and Ruskin

1993).

Because

29

completely synthetic

vtiver oil cannot be manufactured at

realistic
or

price
different oil

(Vietmeyer

and Ruskin
are

1993),

vtiver variants with

more

oil

with

composition germination

of interest. However, since not all vtiver

plants

flower and the

rate of the seeds is

low, it is difficult

to

produce

variants via

traditional sexual

breeding. requires

An alternative is to

produce

somatic variants via

tissue culture. This

can

that in vitro tissue is available, from which such tissue in

plantlets
as

be

regenerated. Generating developed

for

monocotyledons
have not

is not trivial,

the methods suitable for

dicotyledonous species
and this is
can

always
so

proven

monocotyledonous species,

particularly

for grasses
a

(Vasil
of

and Vasil the

1986). Although plants

efficiency
of

be

regenerated

from

wide

variety

expiants,

regeneration depends largely

obtained when

on

the nature of the

or

expiant

used. Best results

are

expiants
as

from immature organs

meristematic and undifferentiated tissue, such

mature and immature

embryos, inflorescences, anthers, apices

are

ovary, the base of young leaves and shoot

used

(Vasil

and Vasil 1994; Vasil and Vasil has been and Vasil form of

1986).
major species suggested
of

Somatic

embryogenesis

reported

for all of the

cereals and grasses

(Vasil

1986),

and it has been

that this

may be the Somatic

the

more common

plant regeneration by

in vitro in Gramineae.
are

embryo

induction is influenced

many factors. The main factors

auxin and

plant

material and the

growth regulators
sources

cytokinin,

but medium

composition (sugars, nitrogen

environment

and

potassium)

and the culture

can

(pH, light, temperature

and the gas

1981 ;

phase)

also affect the

induction of somatic

embryos (Doughall

Hughes

1981 ;

Thorpe 1994)

or

compact calli.
We have found in

preliminary

studies

(R.E. Leupin, unpublished)

structures

that it is the

possible

to induce a

compact callus from vtiver. These

provide
we

best chance to regenerate

plantlets,

but of the various

approaches

have

tested, the compact callus induction

rate has been very low thus far. In this

chapter

we

describe the establishment and maintenance of in vitro

plantlets

of

vtiver and the influence of the medium

sucrose,

composition,

various concentrations of

2,4-dichlorophenoxy
acetic acid

acetic acid

(2,4-D), 6-benzylaminopurine (BA)

and

a-naphthalene

(NAA)

on

the induction of compact calli.

30

MATERIAL AND METHODS

Plant material

The Vetiveria zizanioides from Java

was

from

our

stocks. The

plants cycle

were

grown and maintained in

hours.

green-house

at 25C and a

light

/ dark

of 16 / 8

Media

For all in vitro

experiments,

MS

(Murashige

and

Skoog 1962),

N6

(Chu

et al.

1975)
of

or

modified MS nutrient media

sucrose

supplemented

with various concentrations

growth regulators,

(1-10 %)
were

and 0.65 % agar

(Agar Agar

Powder

Type S1000; adjusted

B & V s.r.l.

Parma)

used. The

pH

of the medium

was

to 5.8 with KOH or HCl before

autoclaving.

The modified MS medium contained 1/2 MS macronutrients, 1/2 MS micro-

nutrients, 0.5 mg I"1 thiamine-HCI, 0.5 mg I"1 pyridoxine-HCI, 0.5 mg I"1 nicotinic acid, 100 mg I"1 myo-inositol, 40 mg I"1 NaFe(lll)EDTA, 200 mg I"1 glycine and
100 mg I1 citric acid.

Establishment and maintenance of in vitro

plantlets

Plants

were was

cut in

separate shoots. Soil

cm.

was

washed off and the

length

of

the shoots ethanol

reduced to 5

The

cuttings

were

dipped
a

for 30

sec

in 70 %

(v/v),

stirred for 20 min in 4 %

Ca(CIO)2

with

droplet

of Tween 20,

rinsed in sterile water and then the outermost leaf

were

was

removed. These steps

repeated

three times, but the sterilization step with

Ca(CIO)2

was

reduced

each time. The second and third time the

in 2.5 %

cuttings

were

sterilized for 15 minutes the

Ca(CIO)2

and 10 minutes in 1 %
were

Ca(CIO)2, respectively. Following

supplemented

sterilization, the cuttings

put

on

modified MS medium

with

25 g I"1 sucrose, 0.65 % agar, 1 mg I1 kinetin and 0.1 mg I1 indole acetic acid

(VRM8)

and cultivated for 1 week in the dark

(23C).

Then the

cuttings

were

transferred to the

light (23C

with 12 hours

photoperiod).

31

For

propagation containing

of the
BA

plantlets,
mg

small shoots acid

were

cultured

on

modified MS acid

medium

(1

I"1), gibberellic
cultured

(0.1

mg

I"1), indolebutyric

(0.1

mg

I"1)

and 25 g I"1

sucrose were

(vtiver propagation medium, VPM).

on

After the

propagation,

the shoots

modified MS medium

supplemented
or on were

with 25 g I"1 sucrose, 0.65 % agar, without any

growth regulator (VRMO) bigger.

The

VRM8,

on

which the shoots

-

produced

roots and grew

plantlets
were

transferred every 6

8 weeks to fresh medium. All in vitro

a

plantlets

cultured at 23C with

12 hours

photoperiod.

Induction of callus

As the used Vetiveria zizanioides from Java does not flower, the
most often used for callus induction

expiants

(immature embryos,

young inflorescences

and

anthers) 1993)

are

not available. Therefore the so-called crown

(Vietmeyer

and

Ruskin

and the basal leaves of the in vitro

plantlets
or

were

used for callus

were

induction. For that purpose, slices

was

plantlets

grown

on

VRMO

VRM8

cut in

(<

mm) starting

at the root

part of the
defined

crown.

As

long

as crown

tissue

still available, these slices

as

were

as crown

slices, the following

between the last the results obtained

slices
crown

leaf slices

(Figure

2.2a

e).

Since the
not

boundary

slice and the first leaf slice

some

was

clearly defined,

with the leaf slices showed These

crown-

variations.
or

(5

7 slices /

plantlet)

leaf-slices
or

(2

3 slices /

plantlet)

were

placed

on

Petri dishes with modified MS, MS

N6 nutrient medium

containing

various concentrations of 2,4-D

(0.4

10 mg

I"1), a-naphthalene
(1
-

acetic acid
All cultures

(NAA,
were

or

0.1 mg

I"1),

BA

(0

2 mg
-

I"1)

and

sucrose

100 g

I"1).

incubated in the dark at 23

25C.

Histology

For

light microscopy,
-

the

samples

were

fixed in

formaldehyde (35 %)

acetic acid
vacuum.

alcohol

water
were

(10:5:50:35)
dehydrated

mix for at least 24 hours under in

a

Fixed tissues

f-butanol series

(Johansen 1940)

and embedded in

paraffin.

Sections

were

cut at 10 urn and stained with

safranine/fastgreen

ortoluidine blue

(Gerlach 1984).

32

RESULTS

Establishment and maintenance of in vitro

plantlets

To obtain in vitro cultures, the disinfected and

plants

from the

greenhouse high,

needed to be

propagated.

The infection rate able to obtain

was

but with the propa material for the

gation

of the

plantlets

we were

enough plant

experiments.
After disinfection of the

plant material, plantlets

it took have
a

some

months until the shoots

rate of about 5-10
on

started to propagate. The small within

VPM
one

propagation

month

on

VPM. No roots

developed during

the first two months

(Figure 2.1b).
were

Shoots from VPM medium

media the shoots

transferred to VRM8

or

VRMO. On these

produced
on

roots and

began

to grow. After 6-18

were

months,
that

depending

on

the size

VPM, the plantlets

big enough

so

they

could

be cut in slices and used for callus induction

(Figure 2.1c).

Within the past 4 years, variations

during propagation
a

of vtiver
a

plantlets,

several

appeared;

we

found

white-green chimera, plantlets

green-light plantlets

green

chimera,

bunch with white dwarf

and several

which had

decussate leaves instead of alternate leaves The

(Figure

2.1

d).
same

plantlets

from the three first variations showed the

phenotype

after

propagation,

while the variant with decussate leaves

yielded

normal

plantlets

with alternate leaves after

propagation.

Induction of callus

On callus induction medium

after
a

containing 2,4-D,
were

calli grew

on

the
or

plant

slices
a

short time in culture. These calli

mass

soft and watery

an

covered with

gelatinous

(Figure 2.2f).
was

After about 4 weeks

additional callus type

structures

was

observed. This callus

compact and contained organized

(Figure

2.2f, g). After 6 weeks in culture, the number of slices with compact callus still increased, whereas the other calli did
not

(data

not

shown).

33

Figure 2.1. Establishment of in vitro plantlets of Vetiveria zizanioides. (a) Vetiveria zizanioides in a green-house; (b) Vtiver plantlets from the propagation medium (VPM); (c) Vtiver plantlets grown on VRM8; (d) Variation appeared during propagation on VPM: vertical section through a vtiver plantlet 1 mm) with decussated leaves (bar
=

From

preliminary

studies

we

knew that these compact calli

provide

the best
was

material to regenerate low

we

plantlets.

As the induction rate of these compact calli

tested the influence of different media,

on

growth regulators

and

sucrose

concentrations

compact callus induction.

34

Figure

2.2. Induction of callus from

crown

and leaf slices of Vetiveria

(a, b) Longitudinal through a vtiver bud; (c) Schematic diagram of a longitudinal section through a vtiver bud; (d) Vertical section through vtiver leaves; (e) Vertical section through a vtiver crown; (f, g) Crown
zizanioides. section slice with callus and compact callus; bar
A:
=

mm.

axillary buds, Ap: apical meristem,

rm:

root
a

meristem,

ss:

side shoot,
cc:

sc:

soft

and watery callus, gc: callus covered with

gelatinous layer,

compact callus

Effect of the medium

composition

on

compact callus induction

To find the
were

optimal 2,4-D
on

concentration to induce compact calli,

sucrose

crown

slices

cultured

modified MS medium with 25 g 11

and various

concentrations of 2,4-D tested the

(0.4

-10 mg

11).

Calli

were

formed at all concentrations the 2,4-D concentration,

2.1

(80

100 % slices with

were

callus),
a

but the

higher

more

calli

covered with

gelatinous layer (Figure

no

d)

and at

concentrations

higher

than 1 mg 11 2,4-D

compact calli

were

induced. A

subsequent experiment

with 2,4-D concentrations between 0.4 and 1 mg 11

was

showed that 0.5 mg 11 2,4-D

most suitable for

inducing compact

calli

(Figure 2.3).

35

20

CO

^
CD

un CD

cd O

r-CD

co CD

a> CD

t^
Q

Figure

2.3. Effect of

2,4-dichlorophenoxy

acetic acid concentrations

on

compact

callus induction. Plantlets grown on VRM8 were cut in slices (120 crown slices / medium) and cultured on modified MS medium supplemented with 25 g I"1
sucrose

D 1.0). The and 2,4-D concentrations between 0.4 -1.0 mg I"1 (DO.4 of crown slices with compact calli was determined after 8 weeks in percentage
-

the dark

(23C)

To compare different basal media, modified MS, MS

200
on crown or

crown

slices

were

cultured for 8 weeks

on

N6 media with 25 g I"1 tested

on

sucrose

and 0.5 mg I"1 2,4-D. About calli

were

slices

were

each medium.

Compact

formed
was

only

the MS and the modified MS medium and compact callus formation

same on

about the

both media

(2

2.4

%).

No compact callus

was

induced

on

N6 medium.

To test the influence of other

crown

growth regulators

on

compact callus induction,

and leaf slices

were

cultured for 8 weeks

NAA

on

modified MS medium
BA

containing 2,4-D (0.5

sucrose.

mg

I1),

(0

or

0.1 mg

I1),

(0

2 mg

r1)

and 50 g I"1

The percentage of leaf and

crown

slices with callus decreased with

no

increasing
be found

BA concentration. Whereas with 2,4-D alone

compact calli could percentage of slices

on

leaf slices, addition of BA resulted in the

crown

same

with compact calli for both leaf and

slices. The percentage of slices with

to 0.5 mg

compact calli increased with BA concentration up

there
was

I"1. At 2 mg I"1 BA,

equal

or

less compact callus induction than with 0.5 mg I"1 BA

(Figure

2.4).

The addition of 0.1 mg I"1 NAA seemed to

improve

the compact callus

36

crown

slices
40
tt

100 90 80

:nj*j::::::Aj5i::::::
.. .

35

^ 70 =i

._

^30

5040-

..

.___

A.___
r-

I
o

25 20

..

.-[*[

t3
CO

3020100o

io 5"-

LU LJ

un o o

un

<M

OJ

un o o

un o

CM

eo

EU

m n

z. n

OJ

Z.

~7 n

-z. Q

eo Q

un o o

un

CM

OJ

m n

m u

LU

un o o

un

c\l

m Eu

Z. u

LU

z.

Z n

7 n

leaf
100 90 80 70 60 50

slices
40
^ s?

35

^30
1 25
o

tS
CO

20

40-f
30-20--

8 iot
5

1o4
0
o

I1
m Q
o

'I'
o

'I1
m Q Eu Q

'I1
Eu -Z.

'I1
o

M1
O

M1
(M

M
EU

0
o

H
EO Q
un o o un o

A
Eu Q

+**4
Eu Q Eu
un o un

VH
(M

EU

m Q

EU

EO Q

ci
Eu

ci
EO

EO

\-

8 weeks
2.4. Effect of various

H
concentrations and the
or

Figure

6-benzylaminopurine
on

addition of

a-naphthalene

acetic acid

the induction of callus

-

compact

callus. Plantlets grown on VRM8 were cut in slices (5 crown and 3 leaf slices / plantlet) and cultured on modified MS medium supplemented with 0.5 mg I"1 2,4D, different BA concentrations (0/ 0.005/ 0.5/ 2 mg I"1), without or with 0.1 mg I"1
NAA and 50 g I"1 sucrose (DB or DNB 0/ 0.05/ 0.5/ 2). The percentage of (a, b) or leaf slices (c, d) with callus (a, c) or compact callus (b, d) was
crown

determined after 8 weeks in the dark

:

(23C) medium),
without DB2

experiment
and DNB2

(95-100

crown

and 21 -30 leaf slices /

A : experiment 2 (114-124 crown and 72-75 leaf slices / medium) D: slices with callus (average of experiments 1 and 2)
:

slices with compact callus

(average

of

experiments

1 and

2)

37

induction
were

on

DB0.5

(Figure 2.4),

but

as

the averages of DNB0.5 and DB0.5

was

within the

experimental

error

for these two media, NAA

omitted from

the callus induction medium.

To test the influence of the callus induction,

crown

sucrose

concentration
were

on

callus and compact

on

and leaf slices

cultured for 8 weeks

modified

MS medium

supplemented
sucrose on

with 0.5 mg I1 2,4-D, 0.5 mg I1 BA and various

-100 g

concentrations of have much effect concentrations

on

(10

I"1).

The

sucrose

concentration did not

sucrose

callus induction
100 g

on crown a

slices, but the higher

effect
on

(50

I"1)

did have

negative

the callus induction from the

leaf slices

(Figure 2.6).
more

The further the used leaf slices

was

were

apical

meristem the

the callus induction

not

inhibited

by high

sucrose

concentrations The For calli

sucrose

(data

shown).
an

concentration had

effect

on

the induction of the callus type.

crown was

slices the

optimal

sucrose

concentration for the induction of compact

was

75 g I"1 sucrose, for leaf slices it

between 75 and 100 g I"1

sucrose

(Figures 2.5, 2.6).

also led to

DB75 not

only

induced

more

slices with compact calli, but

seen

bigger compact

calli after 8 weeks than

for induction

on

DB10.

Effect of the

starting

material

on

compact callus induction

To test the influence of the medium

on

which the

plants

grew before the

compact callus induction experiments,

grown
on

crown

and leaf slices from

on

plantlets

VRM8

or

VRM0

were

cultured for 8 weeks

modified MS medium

containing
sucrose. were

0.5 mg I"1 2,4-D, 0-0.5 mg I"1 BA, 0-0.1 mg I"1 NAA and 50 -100 g I"1

The

plantlets

grown

on

VRM0 formed
on

more

calli and

more

of these

compact calli than did plantlets grown

VRM8

(Figure 2.6).
were

For the callus induction

crown

experiments

crown

and leaf slices

used. The

slices contained root and shoot meristems, whereas leaf slices contained

neither

(Figure 2.2d, e).

calli could be found
crown on

Compact

all

crown

slices

independent
in
some was

of the

origin
an

of

these slices within the

(Figure 2.2c), although

experiments

increase of the percentage of slices with compact calli

detectable for

crown

38

crown

slices
b
30
T l
-

100 90 80

30

-i

25 20

25 20

70 60 50 40 30 20 10 0
o
i-

^
CO

j
"co
co
Q.

J
"c

M
CO
"

*
E
o
o

-A-

tS
co
Q.

15+"
10

10 +

E
o
o

f
"-

--

en

o un

m Q

m Q

m Q

o un

o
1

o un

m Q

CD Q

m Q

m Q

leaf
100 90 80

slices
e

f
30
-i

30

-i

25
co

--

25
co

--

70 60 50 40 30 20 10 0
O
1

^
en

20

--

on
20

--

co

co
o

^
co
"

t
co
Q_

15" 10"

E
o
o

10

E
o
o

--

"-

o un

m u

o un

o un

m Q

m Q

CD Q

m Q

m Q

CD Q

m Q

6 weeks

-w-

12 weeks

Figure

2.5. Effect of the sucrose concentration

on

compact callus induction.

-

Plantlets grown on VRM0 were cut in slices (6 crown and 2 leaf slices / and cultured on DB0.5 media supplemented with different concen plantlet) trations of
crown sucrose

(a, b, c) or f) was determined after about 6 (a, b, d, e) and 12 weeks (c, f) in (23C). : experiment 1 (101 -106 crown and 32-38 leaf slices / medium) : experiment 2 (75-82 crown and 225 leaf slices / medium) D: slices with callus (average of experiments 1 and 2) : slices with compact callus (average of experiments 1 and 2)
e,

DB10 / DB30 / DB50). The percentage of / 30 / 50 g I1 leaf slices (d, e, f) with callus (a, d) and compact callus (b, c,

(10

the dark

39

crown

slices
b
60
-r

100

60

-,

908070-_

50
1

50 40

-{
-

60504030-2010--

|
8

0-I-

I
o un un

eg
"c
o

40

o
CO

30 20

t>

30 20

CO

i"
o
o

10

i 1
o un LO

CO

E
O
O

10

Is-

o o

r^

o o
T-

o un

un

r--

o o

m o

m o

;-

m Q

m q

gg
Q

m o

leaf
10090 80 70

slices
e

60

-r

60 50

-r

50 40

"

o
"

S?

60 50

cg
"r

--

40 30

J
g

o o

30

4030 20 io 0
"

ca

ca

41
O Lo LO

1
r-o o

Q.

E
O
O

2010

o
o

20 10

--

a.
O LO LO

S
o o
-

I
O LO

1
LO

^
o o

r^

f~-

m q

m q

m Q

m Q

m q

m q

m q

m Q

6 weeks

8 weeks

Figure
slices

2.6. Effect of

plant growth

medium and the

on

sucrose or

concentration
crown

on

the

compact callus induction. Plantlets grown

VRMO

VRM8 media

were

cut in

(6

crown-

and 3 leaf slices /

crown

medium) resulting

in 125

and 75 leaf
were

slices for VRMO and 115 cultured

and 69 leaf slices for VRM8. The slices

on supplemented with 0.5 mg I"1 2,4-D and 0.5 DB50 / mg I1 BA with different concentrations of sucrose (50 / 75 /100 g I"1 DB75 / DB100). The percentage of crown (a, b, c) or leaf slices (d, e, f) with

modified MS medium

(a, d) and compact calli (b, c, e, f) was analyzed (c, f) in the dark (23C). D : plantlets from VRMO (125 crown and 75 leaf slices E3 : plantlets from VRM8 (115 crown and 69 leaf slices
callus
8 weeks

after 6
/ /

(a, b, d, e)

and

medium) medium)

40

slices which

originated

in the

region

close to the
on

apical

meristem
on

(data

not

shown).

No compact calli

were

induced

leaf slices cultured

medium with

0.5 mg I"1 2,4-D. Leaf slices could be induced to form

compact calli by addition

of BA

(Figure 2.4).

The number of leaf slices with compact calli decreased with

increasing

distance from the

apical

meristem.

DISCUSSION

In this

chapter

we

showed that it is

possible

to increase

compact callus

formation in Vetiveria zizanioides.

Although

the

reproducibility
was

of compact callus induction

was

poor, with varia

tions up to 20 %, and it

difficult to compare results obtained in

was

experiments
for this

done at different times, the

general tendency

clear. One

reason

variation in the compact callus induction is the

wrote that the
were

plant

itself. Vasil and Vasil of the

(1994)

physiological obtaining
a

condition and

developmental stage plantlets

same

expiants
our

critical in

desirable response, and the

same

used for

different different

experiments

had neither the

age

nor

the

size. Due to the

growth regulators might

in the medium, the

plantlets

used

as

starting

material

for callus induction

be in different

developmental stages,
on

which could

explain why
same

slices from

plantlets

grown

VRM8

or

VRMO did not result in the

callus induction.
our

We have observed in

experiments

with in vitro vtiver

plantlets

that 0.5

mg I1 2,4-D

was

sufficient to induce compact calli.

Higher

concentrations of 2,4-

D did not enhance the induction of

compact calli, whereas for other Gramineae

or even

2,4-D concentrations up

to 10 mg

I"1

higher

have been used

(Flick

et al.

1983).
callus

Often the
or

only plant growth regulator embryos

is the auxin 2,4-D

added to the medium to induce

somatic

(Vasil

and Vasil

1994).

Schenk

and Hildebrandt

(1972)

described that for wheat,

barley,

rice and

bromegrass,

kinetin inhibited callus induction and addition of low levels of kinetin induction of
or

growth,

whereas for other Gramineae,

BAto 2,4-D
et al.

containing

medium

supported

embryognie
1991

callus

(Mathur
as

1988; Sreenath and

Jagadishchandra

).

We found

well that the addition of 0.5 mg I"1 BA to

41

the callus induction medium

was

beneficial for compact callus induction

(Figure

2.4).
Sucrose enhanced compact callus induction;
al.
a

finding

similar to that of Lu et
more sucrose

(1982, 1983),

who found that immature

on

embryos

of maize formed

embryognie

callus

MS medium with 0.5 mg I"1 2,4-D and 120 g I"1

An

than with 60 g I"1

sucrose.

explanation

for this effect could be that osmotic

stress enhances somatic

embryogenesis (Litz 1986; Thorpe 1994).

We conclude, based callus induction is and 75 g I"1

a

on our

experiments,

that the best medium for compact

modified MS medium with 0.5 mg I1 2,4-D, 0.5 mg I1 BA With this medium slices from 0
we

sucrose.

have been able to

-

improve compact possible

to

callus induction

on crown on

20 % to 20

50 %. It is also
now

induce compact calli

leaf slices, and with this the stage is induction of cultures.

set for the

regeneration plantlets
from

of

plantlets,

suspension cultures,

and

regeneration

of

suspension

ACKNOWLEDGEMENTS

The Vtiver

plants

were use

provided by
of the

Mr. Heini

Lang,

Jakarta. We thank FAL,

was

Reckenholz Zrich for

plant sectioning

facilities. This work

supported by

Givaudan-Roure

Forschung

AG Dbendorf, Switzerland and

no.

by

the Swiss Federal Office for Economic

Policy, project

2561.1 of the

Commission for

Technology

and Innovation.

42

43

Chapter

3:

Compact
of
a

Callus Induction and Plant

Vtiver

Regeneration
zizanioides)

non-flowering

(Vetiveria

from Java

Ruth E. Witholt

Leupin,

Marianne

Leupin,

Charles Ehret, Karl H. Erismann and Bernard

Partially published
115-123

in Plant

Cell, Tissue and Organ Culture, 2000, 62(2):

44

ABSTRACT

Crown and leaf slices of in vitro

zizanioides from Java
were

plantlets

of

non-flowering

Vetiveria

used to induce compact calli and to regenerate concentrations

plantlets. light
or

The influence of

sucrose on

(10

or

75 g

I"1),
or

cultivation in
12

dark and cultivation time

callus induction medium

(6

weeks),
plantlets
and

on

the induction of compact callus and the

was

subsequent regeneration

of

studied.

Up

to 75 % of crown slices cultured on modified

Murashige

Skoog

medium

supplemented

with 0.5 mg I"1 and 75 g I"1

2,4-dichlorophenoxy

acetic acid, callus.

0.5 mg I"1

6-benzylaminopurine
of

sucrose

developed compact light

For

subsequent regeneration
on

plantlets,

callus induction in the

for 6

weeks

the callus induction medium transfer to the

on

containing

10 g 11 sucrose, and
was

subsequent

regeneration medium,

the best

procedure,

regenerating plantlets plantlets

per slice.

around 60 % of the

crown or

leaf slices, with up to 100

We have

compared

the

efficiency

of the above mentioned

procedure

with

several other methods to regenerate

plantlets.
best in

Our

findings

indicate that the for the

procedure developed
used vtiver variant.

in this

study

was

regenerating plantlets

INTRODUCTION

The

tropical

grass Vetiveria zizanioides

belongs

to the

subfamily

of

Panicoideae, which includes maize, sorghum, sugarcane and lemongrass

(Vietmeyer

and Ruskin

1993).

The vtiver
as a

plant

contains in its root

an

essential

oil, the vtiver oil, which is used

and
as a

component for perfumes, scenting soaps

of
more

fixative to prevent the Because

a

evaporation

volatile oils

(Vietmeyer

and

Ruskin tured at
more

1993).
a

completely synthetic
and Ruskin
are

vtiver oil cannot be manufac

realistic with
a

price (Vietmeyer
different oil

1993),

vtiver variants with

oil

or

composition

of interest. New variants could be

obtained

by

traditional

breeding (Gupta

et al.

1983; Lai et al. 1998; Sethi 1982;

Sethi and

Gupta 1980)

or, since not all vtiver

plants

flower and the

germination

45

rate of the seed is low

(Vietmeyer
are

and Ruskin

1993) by regeneration

of

plantlets

via tissue cultures, which

putative

somaclonal variants. of vtiver from inflorescence

Several groups have

tissues
et al.

regenerated plantlets

(Keshavachandran

and Khader 1997; NaNakorn et al. 1998; Sreenath

1994),

from leaf material

(Mathur

etal. 1989; Mucciarelli etal. 1993; of young

Sreenath etal.

1994)

and from the

mesocotyl part

seedlings (George

and Subramanian

1999). Except George

plant

and Subramanian

(1999),

all of these

workers used in vivo is disinfected

material to induce calli.

Although

the in vivo material

directly

before the callus induction and

occur

subsequent regeneration,

losses due to infection still do

(Sreenath

etal.

1994). Furthermore,
which also

the

plant

surface is often

damaged by

the sterilization

techniques,
we

influences the
as

experiment.

To prevent such

problems,
were

used in vitro

plantlets

starting

material. The disinfected shoots

were

first

propagated

in vitro and

infected

plantlets study

removed before callus induction.

In this

we

describe the of

optimization
from

of compact callus induction and and leaf slices of in vitro

subsequent regeneration plantlets

of
a

plantlets

crown

non-flowering

vtiver. We have also

compared

the

efficiency

of the

optimized procedure
and Mucciarelli etal.

with those of Sreenath etal.

(1994),

Mathur etal.

(1989)

(1993).

MATERIAL AND METHODS

Plant material

The Vetiveria zizanioides from Java

was

from

our

stocks. The

plants cycle

were

grown and maintained in

hours.

green-house

at 25C and a

light

/ dark

of 16 / 8

Media

For all in vitro

experiments Murashige Murashige

&

&

Skoog (MS) (Murashige

nutrient media
sucrose

and

Skoog

1962)

or

modified

Skoog (modified MS)

supple

mented with various concentrations of

growth regulators,

(1-10 %)

Table 3.1. Media used for callus induction and for

plant regeneration

of vtiver.

Medium

'

Basal medium

Growth

regulators
Kin BA

Additional

compounds

Sucrose

References

2,4-D
Ind.
-

IAA

[mgt1]
DB10 DB75
-

[gt1]
-

modified MS 2.26 2.26 0.45

-

2.22 2.22 4.44 4.44

10

modified MS modified MS modified MS 0.45

-

75
-

Reg.
Ind. Mai Ma2
-

D0.1B1(25) D0.1B1(75)
MS
4.52 5.71 5.71 5.71 9.05
-

25
-

75
-

this ascorbic acid (40) ascorbic acid (40) casein hydrolysate (100) hydrolysate (100)

study
-

1.16 2.32 4.65 0.46 4.65

-

30

Reg.
Ind. Mu1 Mu2

MS MS MS MS
4.52
-

30
-

(Mathuretal. 1989)
-

30 casein
3
-

Reg.
Ind.

30

(Mucciarelli et
-

al.

1993)

Sr1

30

Reg.
MS
-

Sr2(30) Sr2(100)

MS

30
-

100

(Sreenath

et al.

1994)

2 3

Ind.: callus induction medium; Reg.: plantlet regeneration medium 2,4-D: 2,4-dichlorophenoxy acetic acid; IAA: indole acetic acid; Kin: kinetin; BA: 6-benzylaminopurine for this

experiment
was

kinetin

used. Sreenath et al.

(1994)

also used 4.44 uM BA

47

and 0.65 % agar used. The

(Agar Agar

Powder
was

Type S1000;

B & V s.r.l.
or

Parma)

were

pH

of the medium

adjusted

with KOH

HCl to 5.8 before

autoclaving.
Modified MS medium contains 1/2 MS macronutrients, 1/2 MS micro-

nutrients, 0.5 mg I"1 thiamine-HCI, 0.5 mg I"1 pyridoxine-HCI, 0.5 mg I"1 nicotinic acid, 100 mg I"1 myo-inositol, 40 mg I"1 NaFe(lll)EDTA, 200 mg I"1 glycine and
100 mg I1 citric acid.

Establishment and maintenance of in vitro

plantlets

In vitro

plantlets

were

disinfected cultured

as on

previously

described

(Chapter 2).

For

propagation, plantlets

were

modified MS medium with the

growth

regulators 6-benzylaminopurine (BA; indolebutyric gation,

acid

1 mg

I"1), gibberellic
(VPM).

acid

(0.1

mg

I"1)

and

(0.1

mg

I"1),

and 25 g I"1
on

sucrose

After the propa

the shoots

were

cultured

modified MS medium

supplemented
on

with

25 g I"1 sucrose, 0.65 % agar, without any

growth regulator (VRM0),

The

which

the shoots

produced

roots and grew

bigger.

plantlets

were

transferred

every 6
with
a

8 weeks to fresh medium. All in vitro

plantlets

were

cultured at 23C

12 hours

photoperiod.

Callus and compact callus induction

To induce calli and compact calli, in vitro

in slices

plantlets
slices

grown
-

on

VRM0

were or

cut

(<
-

mm) (Chapter 2). plantlet)

These

crown on

(5

8 slices /

plantlet)

leaf

slices

(2

3 slices /

were

placed

different callus induction media and maintained at 23C either

(result

and discussion section and Table

3.1)

under 12 h

daily

illumination

or

in the dark.

Plant

regeneration

After 2, 6, 8

or

12 weeks

on

induction medium, media

crown

and leaf slices Data

were

were

transferred to the different after 6, 8, 12, 16, 18

or

regeneration

(Table 3.1).

collected

24 weeks of culture in the

light (Figure 3.1).

48

C/R
i i i

R
1Nj
1 L
i i

R
v"

R
v"

J-Nl'' jii_i\i

C
i i

C/R
i , i

R
JNJ
1 1 J 1

R
LJNJ
\/

\Z~

C/R

2
v

R
i i

Kl

\/

C/R
r\ .1

R
i i i i

^^^1
I_J1

M 1/

C/R
i i i i

R
"-Ni
v*
1 1 1 1 1 1 1 1

C/R
i i i i

R
1 1 1 1

Kl

\/

1
10

1
15

1
20

weeks

callus induction medium

C:

analysis analysis

of callus and

compact callus induction

=>:

plantlet regeneration
medium

R:

of

regeneration

Figure 3.1. Time sequence of the different callus induction and plantlet regeneration experiments. The following time schedules were used: a,
the influence of the
sucrose

b: to test the
c:

concentration in the callus induction medium

(DB10, DB75)

and cultivation in

light

or

in dark

during

callus induction
-

on

compact callus induction and the subsequent shoot regeneration; c f: to compare the different procedures for callus induction and shoot regeneration;
time schedule for DB10/D0.1 B1 Mucciarelli etal.

(25)

or

DB75/D0.1 B1

(25);

d: time schedule from

(1993);

e:time schedule from Mathur etal.

(1989);

f:time

schedule from Sreenath etal.

(1994)

Histology

For

light microscopy,

the

samples

were

fixed in

formaldehyde (35 %)vacuum.

acetic acid-alcohol-water Fixed tissues

were

(10:5:50:35)
in
a

mix for at least 24 hours under

dehydrated

f-butanol series

(Johansen 1940)

and

embedded in Paraffin. Sections

were

cut at 10 urn and stained with safranine /

fastgreen

ortoluidine blue

(Gerlach 1984).

49

RESULTS AND DISCUSSION

Influence of

sucrose

concentrations and cultivation in

on

light

or

dark

on

compact callus induction and

subsequent plantlet regeneration

Based
we

on our

earlier

experience

with vtiver callus induction

(Chapter 2),

have used modified MS medium

containing

0.5 mg I"1

2,4-dichlorophenoxy
for induction of
was

acetic acid

(2,4-D)

and 0.5 mg I"1

6-benzylaminopurine (BA) plantlets.

/

compact calli

on crown

and leaf slices of in vitro

75 g I"1
sucrose

This DB medium

supplemented

with 10

or

(DB10

DB75).

The

plant

slices
/

were

maintained at 23C either under 12 h

daily
/

illumination

(DBIO(light)

DB75(light))
were

or

in the dark

(DB10(dark)

DB75(dark)).
-

Crown and leaf slices

slices /

prepared

from about 35 in vitro

plantlets (4

crown

plant

and

3 leaf slices /
were were

plant).

After 6 weeks in culture, half of the

crown

and leaf slices slices

transferred to

regeneration

medium
on

(Figure 3.1a).

The

remaining

cultivated for another 6 weeks

the induction media medium. This

(Figure

3.1

b)

and

then also transferred to the

regeneration

experiment

was

repeated

three times.

Callus and compact callus induction

After

short time in culture, soft and

on crown

gelatinous

calli grew
on

on

the

plant

slices.

Callus induction

slices exceeded 75 %, and

was no was

leaf slices it varied

between 40 % and 97 %. There

further callus induction after 6 weeks.

Callus induction
calli
were

on crown

slices

better in the

light,

while

on

leaf slices

more

induced in the dark

(data

not

shown).
containing organized
structures

After about 4 weeks in culture, compact calli

were

observed

on

plant

slices

(Figure 3.2a).
described
1991
as

This compact callus type

resembled

embryognie callus,
and

nodular, hard, yellow, and opaque

(Sreenath
growing

Jagadishchandra
white to

or as

compact, highly organized, slow

and

pale

light yellow
on crown

in color

(Vasil

and Vasil

1994).
on

Most

compact calli

were

induced

slices cultured in the

light

callus

induction medium with 75 g I"1

sucrose.

For leaf slices the effects of the sugar

50

Figure 3.2. From compact callus to regenerated plantlets. (a) Compact callus; (b, c) Sections through compact callus from DB75 with bipolar structures; (d f) Sections through compact callus from DB10, (d) shoot regeneration, (e, f) somatic embryos with shoot (s) and root (r) meristems; (g) Callus with regenerated plantlets; bar: 0.5 mm
-

51

concentration and
some

light

were

less

pronounced (Figure 3.3a, b). Although

after 6 weeks, after 12 weeks the

on

slices compact callus

was

was seen

compact callus

not detectable any more. The number of leaf and crown

on

slices with compact calli still increased after 6 weeks, except

DB10(light)

(Figure 3.3a, b).

On DB75, not
even

only

did the percentage of slices with compact

crown

calli increase, but and

some crown

the amount of compact callus per

were

slice increased

slices

completely

covered with compact calli

(results

not

shown).
To compare the

morphological

characteristics of calli induced

on

DB10

or

DB75, calli

were

sectioned and examined with

light microscopy. Bipolar

compact

structures were found on the sections from DB75

(Figure 3.2b, c).

in addition to

Sections from DB10 contained shoots and somatic

embryos

compact bipolar

structures

(Figure

3.2d

f).

Plant

regeneration

The first

signs

of

regeneration light,

were

already

seen

after 12 weeks

on

callus

induction media. In the and

on

calli of

crown

and leaf slices showed green spots whereas in the dark

DB10
on

even

regenerated plantlets,
on a

plantlets

regen
the

erated

DB10

only

few leaf slices

(Figure 3.3c, d).

After
was

transferring

plant

slices to

regeneration medium,
on

the best

regeneration

achieved for
on

crown

slices induced

DB10 in the

light

and for leaf slices induced The results of

on

DB10

both in the

light

and in the dark

(Figure 3.3c, d).

callus induction
on

Figure

3.3 show

that the influence of

light during
as

subsequent plantlet
as on

regeneration depended

much

the

plant

material used

the sugar

concentration in the callus induction medium. The

regeneration

rate after 12 weeks decreased for crown and leaf

on

slices,
on

except for calli induced

DB10(light)

where

plantlets began

to

regenerate

the callus induction medium

(Figure 3.3c, d). George

and Subramanian

(1999)

reported
medium

that their vtiver callus maintained the

morphogenic potential hydrolysate.

on

containing polyvinyl pyrrolidone compounds

and casein

Therefore

these two

should be tested in the future.

52

Crown

slices

80 70

g
en

60--

^ 50 f
CO
o

40-h

2.30 +

10 0

I
DB10 DB75 DB10 dark

I
DB75 dark
12 1

light

light

time

[weeks]

Leaf

slices

DB10

DB75

DB10 dark

DB75 dark

12

light

light

time

[weeks]
on

6 weeks

on

callus induction

12 weeks

callus induction

medium
-n-/-o-/-

medium
->

DB10(light)

D0.1 B1

(25)

-A-/-0-/-

DB10(dark)->D0.1B1(25) DB75(dark)->D0.1B1(25)
light
or

DB75(light)->D0.1B1(25)
3.3. Effect of
sucrose

Figure

concentrations and cultivation in

dark

on

callus and compact callus induction and subsequent plantlet regeneration. Crown (a, c) and leaf (b, d) slices were cultured on DB10 or DB75 in the light

or

percentage of slices with compact callus was determined after 6 and 12 weeks (a, b). After 6 (open symbols) or 12 (filled

in the dark at 23C. The

symbols) weeks the slices were transferred to regeneration medium D0.1 B1 (25). The percentage of slices with shoots was determined after 6, 12, 18 and 24 weeks (c, d).

53

Influence of addition of medium

a-naphthalene

acetic acid to the callus induction

Earlier callus induction

experiments

on

DB medium

containing
resulted in
were

additional 0.1

mg I1

a-naphthalene

acetic acid

(NAA) (DNB medium)

slightly higher

percentage of slices with compact callus, but the values

error

still within the

of the

repetitions (Chapter 2). Therefore,

on

the influence of NAA addition to

the callus induction media

compact callus induction and subsequent plantlet

regeneration

was

tested.

Crown and leaf slices of 33

plantlets
a

were

put

on

DB10, DB75, DNB10 and

DNB75 and cultured at 23C with slices

were

photoperiod

of 12 hours. After 6 weeks the

transferred to the found

regeneration

medium

D0.1B1(25) (Figure 3.1c).

were

The

changes

by adding

NAA to the callus induction medium

no or a

too

small to be

significant.
crown

On leaf slices, NAA had

slight positive effect,

effect

whereas for

slices, there

was no or even a

slightly negative

(Figure

3.4).

Influence of

starting

material

on

plantlet regeneration

Although
DB10
or

the

same

treatment was used for the

compact callus induction

half the

on

DB75 in the
crown

light

and

subsequent regeneration, only regenerated plantlets

were

percentage of

slices with

obtained
to the

during

the

experiment

with the additional NAA

(Figure 3.4) compared

Vasil and Vasil
as

light/dark
that

comparison experiment (Figure 3.3).

the

(1994, 1986) reported

the

growth

conditions of the donor

plants

well
are

as

physiological obtaining
not

condition desirable
in the

and the

developmental stage

of the

expiants

critical in

response. The in vitro vtiver

same

plantlets

used in this

study

were

always
crown

physiological

state. This seems to have a

larger

effect

on

the

slices
as

than

on

the leaf slices, since the

was

regeneration

of

plantlets

from leaf slices As

expiant

about the

same

in both

experiments (Figure 3.3, 3.4).

crown

slices contain, beside leaf tissue, many other cell types like root and shoot

meristems, this could be

leaf slices
as

a reason

for the differences obtained with

crown

and

expiant

for compact callus induction and

subsequent plantlet

regeneration.

54

Crown
80 70 60 50 40 30 20 10 0
un un

slices
--

80 70 60 50 40 30

--

--

--

CO
o

CO

t>
CO
Q_

8
CO

+
--

E
o
o

12

m Q

m Q

time

[weeks]

Leaf
80 70 60 50
CO
o o

slices

40 30 20 10 0
un un

CO

O
O

12

m Q

m Q

time

[weeks]

DB10-> DB75->

D0.1B1(25) D0.1B1(25)

--:

DNB10->

D0.1B1(25) D0.1B1(25)

-A: DNB75->

Figure
media

3.4. Effect of additional

on

a-naphthalene
were

acetic acid to the callus induction

on

compact callus induction and subsequent plantlet regeneration.

and leaf

Crown

(a, c)

(b, d)

slices

cultured

DB10

or

DB75 without

or

with additional 0.1 mg I"1 a-naphthalene acetic acid (DNB10, DNB75) in the light at 23C. The percentage of slices with compact callus was determined after 6 weeks and D0.1 B1

(a, b). After 6 weeks the (25). The percentage of 18 weeks (c, d).

slices

were

transferred to
was

regeneration

medium

slices with shoots

determined after 6, 12

55

Plantlet

regeneration

from leaf slices

For leaf slices, not all % of the slices

plantlets regenerated
while

from compact calli, since

only
on

20

developed compact calli,

plantlets

were

regenerated

up

to 70 % of the slices.

Thus, either shoots regenerated from compact callus

or

before these

were

detectable,

they regenerated
were

from another callus type cultured in the

or

directly
DB10,

from leaf cells. To test this, leaf slices

light

on

on

D0.1B1(25)

or on

DB10 followed after three weeks

on

by D0.1B1(25).

On

12 % of the

slices, plantlets regenerated directly

on

the callus induction medium

23 % of the slices

DB10, whereas after subsequent cultivation

D0.1B1(25)
were over

regenerated plantlets.
% of the slices, which

Since
was

on

D0.1 B1

(25), plantlets
due to carry

observed

on

only
a

most

probably

of meristems,

callus induction step is necessary to regenerate

plantlets

from leaf slices.

Plantlet

regeneration

from

crown

slices

Despite

higher
of

rate of

compact callus induction

crown

on

DB75

(Figure 3.3),
was more on

regeneration

plantlets

from
on

slices with compact callus than after induction

successful after induction This could be DB75 and

an

DB10

(90 %)

DB75

(20 %).

explained

either

by

regeneration

block for the compact calli from

or

precocious germination

of compact calli from DB10

on

by

induction of

additional callus type with With respect to the first

improved regeneration ability

DB10 in the
on

light.

option,

the compact callus obtained

DB75 had the

appearance of

embryognie

callus described

by

Sreenath and

Jagadishchandra

(1991).
as

Lu et al.

(1983, 1984) reported

that

for immature

embryos

of rye and maize

12 % instead of 3
as was

starting material,

higher

sucrose

concentrations

(6

or

sucrose)

resulted in
our

an

increase of the amount of

on

embryognie callus,

observed in

experiments

compact callus induction. Sreenath (1991,

sucrose

1994) reported
before cious

that at low levels of

(3 %),

somatic

embryos germinate

they

attain the

typical

grass

embryo morphology,

and that this preco

sucrose

germination

could be

suppressed by using higher

concentrations.

Thus, the higher regeneration obtained after induction

be due to

on

DB10 in the

light

may

precocious germination

of shoots.

Looking

at the

microscopical typical
grass

sections of the DB75 calli, the

bipolar

structures do not look like

56

embryos. Therefore,
obtain

the best combination of chemical and

more

physical

stimuli to

embryos

and regenerate

plantlets

must still be found.

Since Sreenath et al. g I1

sucrose

(1994)

obtained very

good plant regeneration

added to

with 100
our

in their

regeneration medium, prior

more sucrose was crown

regeneration
on

medium. After

induction of

and leaf slices for 6 weeks medium medium with

not

DB75 in the

light,

the slices
or

were

transferred to

regeneration regeneration

containing

either 25 g I"1

75 g I"1

sucrose.

On the

75 g I1 sucrose, fewer

crown

and leaf slices

sucrose

regenerated plantlets (results improve

the

shown). Therefore,
regeneration

the

higher

concentration did not

and other stimuli have to be found to regenerate

plantlets.

With respect to the second

two callus

option,

Vasil and Vasil

(1994)

described in maize

types of which type I is compact, slow growing and rapidly loses

whereas type II is soft, friable, fast

regeneration ability, regenerative

Tomes

growing, highly
for
a

and maintains its competence for

regeneration
a

long

time.
was

(1985)

found that the formation and stabilization of

type II callus

strongly
induces

inhibited

by high

sucrose

concentration in the culture medium. If DB75

mainly

callus of type I, while DB10 induces

mainly

callus similar to
to

type II, this could explain why DB75 calli rapidly lose the ability
However, this does
not

regenerate.

explain why

calli from

DB10(dark)

did not regenerate

an

better than calli from DB75 factor in addition to low

(Figure 3.3); light

also appears to be
a

important

sucrose

concentrations for

subsequent high

regeneration efficiency

from

crown

slices. It is not clear whether calli from

DB10(light)

retained the

regeneration ability high

up to 12 weeks

or

whether the
to

regeneration efficiency
while still
on

was so

because the shoots

begin

regenerate

the callus induction medium. This should be tested

more

by comparing
to

the compact calli from DB10 and DB75 in

detail and

by trying

regenerate plantlets after longer times

on

induction medium.

What

procedure might
on

be used to obtain

more

shoot

regeneration

from

compact calli induced

DB75? One

approach

would be to test different

combinations of chemical and

physical

stimuli to

improve regeneration.
medium
as

Increasing by

the

sucrose

concentration in the did not

regeneration

described

Sreenath et al.

(1994)

improve

the

regeneration

from DB75 induced

calli. Another

possibility

is to prevent the compact callus from

reaching

the

57

regeneration

block

or

to reduce the

rapid

loss of

regeneration ability.

Tomes

(1985)

has

reported

that

higher

sucrose

concentrations increased the initial

frequency

of response, but inhibited further maintenance of

embryognie

callus

in maize. Vasil et al.

(1984)
the

were

able to

improve subsequent

culture character of
a

istics

by again reducing

sucrose

concentration. The
on

advantages

high

percentage of compact callus induction

DB75 and the

high regeneration by
first

efficiency

after induction
1
-

on

DB10(light) might
on

be combined

inducing

compact calli for

6 weeks

DB75(light)

followed

by

transfer to then followed

DBIO(light)
by
a

to maximize the

subsequent regeneration potential,

medium.

final transfer to

regeneration

Optimized procedures
plantlets

to induce

compact callus and

to

regenerate

The

optimized procedure
on

to

regenerate plantlets is
slices,

to cut the in vitro

plantlets

grown

VRMO in leaf and

crown

to culture the slices for 6 weeks on

DB10 in the slices to which

light

and to regenerate As the

a

plantlets by subsequently transferring

the

D0.1B1(25).

regeneration
effect

medium D0.1B1 contains 1 mg I"1 BA,

on

already

showed

stimulating

the

propagation,

it

was

not

possible

to determine how many

plantlets

had

regenerated prior

on one

slice and

how many had

simply propagated. However, possible

after

induction from
one

on

DB10(light),
To obtain

it
a

was

to obtain up to 100

plantlets

slice.

lot of compact calli, the

on

optimized procedure

is to cut the in vitro

on

plantlets
the

grown

VRMO in

crown

slices and induce compact calli

are

DB75 in

light. Supposing plantlets given

that the compact calli from DB75 the

able to regenerate

more

right stimuli,

this medium should not be

ignored.

Comparison

of different in vitro

regeneration

methods

After

optimization,

our

procedure

to

regenerate plantlets

was

compared

with

several methods described

previously (Mathur

etal. 1989; Mucciarelli etal.

were

1993; Sreenath etal. 1994). Crown and leaf slices

callus induction media

cultured

on

different

(Table 3.1)

at 23C with a
crown

photoperiod

of 12 hours. After transferred to

2, 6

or

8 weeks

on

induction medium,

and leaf slices

were

Table 3.2.

Comparison
of different

procedures
or

for compact callus induction and

plantlet regeneration.

a) percentage
Crown
Medium Ind.
Reg. ->
'

of slices with callus

slices

compact callus

Leaf callus

slices

Time schedule
2

Time

Slices

Callus

Compact
[%]
33 63

Slices

Callus

Compact
[no]
102 99

callus

[weeks]
6 6 6 8 8 8 208 199 198 220
211 214

[no]
95 97
74

[%]

[%]

[%]

DB10
c c

DB75 Mu1 Mai

e

->D0.1B1(25) ->D0.1B1(25)
->Mu2 ->Ma2
-> ->

73 0 93 90 90
1 1 1

17

49 98 98 102 99

22

d
f f

97

94

Sr1 Sr1
Sr2(100)

Sr2(30)

81

84

b) percentage

of slices with

regenerated shoots
Crown
slices

Leaf

slices

Medium Ind. DB10

c c
Reg. ->

'

Time schedule
2

Slices

Shoots

[%] after3 [no]

214 211

Slices
6w 3 220 0 8w
-4

Shoots
12w 17

[%]
16w
-

after 18w
27

[no]
102 0-8-17 99 0
-

6w 15 98
-

8w

12w

16w

18w

DB75 Mul
->

->D0.1B1(25) ->D0.1B1(25)
Mu2 d
e

36 0 198 0
-

73

0-4-12 0
f f

0
-

Ma1

->Ma2
-> ->

98 199 208 0 0
-

0 0
-

0 102 0
-

Sri Sr1
Sr2(100)

Sr2(30)

0 99

Ind.: callus induction medium

2

(see
1

Table

1); Reg.: plantlet regeneration

medium

(see

Table

1)

Method
see w:
-

Figure

weeks
not

4
:

analyzed

59

the different another 8

or

regeneration
12 weeks

media

(Table 3.1),
-

on

which

they

were

cultured for

(Figure

3.1c

f).
from Java, DB10 and DB75 callus
on

Starting

with in vitro vtiver

plantlets

induction media and effective in

subsequent regeneration

D0.1 B1

(25)

were more

regenerating plantlets

than other methods tested

(Table 3.2).
culture

Constabel and

Shyluk (1994)
not

have

already pointed

out that

published

procedures biological

can

always
as

be

reproduced successfully

because too many

source

factors such

genotype, physiological condition of the

material and differences in culture conditions may interfere. In this

study,

a non-

flowering
used
a

vtiver variant from Java

was

used, while Mucciarelli

et al.

(1993)
from

plant

from Somalia, Sreenath et al.

(1994)
a

described

regeneration

inflorescence and Mathur et al. therefore most

(1989)

used

wild cultivar of vtiver and

probably

flowering
we

vtiver variant. Another factor which

might

influence the results is that whereas the

used in vitro based

plantlets plant

as

starting material, plants

previous reports

were

on

material from in vivo

which had to be disinfected Petersen

prior

to the

regeneration experiments.

Holme and callus

(1996) reported
expiants

difference in the formation of

embryognie

between leaf

from in

wrogrown

shoots and from

greenhouse-grown

plants.
It would be DB75/D0.1 B1

interesting

to test the combinations DB10/D0.1 B1

crown

(25)

and

(25)

with in vitro

and leaf slices from different variants of

vtiver,
al.

to determine whether the differences between the results of Mathur et

(1989),
study

Mucciarelli et al.
are

(1993)

and Sreenath et al.

or

(1994)

and those from

this

due to the vtiver variant

to the in vitro

expiants.

CONCLUSIONS

In summary, two

interesting
of

methods

are now a

available for compact callus vtiver variant from

as

induction and Java

regeneration
crown

plantlets

from

non-flowering

by using

and leaf slices from in vitro vtiver

on

plantlets

starting

material. First, the combination of callus induction

DBIO(light)
from

and
crown

regeneration

on

D0.1 B1

(25)

allows

regeneration

of

plantlets

and

60

leaf slices within

short time

(12-18 weeks). Second,

slices.

DB75

efficiently

induces

compact callus

on

many

crown

Although

thus far the

was

regeneration successfully
used

efficiency
to induce

has been low, the compact callus from DB75

liquid

cultures

(Mucciarelli

and

Leupin,

in

press).

The

procedures

for callus induction and

a

subsequent plant regeneration plantlets

which

described in this

study provide

solid basis to regenerate essential oil

or

might

be somaclonal variants with

more

with

an

altered oil

composition.
obtained

If insufficient variation is found, further variations could be

by inducing

mutations with irradiation

a

or

mutagenic

chemicals. Since it

takes 15 to 22 months until vtiver contains vtiver oil

sufficient amount of the

we

complete

(Roth
can

and Kornmann 1997; Weiss be found among the

1997a),

did not yet test whether

oil variants

regenerated plantlets.

ACKNOWLEDGEMENTS

The Vtiver

plants

were use

provided by
of the

Mr. Heini

Lang,

Jakarta. We thank FAL,

was

Reckenholz Zrich for

plant sectioning

facilities. This work

supported by

Givaudan-Roure

Forschung

AG Dbendorf, Switzerland and

no.

by

the Swiss Federal Office for Economic

Policy, project

2561.1 of the

Commission for

Technology

and Innovation.

61

Chapter

4:

Liquid Culture
of Vtiver

Induction and Plantlet

Regeneration

(Vetiveria zizanioides)

Ruth E.

Leupin,

Karl H. Erismann and Bernard Witholt

Partially published
Maffei M UK

in Mucciarelli M and

Leupin

RE

(in press) Biotechnology.

In:

(ed)

The genus Vetiveria. Hardwood Academic Publishers,

Reading,

62

ABSTRACT

To induce

liquid

cultures and
,

subsequently

to

regenerate plantlets of

Vetiveria zizanioides
sucrose

the influence of the callus induction medium and the

(different (different
were

concentrations)

composition

of the
on

liquid liquid

medium

basal media and different

growth regulators)
were

the
a

culture induction

studied. Three types of cultures with loose calli and cultures

obtained:

mucilaginous type, clumps.

cultures

containing compact
looked most

cell

Since the cultures

we

containing compact clumps

promising

to

regenerate plantlets,

tried to increase the number of these cultures. The induction

on

depended mainly

the

starting
of the

callus and therefore

on

the callus induction medium whereas the

changes liquid

liquid

culture medium itself did not increase the induction of

cultures with compact

we were

clumps.

From two of these

a

liquid

cultures with

compact clumps

able to regenerate

few

plantlets.

INTRODUCTION

Vetiveria zizanioides is

tropical
an

grass. A

major

reason

why

this

plant

is of

interest is that the roots contain

essential oil which consists of

more

than 150

sesquiterpenoids (Akhila perfumes, scenting

volatile oils

et al. 1981
as a

).

The vtiver oil is used

as a

component for
of
more

soaps and

fixative to prevent the

evaporation

(Vietmeyer

and Ruskin

1993).
germination
rate of the seed is

Since not all vtiver

it is difficult to
to

plants

flower and the

low,

produce

variants via traditional sexual

breeding.

An alternative is

produce
In

variants in tissue cultures. it

was

previous experiments
on

shown that it is

possible

to

regenerate plant-

lets via callus

solid medium. As the

occurrence

of somaclonal variations and the extent of the culture would also is found in of shoot
a

increases with the duration of the

disorganized phase
from
a

disorganization (Karp 1994), regeneration

be of interest. Vasil and Vasil cell cultures than in

suspension
more

(1986)

found that much and that

variability

regenerated plants,
of selection is

during regeneration
with the result that

meristems,

some

degree

imposed

only

fraction of the

variability present

in the cell cultures is

actually

recovered in the still be induced

regenerated plants.

If the variation rate is too low, mutations

can

63

by

irradiation
are

or

mutagenic
on

chemicals. Due to the fact that with

shaking,

the cell of

clumps

smaller than

solid medium and often fall apart, the

generation

chimera
In this

plants study,

should be reduced.
we as

describe the influence of well

as

starting

callus from different callus

induction media, establishment of from these

the influence of different

on

liquid

media

on

the

liquid

cultures and

subsequent regeneration

of

plantlets

liquid

cultures.

PLANT MATERIAL

As Java

starting
were

material for the in vitro cultures

non-flowering

Vtiver

plants

from

used. The

plants

were

cut in

separate shoots and disinfected with

calcium

hypochloride (Ca(CIO)2).

The disinfected

cuttings

were

put

on

modified

MS medium

(Murashige

and

Skoog 1962) supplemented

with 25 g I"1

sucrose

and 0.65 % agar

(VRMO).

Modified MS medium contains 1/2 MS

macro-

nutrients, 1/2 MS micronutrients, 0.5 mg I"1 thiamine-HCI, 0.5 mg I"1 pyridoxine-

HCI, 0.5 mg I"1 nicotinic acid, 100 mg I"1 myo-inositol, 40 mg I"1 NaFe(lll)EDTA,
200 mg I"1

glycine

and 100 mg I"1 citric acid. For

on

propagation containing

of the

plantlets,
sucrose

they
the

were

cultured

solid modified MS medium

25 g I"1

and

growth regulators 6-benzylaminopurine (1

mg I"1

BA), gibberellic

acid

(0.1

mg

I1)

and

indolebutyric

acid

(0.1

mg

I"1).

CALLUS INDUCTION

To obtain calli for VRMO

were

liquid

culture induction, the in vitro

plantlets

grown

on

(for

establishment and maintenance of in vitro

plantlets

see

chapter 2)

cut in leaf and crown slices and cultured on different callus induction
were

media. The induction media mented with

composed
acetic acid

of modified MS medium

supple

2,4-dichlorophenoxy
0.5 mg I1

(0.5

mg I"1

2,4-D), benzylaminopH
of the

purine (0
medium
were

or

BA),

sucrose or

(1-10 %)

and 0.65 % agar. The

was

adjusted

with KOH

HCl to 5.8 before

autoclaving.
or

The cultures

maintained at 23C either under 12 h

were

daily

illumination

in the dark.

Calli

formed

on crown

and leaf slices within 6 weeks

on

callus induction and the soft

medium. Three types of calli could be

distinguished.

The

gelatinous

64

calli types

were

observed first, after 2

weeks, whereas

third calli type with

-

compact

structures

(Figure 4.1a) appeared only

after about 4

8 weeks in
-

culture. These calli with compact structures

were

subcultured for another 1 cultures.

months, after which they

were

used to induce

liquid

ESTABLISHMENT AND MAINTENANCE OF LIQUID CULTURES

The calli

were

transferred after 3

5 months

on

callus induction medium to

deep

Petri dishes, The cultures

(5

cm

diameter) containing

10 ml of

liquid

medium

(Table
at 70 rpm

4.1).
and

were

kept

at 23C in the dark on a

rotatory shaker

were

subcultured every second week

soon as

by replacing
dense

the old medium with

were

fresh medium. As transferred to 6

the cultures

were

enough, they

cm

culture vessels
15 ml

(Greiner 967161,
liquid
medium.

190 ml, Greiner

GmbH,

Nrtingen, FRG) containing

As
was

the

case

for the callus induction, three different culture types

were

distinguished

in the

liquid

cultures:

mucilaginous

and thick cultures; cultures cell

with loose, soft calli; and cultures

containing compact

clumps (Figure 4.1b,

c).

In

mucilaginous

cultures the medium became viscous and embedded the interested in

loose calli. Since these cultures month of

we were

adequately growing liquid cultures,

remained

were

discarded when

they

mucilaginous
but the calli

after another
were

subculturing.

Other cultures remained

liquid

soft and

failed to separate in smaller form compact cell

pieces.

It took

long

time until cultures started to

clumps.

Once formed, cultures with compact

a

clumps

grew

faster than the other cultures, with

to 2 months

doubling

time of the fresh

weight

of about 1

(data

not

shown).
were

Plant slices with compact calli from various callus induction media
as source were

used

to induce
on

liquid

cultures in different media

(Table 4.1).

When calli
or

induced

callus induction media with 0.5 mg I1 2,4-D and 10, 25, 50

no

75 g I"1 sucrose,

liquid

cultures with compact structures

were

subsequently
induced
on

established
more

(data

not

shown).

Calli with compact structures

were

slices

by adding
sucrose

0.5 mg I1 BA to the callus induction medium and

increasing
for

the

concentration
are

(Chapter 2). Therefore,

changes

more

compact calli

liquid

culture induction

available. The

of the callus induction

medium also affected the induction of

liquid

cultures with compact

clumps.

65

Table 4.1.

Composition
[mg I"1]:

of the

liquid

media

Basal medium

modified AA Macronutrients Micronutrients

AA

modified N6 N6

MS
29 10
1 1

MS
37 0.1 0.5 0.5 100 30'000 10'000 200 113
-

Thiamine-HCI
Nicotinic acid

NaFe(lll)EDTA (Vit B,) Pyridoxine-HCI (Vit B6)

myo-lnositol
Sucrose Sorbitol

100 20'000 25'000 876

L-Glutamine1

L-Asparagine 1 L-Arginine 1 Glycine

L-Proline
1

H20

300
174

7.5
~

500

Liquid
Basal

media

[ mg I"1]:
AAF

mN6

mN6+B

mN60.5D

mN60.5D0.5B

mN60.1D

medium

modified AA
1
-

modified N6
1
-

2,4-D
BA

0.5 5.8

0.5 0.5 5.8

0.1 5.8

0.5 5.8

pH
AA N6 MS
:

5.8
Mllerand Grfe 1978 Chu et al. 1975

5.8

2,4-D
BA
1

Murashige and Skoog 1 962 2,4-dichlorophenoxy ac etic acid 6-benzylaminopurine

km
no

acids

were

filt

sr

sterilized and added to the autoclaved

edium

Compact

calli from callus induction medium

supplemented
sucrose

with 0.5 mg I1 2,4-D,

30 g I"1 / DB10
or

0.5 mg I"1 BA

(DB)
give

and low concentrations of rise to any

(10

or

DB30)

did not

liquid

cultures with compact

on

clumps.

On the other

hand, if plant slices with compact calli, induced

with 0.5 mg I"1 BAand 50, 75 used
were as source or

callus medium DB75

or

supplemented

100 g I"1

sucrose

(DB50,

DB100),

were

to establish

liquid cultures, liquid

It
was

cultures with compact

clumps

produced (Table 4.2).

observed that,

by increasing only
on more

the

sucrose

concentration of the callus induction medium, not calli

were

slices compact

induced, but there

were

also

more

compact

structures per callus. Per

plant slice,

the amount of compact callus thus increased,

compared

to the

amount of soft callus. This

change

of the ratio between compact and soft callus

may

explain

the observed difference in establishment of

liquid

culture with

compact clumps.

66

The difference in the induction of modified AA basal medium with compact

was

liquid

cultures with the modified N6

more

or

the

minor. However, since somewhat

on

cultures

clumps

were

induced

mN6 than

on

AAF,

we

selected the

modified N6 basal medium for further

experiments (Table 4.2).

Table 4.2. Establishment of

liquid

cultures

callus induction medium

1

development
medium
2

of the cultures induction

time

on

liquid

medium

time

cultures

compact
0 0 18
11

appearance of the cultures loose mucilaginous

[months]
DB10 DB30 DB50 DB75 DB100 DB75 DB75 DB75 DB75 DB75
1

[numbers]
mN6 mN6 mN6 mN6 mN6
AAF

[months]
10 10 10 6-14 7-8 8-14 8-14 6 9 9

[%]
20 0 0 13
-3

4 4 4

10 8
11 124 24

80 100 82 76
-3

3-5 8-10 4-5 3-5 3 3 3

13 5 10 0 8 0
acetic

38 62 13 25 25
with 0.5 g 11

11 14

84 76 0
-3 -3

mN6+B mN60.1D mN60.5D mN60.5D0.5B

100
-3 -3

DBx: modified MS medium

supplemented
'

2,4-dichlorophenoxy

acid,

0.5 mg 11

benzylaminopunne

and

g I

sucrose

2: composition of the media 3: no data

see

Table 1

As the effect
on

growth regulator

BA in the callus induction medium had

beneficial
on

the induction of calli with compact structures establishment of

(Chapter 2)
was

and

the

subsequent liquid

liquid cultures,

0.5 mg 11 BA

added to the beneficial

culture medium.

Unfortunately,

BA did not have the

same

effects in
nor

liquid medium;

it influenced neither the induction of compact

clumps
BA

the induction of the other

not further added to the

liquid liquid

culture types

(Table 4.2). Accordingly,

was

culture medium.

In earlier

experiments
more

with callus induction media, calli

higher 2,4-D

concen

trations induced number of the

gelatinous

(Chapter 2). Therefore,

maybe
obtain

to reduce the
more

mucilaginous liquid

cultures and
was

cultures with
or even

compact clumps, the 2,4-D concentration

0.1 mg 11. This resulted in fewer

lowered from 1 to 0.5

to

mucilaginous cultures,
did not increase
no

but the percentage of The addition of

liquid

cultures with compact

clumps

(Table 4.2).
effect
on

0.5 mg 11 2,4-D to the

liquid

culture medium had

the percentage of

cultures with compact

clumps,

but with 0.1 mg 11 2,4-D in the

liquid

culture

medium,

none were

induced and the cultures turned brown. The reduction of

67

the 2,4-D concentration in the medium also had another effect: in root-like structures
were

some

cultures
even

found

(Figure

4.1

d).

Some of these structures

produced something

like side-roots.

Histological growing
in
a

observation showed that these root-like fashion

structures were not roots, but callus

(Figure

4.1

e).

In time

some

of these structures reverted back to callus

more

clumps.

Addition of 0.5

mg 11 BAto the mN60.5D medium resulted in

structures and no

cultures with root-like

compact clumps (Table 4.2).

Liquid culture induction and plantlet regeneration of Vetiveria (a) Compact callus (cc) was used as starting material for liquid 2 mm); (b, c) Liquid cultures with compact clumps after culture induction (bar about 1 year on mN6 (b: bar 5 mm, c: bar 50 urn); (d) Root-like structures from mN60.5D0.5B (bar 5 mm); (e) Section of a root-like structure from 2 mm); (f) Regenerated plantlets from the liquid culture on mN60.5D0.5B (bar mN60.5D (bar 3 mm). Figure
4.1.

zizanioides.

68

PLANT REGENERATION FROM LIQUID CULTURES

For the
were

regeneration experiments compact clumps

from the
or

liquid

cultures

transferred to solid callus induction media DB75

DB10

solid modified

MS medium with 0.5 mg I"1 2,4-D, 0.5 mg I"1 BA and 75

or

10 g I"1

sucrose).

The

resulting ability
tration

calli

were

subsequently

transferred to

regeneration regeneration

media to test the the 2,4-D

was or concen

of the culture to regenerate

was

plantlets.

For

either reduced to 0.1 mg I"1 and the BA concentration

or

increased
1 mg

to 1 mg

I"1 (D0.1 B1 ),

2,4-D

was

omitted and 0.5 mg I"1 BA

(B0.5)

I"1

kinetin and 0.1 mg I"1 indole acetic acid

(VRM8)

were

added. For all three with 25 g I"1

regeneration
sucrose

media the modified MS medium

was

supplemented

and 0.65 % agar.

Some of the

clumps
a

became brown, others remained white and grew

as

fine

granular callus,

few

produced bigger compact

we were on

structures which became

a

green, and from two cultures

4.1

able to regenerate

few

plantlets (Figure by

f).

Both cultures

regenerated

DB75 callus induction medium followed

D0.1 B1

regeneration

medium. The two calli which

regenerated plantlets
from
a

came

from two different cultures. One callus

developed

9 months old mN6

liquid culture,

the other from

were a

6 months old mN60.5D

liquid

culture. At this

stage these cultures

mix between the

no more

original calli,
were

loose calli and from these from the

compact clumps. Afterwards

cultures, thus it is

plantlets

regenerated

not clear whether the

plantlets

were

regenerated
from the

liquid

culture

or

simply represented

carry-over

primordia

original

material. It took 6-14 months before compact structures and

even

developed

in

liquid

cultures

longer
so

until that

enough compact clumps only

a

were

available for

regeneration
a

experiments,

few

regeneration experiments
were more

could be done. At

later stage, when

enough compact clumps

To regenerate

available in the

liquid cultures, liquid

the

they

no

longer regenerated.

plantlets
or

from the

cultures, faster methods

to obtain

liquid

cultures

methods to

prolong

regeneration potential

of

liquid

cultures must be found.

Several factors influence the

production

of

liquid

cultures: the

starting

material, the composition of the liquid medium, the

treatment of the cultures

69

and environmental conditions

(temperature, shaking speed,

cultivation in dark/

light,...).
The it
was

starting

material

seems

to be an
on

important factor,

as

in the

experiments
an or

shown that the calli induced

on or

different callus induction media had

effect

the

liquid

culture induction. The

changes

of the

liquid

media

(mN6

AAF)
0
or

different concentrations of

growth regulators (1
on

or

0.5 mg I"1 2,4-D and

0.5 mg I1

BA)

did not show any effect

the induction of

liquid

cultures with
to

compact clumps. The first step in the improvement of the liquid culture is

improve

the

starting

callus. One

possibility
material

is to

change

the ratio between soft

and compact callus in the and

starting

by tearing

the calli into small is to further

pieces

discarding

any soft callus present. Another

possibility

improve liquid

the callus induction medium to obtain better callus types with which the culture
can

be established faster.
in
our

One

problem

process

was

that the callus

clumps

used for inoculation

remained with and

more or

less intact and did not

split

up. Patnaik etal.

(1997)

were

able

palmarosa

to establish

suspension

cultures with cell aggregates

by sieving

resuspending

the culture in fresh medium. This method also has the

advantage
time. As

that carry-over

primordia

from the

original

material will be removed in

soon as

the

starting

material is

improved

and the subculture method is

optimized

it

might

be worth to make
sucrose or

changes

to the

liquid

medium

composition

(growth regulator,

sorbitol concentrations, additional

compounds,...)

and the environmental conditions.

In this

study

we

showed that the induction of

we

liquid

vtiver cultures

containing
from

compact clumps is possible and

obtained

some

regenerated plantlets liquid

cultures and

these cultures. However, before the establishment of

subsequent plant regeneration provide plantlets

which

useful method to

efficiently regenerate
is

might

be somaclonal variants, additional

optimization

necessary.

70

ACKNOWLEDGEMENTS

The Vtiver

plants

were

provided by Wang

Mr. Heini

Lang,

Jakarta. We thank Dr.

G.

Spangenberg

and Dr. Z.Y.

from the

plant

science group of Prof. Dr. I.

use

Potrykus,

ETH Zrich for advice and FAL, Reckenholz Zrich for

of the

plant sectioning Forschung

Economic Innovation.

facilities. This work

was

supported by by

Givaudan-Roure

AG Dbendorf, Switzerland and

the Swiss Federal Office for

Policy, project

no.

2561.1 of the Commission for

Technology

and

71

Chapter

5:

Comparing analysis
and

methods for

detecting

quantitative

qualitative changes

in the vtiver oil

composition

Ruth E.

Leupin,

Charles Ehret, Karl H. Erismann and Bernard Witholt

72

ABSTRACT

Several methods to

assess

quantitative

and

qualitative changes
were

of

large

numbers of small scale

samples

of vtiver extracts

compared:

via

olfaction, inhibition of microbial growth and analysis by thin layer chromato

graphy (TLC)

and gas

chromatography (GC). By smelling, though

the results
are

it is

possible

to

detect if oil is present, Vtiver oil color

subjective

and

only approximate.

efficiently
of

inhibits

growth

of three

actinomycetes

and reduces red

production high

one

actinomycete,

but the amount necessary to obtain

inhibition is red color

and it is not known which

compound

inhibits the

growth

or

the

production. Analysis by only

TLC

or

GC has the

advantage

that these

methods not

detect whether the

sample

contains oil, but also separate

components and therefore make it possible

contrast to

to detect

qualitative changes.

In

GC, several samples

can

be

analyzed simultaneously by

TLC and
-

non-volatile

compounds

can

be detected. The detection limit for TLC is 5 and the

10

ug vtiver oil, which is

high,

analysis provides

limited resolution with

only

about 15 spots for

more

than 300 components. Nevertheless, it is

possible single
of

to detect

changes

in oil

composition. separation

GC also fails to resolve the oil in

components, but
the oil

as

the

is better, the amount of oil and

more

changes

composition

can

be determined

exactly.

For

GC

analysis only
run.

about 0.5 ug oil is necessary. The

analysis

time is 90 minutes per GC

Since GC

analysis

allows

higher sensitivity
more

and resolution than TLC, but the

in

latter enables

analysis

of far

samples

parallel,

TLC is

preferred

for

preliminary analysis

of many

samples,

whereas GC

analysis provides

more

detailed information for smaller sets of selected

samples.

INTRODUCTION

The

tropical

grass Vetiveria zizanioides

belongs

to the

subfamily

of

Panicoideae, which includes maize, sorghum, sugarcane and lemongrass

(Vietmeyer

and Ruskin

1993).

One

reason

why

vtiver is cultivated, is that the than 300

roots contain an essential

oil, consisting of
which is used

more

sesquiterpenoids (de

Guzman and

Oyen 1999),

as a

component for perfumes, scenting

73

soaps and

as a

fixative to prevent the

evaporation
a

of

more

volatile oils vtiver oil

can

(Vietmeyer

and Ruskin

1993).

Because

completely synthetic
and Ruskin
are

not be manufactured at a realistic

price (Vietmeyer

1993),

vtiver

variants with

more

oil

or

with

different oil

composition

of interest. New

variants could be obtained

by

traditional

breeding (Gupta

et al.

1983; Lai et al.

1998; Sethi 1982; Sethi and Gupta 1980) or, since not all vtiver plants flower
and the

germination
of

rate of the seed is low

(Vietmeyer (George
et al.

and Ruskin

1993) by

regeneration

plantlets

via tissue cultures

and Subramanian 1999;

Keshavachandran and Khader 1997;

Leupin

2000; Mathur et al. 1989;

Mucciarelli tal. 1993; NaNakorn tal. 1998; Sreenath tal. To find oil variants, all

1994).

plantlets regenerated
or

via tissue culture have to be Plants

tested for increased oil content

22 months to

altered oil

composition.

require

15 to

produce

the

complete

vtiver oil

(Roth

and Kornmann 1997;

even

Weiss

1997a). Starting

from in vitro

plantlets,

it will take

longer

before all

regenerated plantlets
soil and grow

can

be screened since
an

they

have first to be established in would be of interest.

larger. Clearly,

earlier

pre-screening

However, without being able

to induce the oil in an earlier

stage, this will

not be

possible. Consequently, regenerated plantlets analyses.

the oil induction

experiments

and the

screening

of all

involve

large

numbers of small scale extractions and

a

For oil induction

experiments,

fast

screening

method to test whether

the oil is induced is needed, whereas for

pre-screening
a

of the

regenerated
method

plantlets

and minimization of the extraction

miniaturized

analysis

which detects

qualitative

and

quantitative changes

with small amounts of

essential oil has to be found. Different methods like gas

chromatography (GC),
and

high
thin oils

pressure

liquid chromatography (HPLC),

were

column

chromatography (CC) analysis

of essential

layer chromatography (TLC)

described for the

(Banthorpe

1991 ; Croteau and Ronald 1983; Harborne 1984b; Kubeczka

1985).
In this

study

we

tested and

compared
and

olfactive detection, bacterial inhibition, differences for sets of small

TLC and GC to detect

quantitative

qualitative

samples.

74

MATERIAL AND METHODS

Distillate,

extract and vtiver oils

To test the different

analysis methods, mainly

vtiver oil Bourbon

(Givaudanwere

Roure),

and distillates and solvent extracts of vtiver roots from Java

used. In addition, different

commercially

available vtiver oils

were

also

analyzed (Table 5.1).

Table 5.1. Different

commercially

available vtiver oils

origin
vtiver oil, Bourbon
1
_

company

Givaudan-Roure,
Dbendorf, Switzerland
PRIMAVERA Life,

vtiver oil

El Salvador

Sulzberg, Germany
vtiver oil, Bourbon West India

ELEXISIS, Graz, Austria

vtiver oil

La Reunion

Seidenberg Collection, Hagenbuch,

Switzerland

vtiver oil

MIGROS,

imp. Germany
Peti's vtiver oil
-

Duftschloss

zum

Wolkenstein, Wolkenstein,

Eschenz, Switzerland
Peti's vtiver

fragrance

Duftschloss

zum

Eschenz, Switzerland
artificial vtiver oil
-

neoLab, Labor Spezialprodukte,

Heidelberg, Germany
1

-,

not indicated

Distillate:

Dry

vtiver roots from Java

was

were

distilled with

phosphate

buffer

(0.5M, pH 8).
ether

The oil

extracted from the distilled water with

methyl ferf-butyl

(MTBE) (see Chapter 6).

Dry
vtiver roots from Java
acetate or
were

Solvent extract:
solvents

extracted
at room

overnight

with

(hexane

MTBE, ethyl

ethanol)

temperature (see

Chapter 6).

75

Inhibition of bacteria

Strains and medium

To test the

inhibitory

effect of vtiver oil Bourbon

aureus

(Givaudan-Roure)
and 3

on

bacteria, Staphylococcus
strains
were

Cowan I grown

(ATCC12598)
LB medium

actinomycete
etal.

used. S.

aureus was

on

(Sambrook

1989)

(1

liter contains 10 g Bacto trypton, 5 g yeast extract, 5 g NaCI and 15 g

agar)

and the three

actinomycetes

were von

cultured

on

M65

Streptomyces

medium

(DSMZ:

Deutsche

Sammlung

Mikroorganismen

und Zellkulturen GmbH,

4 g

Braunschweig, Germany) (1
malt extract, 2 g

liter contains 4 g
or

glucose,

yeast extract, 10 g

CaC03,

with

without 15 g agar,

pH 7.2)

Filter paper disc diffusion method

To test the influence of vtiver oil diffusion method

was

on

bacterial

growth,

filter paper disc

used

(Vincent

and Vincent

1944).
to

The spores of the medium

actinomycetes
the
on

were

transferred from solid beads

plates

liquid

by shaking adhering

plate
the

with

glass

(0

2-3

mm)

and

resuspending

the spores

glass

beads in water. These of S.

aureus were

resuspended
diluted

spores of the

actinomycetes
units

and

pre-cultures
per Petri
LB

(=108, =106, =104 colony forming

on

(cfu)

dish)

and

layered
or

in soft agar

Petri dishes

(5

cm

diameter)

containing

(S. aureus)

M65 medium
or

(actinomycetes).
dimethyl sulphoxide (DMSO)
was

15 ul of the vtiver oil, pure

diluted in

added to filter paper discs

(6.5

mm

diameter), resulting

in total amounts of

around 15, 7.5, 0.8, 0.08, 0.008

or

0 mg vtiver oil per paper disc. Each filter

were

paper

was

transferred to

separate Petri dish. The Petri dishes

-

incubated

at 37C and the inhibition zones were measured after 1

days.

Influence of the vtiver oil concentration

on

the red color

production

of the

actinomycete

3 in

liquid

culture

Spores
1 ml of the

of the

actinomycete

were

transferred to M65
was

liquid

medium. To
were

culture, 10 ul diluted vtiver oil

added and the tubes

76

incubated at 37C in

rotation wheel.
-

After incubation for 2

days

at

37C, the cultures

were

harvested

by

centrifugation (5
538
nm.

min at 14'000

rpm)

and the supernatant

was

measured at OD

Thin

layer chromatography (TLC)

Extracts

were

separated

on

silica

gel plates (Silica gel

60 F254,

MERCK),

using

the solvents hexane, isooctane,

isopropanol, diethyl ether, acetonitrile,

or

methanol, methylene chloride, chloroform, toluene

the solvent combinations

chloroform-methanol, ethyl acetate-hexane, petroleum ether-diethyl ether-acetic

acid. To detect the different

compounds,

UV

(254 nm),

iodine vapor, and different

staining

solutions like

anisaldehyde-acetic
-

acid-sulfuric acid

(2

ml

40 ul

20

ul),

vanillin-sulfuric acid acid and

(1

5 g vanillin per 100 ml sulfuric

acid), phosphomolybdic
were

2,6-dichlorophenol-indophenol (0.1

% in

ethanol)

tested
were

(Cosicia
sprayed

1984; Gibbons and Gray 1998; Merck 1970). To stain, the plates
with
a

staining

solution and, after

drying,

heated until the color spots

were

visible. If not mentioned otherwise, UV, iodine vapor and acid-sulfuric acid As spots often

anisaldehyde-acetic

staining

were

used
was

consecutively
not

to detect the vtiver oil.

overlapped,

it

possible
we

to determine the

midpoint
zones

of

the spot to calculate the Rf value. Therefore,

describe spots with

(Rf*(start)-Rf*(end)).

distance of Rf
=

zone

(start

or

end) from

origin ^

solvent front distance from

origin

1: when hexane and chloroform

front of chloroform
was

were

used

as

consecutive solvents, the

running

used

as

reference.

Due to the different amounts of oil loaded and

slightly

different

running

conditions, Rf* values varied up

to 10 % within the

experiments.

77

Gas

chromatography

A Hewlett Packard 5890A gas

Chromatograph equipped

with

flame
urn

ionization detector and

DB-WAX column
was

(25

m x

0.32 mm, 0.25

film
as

thickness, J & W Scientific, FISONS)

carrier gas at mode.
a

employed. Hydrogen
were

was

used

rate of 2 ml

min1. The samples

were

injected

in the

splitless

Injector

and detector temperatures

oven

maintained at 200C and

was

300C, respectively. The column

temperature

programmed
was

for 80C

(after

min)

to 220C at 2.5C
areas

min1 and the final temperature

were

held for

20 min. Peak

and retention times

measured

by

electronic

integration

(Hewlett

Packard 3390A

integrator).
GC, internal standard
was

Before

injecting

in the
-

added to all

samples

resulting

in around 0.01

0.05 ug

dibutyl phthalate

per

injection.

Fractionating
and

of the

sample (vtiver

oil

or

extract)

in

hydrocarbons,

acids

remaining components (alcohols, ketones,...)

To obtain

hydrocarbons,

hexane extract of vtiver roots

was

loaded

on a

silica column. The eluent contained the

hydrocarbons (Croteau
on

and Ronald

1983; Kubeczka 1985) whereas the rest stayed

The acids
were

the silica column.

separated by extracting

hexane extract with base

(1M
was

KOH) (Kubeczka 1985;

Shibamoto and Nishimura

1982).

The KOH

phase

acidified with concentrated HCl and extracted with hexane.

Scrape-off experiment

A hexane extract, after extraction of the acid

compounds

with 1M KOH,

was

separated by
a

TLC with hexane and chloroform. On both sides of the TLC

plate,

strip

was

cut off and stained. The rest of the zones,

plate

was, with the

help

of these

two

lanes, divided in

starting

at the

origin (sample
fronts

up to

a zone

between the hexane and chloroform each of these

were

running

(sample 34).

The silica of

samples

was

scraped off,

eluted with MTBE, and the eluates

analyzed by

GC and TLC.

78

RESULTS AND DISCUSSION

In this

chapter,

we

describe

experiments

to

optimize analysis
was

methods for

small

samples containing rapid analysis

and

less than 1 mg vtiver oil. This

necessary to

enable

screening

of

large

numbers of small

plant samples,

providing

information about the

quantity

and the

quality

of the vtiver oil.

Description

and

optimization

of

analysis

methods

Olfactive detection

As the vtiver oil is volatile, the

nose can

be used to determine whether

plant

samples

contain vtiver oil. No extraction is necessary and detection of the oil is

a

easy and fast. In

small
was

experiment,

the variation in

sensitivity
was

within

test

panel

of 12 persons

determined. 5 ug vtiver oil

still detected

by

all

test persons.

However, fewer persons detected lower

was

amounts of oil and 0.005

ug vtiver oil
nose are

detected and

by only
on

2 persons

(Table 5.2).
on

Results obtained

by

subjective

depend

the person and

his / her state of health

(Gardner

and Bartelett

1999).
to

For

an

untrained person, it is difficult to determine the

the exact amount

or even

recognize

compounds

of the oil. Therefore, this contains oil. The other

analysis

method is useful

only

to indicate whether a

sample

test is non-destructive and the

sample

can

then be

analyzed by

analysis

methods.

Bacterial inhibition

by

vtiver oil

Vtiver oil has antibacterial and

antifungal properties (Chaumont

and

Bardey

1989; Dikshitand Husain 1984; Gangrade et al. 1991; Gangrade tal. 1990;
Hammer et al. 1999; Maruzzella and Sicurella

1960).

This fact
or

can

be used to

develop

an

analysis

method to

screen

plant

material

extracts for the

presence of vtiver oil.

79

Table 5.2.

Comparison

of different olfactive detection

a

analysis

methods for vtiver oil

inhibition of bacteria
b

TLC

GC

amount per

analysis

0.005
1

5-80c

5-10 -15/

0.5
1

[ug]
simultaneous

many

samples
time needed
~

(10
1 min
+

cm

plate wide)
+

break

before next

days (growth and analysis)

3
-

2h

1.5 h

(run)

(separation and staining)

data

analysis

sample
detection of

compounds:
-

volatile non-volatile

d d

+ +

oil

changes
-

detectable:

quantitative qualitative reproducibility

-

rough rough
a

d d

rough rough
+

+ + +

low

: : :

b
c

test

depends on the testing person organism: actinomycete strain

(Erismann, unpublished)
-

d
e

: :

Filter paper disc diffusion method: 80 ug vtiver oil per filter Inhibition of red color production in liquid cultures: 5 50 ug vtiver oil per ml culture not known which of the oil compounds inhibit the growth or color production several

compounds

at same

spot, therefore small changes

are

not detectable

Filter paper disc diffusion

plate

method

The

inhibitory

effect of different amounts of vtiver oil

was

tested with the

filter paper disc diffusion

plate

method

(Vincent

and Vincent

1944).
etal.

Staphylococcus

aureus, which is

susceptible

to vtiver oil

(Gangrade

1990; Hammer etal. 1999), and three pre-selected actinomycetes (Erismann,

unpublished data) screening

method.

were

chosen to test and to

optimize

the usefulness of this

For S. aureus, 0.8 mg vtiver oil still

was

the lowest amount of vtiver oil which

zone: 0

slightly

inhibited bacterial

growth (inhibition

7.5

mm) (Table 5.3).

day
The

For the

actinomycetes

1 and 2 the inhibition

zone was

measured after 1 after 2

in culture. For

actinomycete
was

3 the

analysis

was

only possible
a

days.

optimal

cell number

around 106 cfu per Petri dish. With

higher inoculum,

80

the filter paper

was

rapidly

overgrown, and with

lower inoculum, the bacteria

did not grow well. We observed that the inhibition

7.5 mg vtiver oil
was zone

around the filter paper

containing
and that

larger

than that of about 15 mg

(non-diluted oil),

DMSO itself did not inhibit the

vtiver oil intensified the inhibited

growth. Apparently,

the addition of DMSO to the

inhibitory

effect. The lowest oil concentration that still

growth

was

about 0.08 mg for all three

actinomycetes (Table 5.3). actinomycetes,

it also

Vtiver oil did not

only

inhibit the

growth

of the tested

influenced the spore formation of vtiver oil For

a

actinomycetes

1 and 2. With 15 and 7.5 mg

spore-less

zone was

observed after 3

days

incubation

(Table 5.3).

actinomycete 3,

vtiver oil also inhibited the

production
red

and excretion of red still observed

color to the medium. After 6 with 0.08 mg vtiver oil

days

in culture

lighter

zone was

(Table 5.3).

Table 5.3. Antibacterial

activity

of vtiver oil and its dilutions tested

by

filter

paper disc diffusion method

0

inhibition

zone

[mm]a
Actinomycete
2

vtiver oil

S.

aureus

Actino mycete 1
1

Actino mycete 2
1

overnight
b

day
13 15.5 13.5 9.5 ni ni

days

day
11

days
10

days
30 34 26 16 ni ni

days
28 28

[mg]
15 7.5 0.8 0.08 0.008
11 11

(13)d
8 ni ni ni

10 7.5

12(14)

16 12.5 9 ni ni

12(13)
7

8 ni

[20] [10]
ni ni

nic
ni ni

ni ni ni

DMSO
a

: :

filter paper 0 6.5 mm 15 pi not-diluted vtiver oil

no

c:
d
e

inhibition
zone zone

: :

( )

with

no

spores

[ ]

with less red color

81

Correlation of red color production

in the

by actinomycete

3 and vtiver oil content

liquid

medium

Since vtiver oil inhibits the red color

production by actinomycete 3, spectrophotometrically.

As the

we

used

liquid

cultures to

measure

this inhibition

analysis

method should also be suitable for solvent extracts of vtiver, the effects of

hexane, ethyl
In

acetate and MTBE on red color formation were also tested.

liquid culture,

DMSO alone inhibited the red color

production.
was

This made it due to

difficult to determine whether the inhibition vtiver oil

or

by

vtiver

samples ethyl

due to DMSO. Since MTBE, hexane and

was no

acetate were not

inhibitory,
After 3

the vtiver oil

dissolved in MTBE for further tests.

days

in culture,

growth

was

observed with 0.5 mg vtiver oil per ml

was

culture. With 0.05 mg little It

growth

was

observed and the medium

light

red.

depended

on

the

experiment,

whether lower concentrations could still be

0.005 mg per ml culture
were

detected: in

some

experiments

detectable,

whereas in others 0.025 mg did not show any

inhibitory

effect

(results

not

shown).

Therefore the detection limit is between 0.005 and 0.05 mg vtiver oil The lack of

(Table 5.2).

reproducibility

could be

explained by

the

difficulty
on

to

inoculate constant numbers of spores and medium the

by

the observation that with time:

some

solid

phenotype

of

some

colonies

changed

remained

dark red whereas others did not. With these red color

experiments,
were

it

was

shown that

growth

of the

actinomycetes

and

production

inhibited

by

vtiver oil. However, the amount of oil

was

needed for inhibition of red color

production

still

high

and it is not known other

which components inhibit the bacteria and whether the

perhaps

compounds

in

plant

extract are inhibitors.

or

Summarizing,

it is difficult to determine the

presence, the amount

bacterial

the

composition

of the vtiver oil from the inhibition of

at least with the tested
or

growth

or

color

production. Therefore,

strains,
of the

this method is not useful to determine vtiver oil.

quantitative

qualitative changes

82

Thin

layer chromatography (TLC)

TLC is

simple

and fast

analysis

method and several

samples

can

be

analyzed simultaneously

on one

TLC

plate.

Staining

As the vtiver oil itself is colorless and detectable

as

only
a

few components

are

by

UV

(vetivones (Andersen 1970)),

staining

method had to be

chosen. Several detection methods for

terpenoids

have been described

(Cosicia 1984;

Croteau and Ronald 1983; Gibbons and

methods
are

Gray 1998;

Merck detect

1970). However, staining

compounds solely
of the

seldom very

specific. They rarely

given

class and often will not detect every

single

compound (VanMiddlesworth
methods like

and Cannell

1998). Therefore,

different detection
or

anisaldehyde-acetic
acid

acid-sulfuric acid, vanillin-sulfuric acid

phosphomolybdic staining
After for

staining

for

terpenoids, 2,6-dichlorophenol-indophenol
and iodine vapor
were

organic acids, UV(254 nm)

tested.

anisaldehyde-acetic

acid-sulfuric acid

staining, pink,

dark and

light

violet, yellow and brown spots appeared. After vanillin-sulfuric acid staining pink
and brown spots
were

visible and

phosphomolybdic

acid

staining

resulted in

green-black spots staining

on a

yellow background (results

not

shown).

The three color range chosen for

methods resulted in similar spot patterns. Due to the

bigger
was

of the spots, the further

use.

anisaldehyde-acetic

acid-sulfuric acid

staining

With

2,6-dichlorophenol-indophenol,

the lowest
not

smear

of the solvent The

extract could be identified as acidic

compounds (result only

a

shown).

detection

by

UV and

by

iodine vapor resulted in

few spots

(Figure 5.1a,
with

b).

Since these two detection methods did not influence the

acid-sulfuric acid and

staining

anisaldehyde-acetic they
acid
were

give

some

additional information, acid-sulfuric

used further in combination with the

anisaldehyde-acetic

staining (Figure 5.1).

83

layer chromatogram of different vtiver oil extracts. Vtiver oil (Givaudan-Roure) (lanes 1 and 5), a distillate of vtiver roots from Java (lane 2), a MTBE extract of the distilled roots (lane 3) and a MTBE extract of vtiver roots from Java (lane 4) were separated on a silica plate with hexane (Hex) and chloroform (Chi) as consecutive solvents. The spots were visualized with UV (254 nm) (a), iodine (b) and stained with anisaldehyde-acetic acidsulfuric acid (c). The Rf* zones of the spots are indicated on the right side of the plates. Figure
5.1. Thin

Bourbon

Influence of the mobile

phase

on

the

separation

TLC separates the

compounds

on

silica

plates according

to their relative
com

polarities (Gibbons pounds ranging

and

Gray 1998).
acids to

As vtiver oil contains about 300 it is not

from

polar

apolar hydrocarbons,

possible

to
one

separate all compounds within

run, a series of mobile

one run.

To obtain maximum information in from

phases, varying

polar

to

apolar,

were

tested.

With

an

apolar

solvent like hexane and the

or

isooctane, the major part of the oil

run

remained at the With

1

origin

hydrocarbons

up

(Croteau

and Ronald

1983).

polar

solvents like methanol,

diethyl ether, ethyl acetate,

was

acetonitrile and

-butanol, the major part of the oil

found in the upper half of the

plate.

The

best

separation

over

the whole

length

of the

plate

was

found with chloroform,

84

methylene

chloride and
was

combination of

hexane-ethyl

acetate

(7:1) (data

not

shown).

There

tailing

of the acid spot with all solvents.

By adding

acetic the

acid to the mobile

phase (petroleum ether-diethyl

ether-acetic acid,

8:2:1),

tailing

of the acid could be avoided

run

(Gibbons

and

Gray 1998).

As

a conse

quence, the acid components

the

with the other oil did

compounds

and disturbed
no

analysis

more

than the

tailing spot

(data

not

shown). Therefore,

acetic acid

was

added to the mobile

was or

phase.

As the

overlapping

of the acid spot

with the darkest oil spot with

smaller after

development

with chloroform than

was

methylene phase

chloride

hexane-ethyl acetate,

chloroform

used

as

the

mobile

for TLC in further used

as

experiments. However,

when hexane and

chloroform

were

consecutive solvents to separate vtiver oil, the

in two

highest spot

could be

separated
was

spots (Rf*: 0.68

0.73, 0.81

0.85). ).

Therefore this combination

used in further

experiments (Figure

5.1

Detection limit

To determine the lowest amount of vtiver oil still detectable

amounts of a vtiver oil Bourbon

by TLC,

different

(Givaudan-Roure) (0.7/1.713.4/
developed
on a

5.1/ 6.8/ 8.5 /

10.2/11.9/13.6/15.3/17 3.4 ug vtiver oil, faint

was

ug)

were

TLC

plate (Figure 5.2).

With

spots

were

still detectable

by

UV. With iodine, 6.8 ug oil

necessary to detect all spots stained with iodine. After

acid-sulfuric acid, the darkest spot

staining
0.13
-

with
was

anisaldehyde-acetic faintly

(Rf*

0.21)

visible with 0.7 ug vtiver oil, whereas all spots

were

were

faintly

visible with 5 Table but To

ug and from about 10 ug on, all spots

clearly

detectable
was

(Figure 5.2,

5.2).
with

From 0.7 to 5 ug the increased spot

intensity

clearly detectable,

higher

amounts the differences were no

an

longer

obvious

(Figure 5.2).

determine the amount of oil in have to be added

on

extract, different concentrations of the control

the

same

TLC

plate

since the color

intensity
a

varied of

between individual

plates.

In conclusion, TLC is useful for

rough analysis

many
much.

samples,

to determine whether oil is

present and approximately how

85

Figure

5.2. Detection limit of vtiver oil

Bourbon

(Givaudan-Roure)

were

by TLC. separated on

Different amounts of vtiver oil

a

silica

plate

with hexane and

chloroform

nm) (a),

as consecutive solvents. The spots were visualized with UV (254 iodine (b) and stained with anisaldehyde-acetic acid-sulfuric acid (c).

Gas

chromatography (GC)

Gas

chromatography (GC)
more

has been the classical tool for

analysis

and

isolation of the lower,

volatile
a

terpenoids (Banthorpe 1991).

GC
run

With the

temperature program used,

complete
not

required
but

90 min per
more

sample

(Table 5.2).

The

separation

was runs

complete, longer

by using

shallow

temperature gradients, the

took

and the

separation

did not

improve.

Changes
obtain
a

in amount and

composition

should nevertheless be detectable. To

reasonable the

chromatogram

which shows minor components without about 0.5 ug vtiver oil should be

overloading

major components,

injected

(Table 5.2).
One

major problem

with GC is that non-volatile

compounds
non-volatile

cause

base line
must

shifting

and increased noise.

Samples containing

compounds

therefore be cleaned

(column chromatography, TLC, etc.) (Croteau

must be carried out between

and Ronald

1983)

or

cleaning

runs

sample injections.

To compare extract

chromatograms,

an

internal standard must be added.

so

The internal standard should not be too volatile,

that it does not evaporate

during

concentration of the

sample,

nor

should it be soluble in water the internal standard

(i.e.,

in the

rest water after

distillation). Additionally,
At the
same

peak

should not

overlap

with

sample peaks.

time, for TLC the internal standard spot

86

should

run

among the oil spots,

so

that it

can

be

scraped methyl

off

together

with the

oil. Of the tested chemicals,

dibutyl phthalate
met these

and

vanillate

(4-hydroxy-3-

methoxy-benzoic acid-methylester)
vanillate gave the
a

conditions, except that methyl

with UV, which disturbed

bright spot

on

the TLC

chromatogram
was

analysis

of the oil. Therefore

a

dibutyl phthalate

generally

used

as

internal standard. If
was

second internal standard

was

needed, methyl vanillate

used

as

well.

GC

chromatograms
A contained

were

subdivided in three segments for

analysis. mainly
acidic

Segment

mainly hydrocarbons, segment

C contained

components and segment B contained the remaining components (alcohols,

ketones,..) (Figure 5.3).

were

To

analyze changes

in

more

detail, segments A and B

subdivided in

peak

groups

(Aa,

Ba

Bf).

Figure

5.3. Subdivision of

(4) was (2) and a hexane extract without acid fraction (3). For the analysis of the data the chromatogram was divided in segment A containing mainly hydrocarbons, segment C containing mainly acids, and segment B containing the remaining components (i.e. alcohols, ketones,...). The segments A and B were subdivided in peak groups (Aa, Ba Bf). IS: internal standard
of vtiver roots fraction
-

gas chromatogram of vtiver oil. A hexane extract fractionated in a hydrocarbon fraction (1), an acid
a

87

Comparison

between TLC and GC

Analysis by
as

TLC and GC has the

advantage
TLC

that these methods fractionate the vtiver oil in several

well

as

detect oil in the test

samples.

separated

spots, while GC resulted in

should be

more

than 150

peaks.
and

With these two methods it

possible

to determine

quantitative

qualitative changes

in the oil

composition.

Comparison

of the

separation

methods

TLC separates the vtiver oil components

according

to their

polarities
to their

(Gibbons
polarities

and

Gray 1998),

whereas GC separates them To obtain

were a

according

and volatilities

(Harborne 1984a).

correlation between

TLC spots and GC

First the vtiver oil

peaks,
was

different fractions

analyzed by

GC and

by

TLC.

fractionated in

hydrocarbons,

acids and

remaining

components (i.e. alcohols, ketones,...) (Figure 5.3). Later,

exact correlation between TLC and

to obtain a more

GC,

hexane extract without acidic off

compounds

was

separated

on a

TLC

plate. Scraped

samples

were

analyzed

with both methods

(Figure 5.4).

Figure 5.4.A Correlation chromatography peaks.

of thin

layer chromatography spots

and gas

88

-J.J.. l<

ij.._

JoU^Ji>-jJ,
Ba
Bb

w
Be Bd Be Bf

Jjyxj^-

ivJ^y*W

^L-*-

LJJ U_J'
10

ft

Jjl___L_n_Ju,

11

12

aJL_
13

Vk.

jt_ilA__

JVA-

JL_
14

L^

1J

Ai

.,

-Lx.

.I

JOc^_ -^

15

n
L_

16

jiM^-j.

17

n
_.L._

19

_^L

Figure 5.4.B. Correlation chromatography peaks.

of thin

layer chromatography spots and gas

89

-L-

TJ*>

^-^j-

A._

*__

^^A.

_iJliJ
;c|
Bd

U
I
Be

Bf

c-

Figure

5.4.B.

(continued)

Figure 5.4. Correlation of thin layer chromatography spots and gas chromato graphy peaks. A hexane extract of vtiver roots without the acid fraction (see Figure 5.3(3)) was separated on a TLC plate and the plate was divided in 34 zones (samples 1 34). The re-eluted samples were analyzed by TLC (panel A) and GC (panel B). Of the GC chromatograms only the samples containing peaks are presented. E: vtiver oil; IS: internal standard
-

90

The

separation

in

hydrocarbons,

acids and

remaining components
Rf* 0.81
-

was were

similar for both methods: the followed acids

hydrocarbons (segment A,

0.85)
and

by

the alcohols, ketones,... Rf* 0

in
-

(segment B,

Rf* 0.13

0.81

finally

the

(segment C,

0.13) (Figure 5.1, 5.3). However,

peak

the subdivision of

the GC

chromatogram

groups did not correlate with the TLC spots

(Figure 5.4).
possible
but the the GC
to

These differences in
some

separation

between GC and TLC made it that have For

a

separate

compounds by TLC, analysis.

different Rf*-value,
in

same

retention time in the GC

some

example,
more

segment Bf of
one

chromatogram,
in

bigger peaks

contained

than

compound

(found
TLC:

samples
off

4 and

12, Figure 5.4 panel B). The

showed spots with the
or

same was

found with

scraped

samples

same

Rf* zone, but

they

did
10-

not contain the same

composition

ratio of

compounds by

GC

(i.e. sample
are

14). Therefore,

we

concluded that either not all vtiver components masked

stained

by anisaldehyde

or some are

by

more

strongly staining compounds.

Comparison

of detection methods

Another difference between TLC and GC detects volatile visualized

was

the detection method. GC all

compounds,

whereas

on

TLC

plates

compounds

which be

are

by UV, iodine, anisaldehyde anisaldehyde-acetic phenols

off

or

other

staining methods,

can

detected. Since

acid-sulfuric acid stains many other

compounds

like sugars,
some

and steroids besides

1

terpenoids (Gibbons
-

and

Gray 1998),
showed
no

scraped

samples (i.e. samples

3)

of TLC spots

GC

peaks (data
-

not

shown).
that could be observed and

A violet

spot (Rf* 0.67

off in two but

0.77),

by TLC,

was

separated peaks

during scraping
in the GC

samples (29

30). Sample

30 showed
can

some

analysis,

sample

29 did not. Therefore, it

be concluded that either not volatile

the
or

compounds giving

rise to the violet color in

sample

29

are

not detectable with the GC method.

Detection of

qualitative changes

The

scrape-off experiment
with either TLC
or

showed that the

separation
so

of the oil is not

a

complete

GC. This is

especially

for TLC, where

few

91

spots

are

detectable while the oil contains

more

than 300 components. To

see

whether these two different

analysis

methods

are

sufficient to detect
as

qualitative changes,
a

commercially

available vtiver oils

well

as a

distillate and

hexane

extract of our roots from Java were

analyzed. plant

Since the vtiver oil

composition

depends

on

the type and

origin

of the

material

(Dethier

et al.

1997),

the

harvesting time,

the treatment of the roots and the distillation conditions differences in oil

(Anonymous 1976),

compositions
observed

were

expected.
The hexane and many
was were

With both methods differences

extract was the

were

(Figure 5.5).

only sample showing

our

an

acid spot

(Rf* 0-0.13)

peaks

in

segment C. In
since

distillate, fewer acids

with
a

were

found. This

expected,
extracted but
no

by distilling

phosphate samples
on

buffer had
a

(pH 8)

fewer acids
in this

(Chapter 6).
were

The other oil

few

peaks

segment,

acid spots

recognizable

TLC.

In the hexane extract and the distillate of roots from Java, the

highest spot

(Rf*

0.81

0.9)

was

not detectable.

Moreover, fever segment A hydrocarbon

The two extracts contained

peaks
the Aa

were

found in the GC

chromatograms.

only

peaks
The

whereas the other vtiver oils contained also other segment A of the
in

peaks.

intensity

hydrocarbon spot

correlated well with the

area

and

the amount of the

peaks

segment A. For example vtiver oil Bourbon

(Givaudan-Roure),
resulted in
a

Bourbon

(Elixisis)

and from El Salvador

(PRIMAVERA Live)
peaks,
whereas
a

fainter TLC spot,

showing
a

fewer segment A GC

the "artificial vtiver oil" resulted in number of segment A

strong TLC spot, containing also

large

peaks (Figure 5.5). running

were

Except

for the "artificial vtiver oil", all oils contained components

on

between Rf* 0.15 and 0.30

TLC. In the "artificial vtiver oil", these spots

were seen

completely

absent and

no

segment B peaks

in the GC
one

chromatogram.
showed

TLC of Peti's vtiver

in in

fragrance

showed

TLC spot and GC

peaks peaks

segment Ba

Be, but the main TLC spots and the correspon

-

ding

GC

segments Bd

Bf

were

absent. Within the Rf*

zone

0.5

0.8,

the TLC pattern of the oils varied, but without this

zone

scraping
it is not

off the TLC spots within

and

analyzing

each of these

by GC,

possible

to find the

correlation between the TLC spot pattern and

changes

within the GC

chromatogram.

92

With both methods it

was

possible
more

to detect

changes

in oil

composition,
of the oil

with

TLC

only roughly

and with GC

exactly.
more

When

changes
a

composition

have to be determined methods

precisely,

combination of different

chromatographic separation (i.e. comprehensive

(Wolf 1996)

or a

high-resolution chromatographic
mass

gas gas

chromatography-tandem

spectrometry

or

chromatography (GCxGC)) (Cazaussus

etal. 1988; Marriott

etal. 2000; Sellier etal.

1991)

of the crude oil will be necessary. However, to

compare miniaturized extraction methods and to pre-screen

regenerated

in vitro

plantlets,

one-step analysis that generates significant information is highly

are

desirable. Therefore, TLC and GC

useful

analysis

methods for this purpose.

Figure

5.5.A

Figure 5.5. Thin layer chromatography and gas chromatography of commercially available vtiver oils. Different commercially available vtiver oils (samples 3 -10) as well as a distillate (samples 2 and 11 ) and a hexane extract (sample 1) of our roots from Java were separated by TLC (panel A) and GC (panel B). Panel A: TLC was carried out on a silica plate with hexane (hex) and chloroform (chl) as consecutive solvents. The spots were visualized with UV (254 nm) (a), iodine (b) and stained with anisaldehyde-acetic acid-sulfuric acid (c). Panel B: GC analysis. IS: internal standard sample 1 : hexane extract of roots from Java sample 2: distillate of roots from Java sample 3: vtiver oil from La Reunion (Seidenberg Collection) sample 4: vtiver oil Bourbon (Givaudan-Roure) sample 5: vtiver oil Bourbon from West India (ELEXISIS) sample 6: Peti's vtiver fragrance (Duftschloss zum Wolkenstein) sample 7: Peti's vtiver oil (Duftschloss zum Wolkenstein) sample 8: vtiver oil from El Salvador (PRIMAVERA Life)

93

u
Bb
Be

lAw
Be

Bd

Bf

-A-

-B-

I
iU'
u

louJW
UM

yw li

J^1
xjM U1/ \mJILaaAa
iJL.

UJ

(/UuJ

_L<>

jjUkkj-lL*- juliwJ1

h .^a^JJ

.AH

iLjj.Lu)J iJWJUL

WW

J M'JP/.P
ui
.

J-

LjUJUL^JUJllw

MiJJUWl^

---^jJ-UkA.

u L*

U
All'

|Uo~

U^uJ^w

ta^

JuILjl.-

Ai
ilJWVtJ

J^

IJ4JJlLy.ut
Wu

jLJ*--

w
10

JlullPljjl

_J_-~ w_^

V.J

J^aJJWLAJuJL A-^/l^AJ~^AJ^\^^LU^iiJ^J

'"

Figure

5.5. B.

Figure 5.5. (continued) sample 9: vtiver oil (MIGROS) sample 10: "artificial vtiver oil" (neoLab, sample 11 : only on TLC plates: 1.5 times

Labor

the amount of

Spezialprodukte) sample 2

loaded

94

Artifact

production

During preparation
oxygen sensitive
reason

for TLC the vtiver oil components could therefore be

are

exposed

to air and

compounds scraped
off

decomposed.

This could be the

on

why

some

samples

showed additional spots i.e.

TLC and
23
-

additional

peaks

in the GC

chromatograms (Figure 5.4,

samples

34).

Moreover, during the evaporation of the solvent, volatile hydrocarbons may be

lost

(Kubeczka 1985).
the hot

Similarly,

injection port

of the GC may introduce several artifacts like

isomerization, dehydration and polymerization (Croteau and Ronald 1983).

Such artifacts
can

influence the identification of the separate

compounds.

Comparison

of the different

analysis

methods

To

optimize

the extraction of the vtiver roots, TLC is useful for

an

initial

analysis

to detect non-volatile

compounds

and to

roughly

determine the amount

of oil in individual For the

more

samples,

to estimate the amount of

sample

necessary for GC.

exact

analysis,

GC should be used.
the
nose

For the oil induction first

experiments,
but for
or

provides

very useful and

rapid

analysis (Table 5.2), compounds,

yield

determination and identification of the

induced

TLC

GC should be used. Also here, TLC is useful to

and to estimate the amount and the compo
as a

detect non-volatile

compounds

sition of the oil, but GC is necessary

final and definitive method.

a

Finally

for the

pre-screening

of the

plantlets only

small amount of oil is

available. GC is best suited for this purpose.

ACKNOWLEDGEMENTS

The Vtiver roots

were

provided by

Mr. Heini

Lang,

Jakarta. This work

was

supported by

Givaudan-Roure

Forschung

AG Dbendorf, Switzerland and

no.

by

the Swiss Federal Office for Economic

Policy, project

2561.1 of the

Commission for

Technology

and Innovation.

Chapter

6:

Comparison

of Vtiver oil extraction

by

water distillation and solvent extraction

Ruth E.

Leupin,

Charles Ehret, Karl H. Erismann and Bernard Witholt

96

ABSTRACT

The essential oil of Vetiveria zizanioides and solvent extraction, to

extracts.

was

extracted

by

water distillation

optimize

the methods and to compare the

resulting

With water distillation,

only

volatile

compounds

are a

extracted from the roots, few

however due to the necessary distilled

cooling system, only

samples

can

be

simultaneously
buffer at

and the process is time

8 and

consuming. By using

0.5 M

phosphate

pH

by rinsing dry

the cooler with solvent, the time

necessary for the distillation of 6 g

roots was reduced from an initial 3

days

(3

litre distilled

water)

to about 5 hours
room

(100

300 ml distilled

water).
can

With solvent extraction at

in

temperature, many samples

are

be extracted

parallel. However,

non-volatile

compounds

also extracted from the root

material.

Analysis by
components

gas

chromatography

showed that similar amounts of the volatile

were

extracted with hexane,

methyl ferf-butyl ether, ethyl compounds Although

and
more

acetate

and ethanol. Hexane extracted less of the non-volatile other solvents and
was

than did the the hexane acidic and

selected

as

preferred

extractant.

extract contained less of the alcohols and

hydrocarbons

non-volatile

compounds

than the

phosphate

buffer distillate, the gas chromatowere

grams of the distillate and the hexane extract

concluded that hexane extraction of small
an

comparable. Thus,

we

plant

root or tissue

samples permits

adequate

initial

analysis

of the oil

composition.

INTRODUCTION

Vtiver oil is

an

essential oil from the roots of the

tropical

grass Vetiveria

zizanioides. The vtiver extracted from the roots

plant by

is harvested after 15-22 months and the oil is

steam distillation

(Roth
yield

and Kornmann 1997; Weiss varies between 0.1-3.3 % oil

1997a). Depending (Akhila

more

on

the vtiver variant, the

et al. 1981 ;

Anonymous 1976;
and

Roth and Kornmann

1997).

It contains

than 300

bicyclic

tricyclic sesquiterpenoids (hydrocarbons, alcohols,

97

ketones, esters, aldehydes and carboxylic acids) (Akhila etal. 1981

Guzman and

; de

Oyen 1999;

Roth and Kornmann 1997;

Vietmeyer

and Ruskin

1993).
Several methods have been described to extract essential oils from

plant

material, including

steam or water

distillation, solvent extraction, enfleurage,

expression, supercritical

carbon dioxide extraction and microwave extraction

(Craveiro
tion of the

etal. 1989; Roth and Kornmann 1997; Weiss

1997b).

The

composi

resulting

oils

can

vary

significantly (Boutekedjiret

etal. 1997; Pino et the

In

al. 1996; Scheffer 1996; Simndi etal. and the

use

1999). Depending
are

on

plant

material

of the oil, different extraction methods

in

use.

industry,

vtiver oil is extraction

produced by

steam

distillation, although other methods like solvent

Naves 1974; Weiss

(Anonymous 1976; Hegnauer 1986;

1997a)

and

supercritical

fluid extraction have also been used

(Blatt

and Ciola 1991 ; Weiss

1997a).
As the vtiver oil is with
a a

valuable

raw

material in

perfumery,

new

vtiver variants ratio of the

higher

essential oil
are

yield

or a

different odor

tonality (another

different

components)

of interest. New variants have been obtained

by

traditional

breeding (Gupta

etal. 1983; Lai etal. 1998; Sethi 1982; Sethi and

Gupta 1980).
attempts
culture
to

Since several vtiver cultivarsdo not flower, there have also been
somaclonal variants

produce

by regenerating plantlets

via tissue

(Chapter 3) (George
Leupin

and Subramanian 1999; Keshavachandran and

Khader 1997;

etal. 2000; Mathur etal. 1989; Mucciarelli etal. 1993;

NaNakorn etal. 1998; Sreenath etal.

1994).
plantlets
contain the

It takes at least 15-22 months until such vtiver oil, and it would therefore be

complete

advantageous

to assay

regenerated producers

plantlets

earlier for

changes

in oil

composition

to eliminate poor oil

and thus reduce the space necessary to cultivate

only

the most
a

promising

plants. Therefore, samples

In this

suitable method to extract oil from

large

number of small

has to be

developed. optimized
to reduce the distillation
room

chapter,

water distillation was

were

time,

different solvents the

compared

for extraction at

temperature and finally,

were

optimized

distillation and solvent extraction methods

compared.

98

MATERIAL AND METHODS

Plant material

The

dry

vtiver roots

were

provided by
were

Mr. Heini
mm

Lang,

Jakarta. For the Fresh roots from

were

extraction

experiments,

the roots

cut in 5

pieces.

harvested from the Vetiveria zizanioides from Java. The

were

plants

our

stock

grown outside

during

summer

and in

greenhouse

at 15C in winter.

Distillation

Dry

or

fresh vtiver roots

(5

g)

were

extracted

by

water distillation in an oil

bath at 140C

(Figure

6.1

).

To examine the influence of the

pH

on

the

distillation,
different

water

(H20)

and

phosphate

buffer

(P-buffer,

0.05 M and 0.5

M)

at

pH (4.6, 7, 8, 9)

were

used to distill the essential oil. After every

fraction of 100 ml distilled water, another 100 ml

to the roots.

pre-warmed

water was added

Depending

on

the

experiment,

up to 30 fractions of 100 ml distilled

water were collected. Since the distillation was

stopped overnight, longer day.

distillation

experiments

were

divided in batches of at most 10 fraction per

or

The 100 ml fractions and the residual water

P-buffer, which remained after distillation,

10 ml
were

extracted with ether

methyl ferf-butyl
1
-

cooling

water

(MTBE) (sample R).

x,

sample

To extract all acids, the

or

residual water

buffer

was

acidified with concentrated HCl and extracted

a

second time

with 10 ml MTBE

(sample

acid).

The distilled roots

were

extracted
MTBE at

overnight
room

in 50 ml

temperature
All

Figure 6.1. Distillation apparatus this study to distill vtiver roots.

used in

(sample roots).
were

samples
GC.

analyzed by

99

Solvent extraction

5 g vtiver roots,

pre-wetted

with 10 ml water

or

dry,

were

extracted with 50 The

ml of different solvents
were

(hexane, ethyl acetate, MTBE, ethanol).

samples

stirred

overnight

at room
was

temperature. After removing the solvent (extract

1),

new

solvent

(50 ml)

added. After another 24 hours This

was

stirring,
twice

the extract

was

again

removed

(extract 2).

repeated

once or

more

(extract

3, extract 4).

Comparison

of distillation and solvent extraction

Hexane extract: 10 g
3 times
extract

dry

roots

pre-wetted

with 20 ml water

were

extracted

overnight
1, 2, 3).

at room

temperature with 100 ml hexane each time (hexane

Distillation:
extract

Dry

vtiver roots

(5 g),

hexane extracts
were

(50

ml of each hexane

1, 2 and 3) and the extracted roots

distilled in 0.5 M P-buffer

(pH

8).

The hexane from the hexane extract

was

distilled off first

was

(sample hexane).
stopped
and the

After every 100 ml distilled water fraction, the distillation cooler

was

rinsed twice with 10 ml MTBE

(sample

/1

b).

The 100 ml distilled

water fraction was extracted three times with 10 ml MTBE

(sample 1,

', 1 ").
was

After three fractions of 100 ml distilled water

(sample 1,2,3),

the distillation

stopped.

All

samples

were

analyzed by

GC.

Gas

chromatography

A Hewlett Packard 5890A gas

Chromatograph equipped

with

flame
urn

ionization detector

(FID)

and

DB-WAX column
was

(25

m x

0.32 mm, 0.25

a

film

thickness)

was

employed. Hydrogen
were

used

as

carrier gas at

rate of 2 ml

min1. The samples

injected

in the

splitless

mode.

Injector

and detector The column

temperature
oven

were

maintained at 200C and 300C,

was

respectively.
4

temperature

programmed
was

for 80C

(after

min)

to 220C at 2.5C
areas

min1 and the final temperature

times
were

held for 20 min. Peak

and retention

measured

by

electronic

integration (Hewlett

Packard 3390A

integrator).

100

Before all

injecting

in the

GC, internal standard (dibutyl phthalate)

was

added to

samples.

To compare the
was

composition

of the extracts, the GC

chromatogram

(Figure 6.2)

divided in three segments

(A, B, C). Segment

mainly
the acidic

A contained

mainly hydrocarbons, segment

C contained

compounds

and

segment B contained residual compounds (alcohols, ketones,...) (Chapter 5).

To detect the effects of the extraction methods
on

the oil Ba
-

composition

in

more

detail, the segments A and B

were

subdivided

(Aa,

Bf; Figure 6.2). In the

within segment X

following text,
of the GC

compounds

refer to the

compounds running

chromatogram.

Figure 6.2. Gas chromatogram of vtiver oil on a DB-Wax column. For the analysis of the data the chromatogram was divided in segments A, B and C. Segment A contains mainly hydrocarbons, segment B contains mainly alcohols, aldehydes and ketones and segment C contains mostly acids. The segments A Bf. IS: internal standard (dibutyl and B were subdivided in Aa and Ba phthalate)
-

Estimation of the amount of oil based

on

GC

analysis

To determine the amount of any

compound

Z from

GC

chromatogram,

the

response factor of that

compound

Z and the internal standard

(IS)

has to be

determined. The FID response is

atoms

roughly proportional

to the number of carbon

present in the compounds, however the response is also affected by

hetero atoms and various functional groups each

(Flanagan 1993). Therefore,

for

compound

the response factor

can

be different and has to be determined

separately.

response factor

peak

area

amount

101

Using

this response factor, it is

Z from
a

possible

to estimate an unknown amount of

compound

GC

chromatogram.

amount of

compound

[ug]

peakareaZ
peak
area

response factor IS
response factor Z

IS

As many vtiver oil components

are

not isolated and the structure not

elucidated, it is

not

possible

to determine the response factor for each

are

single

component. Since vtiver oil components

expected

to be all

sesquiter-

penoids, they
are

should have C15 and

exceptionally

C14. The main differences

the number and the

we assume

position

of the functional groups. To

simplify

the

calculation,
as

that all vtiver oil components have the This results in:

same

response

dibutyl phthatate (C16H2204).

amount of compound

_,

ug

peakareaZ
=

peak area IS

amount IS

The amount of oil in each segment X standard

was

estimated relative to 1 ul internal

containing

0.025 ug

dibutyl phthalate by summing

up all

approximate

amounts of the

compounds

within segment X.

material in

segment X[ug]

X peak areas
=

in

segment X

; peak area IS

0.025

uq

Ma

It has to be noted that the calculated amount is of oil, based

on

only

an

approximate
on

amount

the

assumption

described above.

Depending
was

the commercial

vtiver oil tested the amount used

(see chapter 5), (results

not

the calculated amount

up to 1.5 times of

shown).

Dry weight

of the extracts

The solvent of the solvent extracts and of the distilled

samples

was

removed
were

by

distillation and for several

by evaporation
3
-

with

nitrogen.
a

The

glass

vials with extract

kept
or

days (typically dry weight

7)

in

desiccator to

remove

traces of solvent

water before the

was

determined.

102

RESULTS AND DISCUSSION

In this

study,

we

describe

experiments

to

optimize

the oil extraction

by

water

distillation and
extracts

by

solvent extraction. Water distillation has the

advantage

that it

only

volatile
a

compounds,
can

but it is time

consuming

and laborious

(Weiss

1997a): only

few

samples

be distilled

simultaneously

due to the limitation

of the necessary useful the

cooling system. cooling

can

Solvent extraction at

room

temperature is
can

option,

as no

is necessary and many be

samples

be extracted at

same

time. Extracts

injected directly

in the GC for

analysis (Chapter
volatile
com

5).

One

problem

of solvent extraction however is that not

only

pounds

but also non-volatile

compounds
GC

are

extracted, which may give rise

to

problems during subsequent

analysis (Scheffer 1996).

Distillation

Comparison

of water and

phosphate

buffer distillation

Essential oils

are

mainly

extracted

by

steam distillation or water distillation.

We therefore tested water distillation

(H20 distillation)
to 4.6

for three

days. During

the

distillation, the pH of the

water

dropped

(Figure 6.3a). Banthorpe (1991 )

plant
vacuoles

explained during

this

drop

in

pH by drop

liberation of the acid material from of

distillation. This

pH

can, in combination with

high temperature, terpenoids

or

lead to artifacts such other modifications

as

elimination of water, rearrangements of As

a

(Banthorpe 1991).

remedy, Banthorpe (1991) proposed

to conduct steam distillations in the presence of a near neutral buffer. We tested

the influence of the

pH

on

the distillation of

samples containing phosphate

Figure 6.3. Water distillation of vtiver roots with water or phosphate buffer (0.05M) at different pH. Dry vtiver roots were distilled with water (H20, X) or 0.05M phosphate buffer (P0.05) at pH 4.6 (O), 7 (O) and 9 (A) over three days (samples 1 -10/11 -20/21 30). To observe pH changes, the pH was measured every day once (a). All samples were analyzed by GC. The yield of material A, B and C was followed during the distillation (b, c, d). Additionally, the dry weights of the samples were determined (e). app: cooler rinsed with MTBE; R: remaining water or buffer extracted with MTBE; R acid: remaining water or buffer acidified before extraction with MTBE;
-

roots: distilled roots extracted with MTBE

ecu

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0"6 0"8 0"Z -6 0"9 0"9 0mP

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en

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Q-3D-0

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104

buffer

(P-buffer)

at

pH 7.0,

8.0 and 9.0. To exclude that observed

changes
as

are

due to the P-buffer and not to the the first effect

pH,

P-buffer at

pH

4.6

was

tested

well. In

experiments,
not

0.05 M P-buffer

(P0.05)

was

used, but the buffering

was

strong enough and the pH dropped during the distillation (Figure

6.3a).

For later

experiments
a more

0.5 M P-buffer

(P0.5)

was

used, which

was

sufficient to maintain We found

no

constant

pH (Figure 6.4a). compounds only

than for the

differences in extraction behavior for B and C

after distillation with material A:

H20

or

P0.05/pH4.6.

Differences
more

were

found

H20

distillation resulted in

slightly

hydrocarbons

P0.05/pH4.6

distillation

(Figure 6.3).
are

We concluded therefore that observed

differences in material B and C buffer. As

due to the

pH

and not to the

phosphate

expected

for

distillation with
were

pH higher

than

or

equal

to

7, fewer acidic
were

compounds (segment C)
extracted faster than with
A

distilled.
or

Additionally,

the B

compounds
The

H20

P0.05/pH4.6 (Figure 6.3).

the

composition

of

compounds

was or

influenced

by

pH

of the distillation, since distillations with

pH

lower than 7

pH

9 resulted in additional
not

peaks

which

were

not

present in

the other distillates

(data

shown).
the

These additional distillation time,

peaks
or

could be due to
a

artifacts, caused by the pH

extraction of
some

or

long

from

selective

hydrocarbons.
or

Since after 6
material B had buffer
were

(P0.05/pH9)
already

10

(P0.05/pH7)

fractions

more

than 95 % of the

been extracted,

subsequent

distillations with 0.5 M P-

reduced to 10 fractions

(Figure 6.4).

Due to the 0.5 M P-buffer the

pH during

the

P0.5/pH7

distillation remained constant and the distillation

as

showed the

same

behavior

with P-buffers at

pH

or

(Figure 6.4).

phosphate buffer (0.5M) at pH. Dry phosphate buffer (0.5M, P0.5) at pH 7 (O), 8 (D: 10 samples, : 5 samples) and 9 (A, A) (samples 1 10). The pH of the buffer was measured before and after the distillation (a). All samples were analyzed by GC. The yield of material A, B and C was followed during the distillation (b, c, d). Additionally, the dry weights of the samples were determined (e). app: cooler rinsed with MTBE; R: remaining water or buffer extracted with MTBE; R acid: remaining water or buffer acidified before extraction with MTBE; Figure
6.4. Water distillation of vtiver roots with

different

vtiver roots

were

distilled with

roots: distilled roots extracted with MTBE

material C

material B material A

CO"

dry weight [pg mg-1dry roots] [pg mg"1dry roots] [pg mg1 dry roots]
co
en en o o o o o o o

co co

[pg mg1 dry roots]

'-L '-L

pH
^
ai ai

CD
ocnocnocnocn

k>

k>

si

id

iQ)

I
CO

iT3

CD
CO

2--

CD

> 4--

5--

7'

O Ol

8'

9'

10'

app'

R'

R acid

roots

106

We tested in
on

more

detail whether the

pH

of the distillation buffer has

an

effect
-

the
to

composition

of the oil

(Table
as

6.1

).

Some variations in the fractions Ba

were were

Bf

(up

0.05)

were

found, but

such variations

also observed for the

most

repetitions
the

of

P0.5/pH8

and 9 distillations,
errors

they

probably by
the

caused of the

by

plant

material and

integration

of the GC and not

pH

P-buffer.

Table 6.1. Oil

composition

of different distillates,

as

determined

by

GC

Ratio of material X normalized to material B

H20
segmenta
A

0.05 M P-buffer

0.5 M P-buffer

pH4.6
0.051 0.013 1.000 0.048 0.218 0.064 0.193 0.142 0.335 1.805 0.039 0.010 1.000 0.048 0.223 0.055 0.187 0.146 0.336 1.975

pH7
0.033 0.012 1.000 0.033 0.247 0.064 0.178 0.143 0.305
1.177

pH9
0.024 0.009 1.000 0.024 0.245 0.091 0.183 0.134 0.285 0.339

pH7
0.021 0.008 1.000 0.058 0.264 0.072 0.183 0.130 0.294 0.359

pH8c
0.014 0.005 1.000 0.060 0.248 0.104 0.182 0.129 0.277 0.093 0.015 0.006 1.000 0.056 0.268 0.074 0.185 0.118 0.299 0.065

pH9c
0.014 0.006 1.000 0.052 0.222 0.115 0.177 0.136 0.301 0.073 0.020 0.006 1.000 0.054 0.269 0.071 0.178 0.139 0.289 0.085

Aa
B

Ba Bb Be Bd Be
Bf

a: Division of the segments

see

figure

6.2

b:

To

directly

were

compare the oil composition of the different distillates the material normalized to the material B

A, B, C, Aa and Ba

to Bf

c: two repetitions

Distillation with

H20, P0.05/pH4.6

and

P0.05/pH7

extracted between 12.3 and the P0.5

and 14.9 mg vtiver oil per gram roots, whereas distillations This

P0.05/pH9

yielded

at most 6.3 mg vtiver oil per gram roots

(Figure 6.3e, 6.4e). (Figure 6.3d,

nicely

reflects the observed differences in the material C

6.4d).

The total

dry weight including

the distilled about 20

samples,
-

the extract of the

residual water and the root extract distillations vtiver oils

was

23 mg per gram roots for all

(Figure 6.3e, 6.4e). By comparing

we

different

commercially

available We

found that

they they

contain few acidic components

are

(Chapter 5).
vtiver odor.

concluded therefore that

not

important
a

for the

typical
was

Accordingly,

buffered distillation with

or

pH higher

than 7

chosen for further

separations. P0.5/pH8
the A, B and C
too

P0.5/pH9

showed the

same

distillation behavior for

compounds.
chose

To avoid

changes

of the oil

composition

due to

high pH,

we

pH8

for further distillations. Since after 5 distillation

107

fractions

more

than 95 % of the total material B

was

was

already

distilled from the

roots, the distillation of dry roots

reduced to 5 fractions

(Figure 6.5).

Comparison

between

dry

and fresh root distillation

Since for the

we

analyzes

of the oil content, fresh material could also be used,

a

compared

the distillation of fresh and dried roots. Fresh roots from

were

vtiver and

plant

from Java
was

harvested. One part

was

distilled

directly (fresh roots)

the other

dried for 7

days

at room

temperature (dried roots). For these

freshly

harvested roots, 10 fractions

were

again

collected. The distillation of the than the

fresh harvested roots with

(fresh

or

dried)

took

longer

previous

distillations

dry

roots: the distillation of 100 ml water took about 1.5 to 2 hours for each

of the first 5 fractions for the fresh harvested roots, whereas for the

dry

roots

only

the distillation of the first 100 ml needed up to 2 hours, the

following

100 ml

fractions

requiring only

about 0.5 to 1 hour. Therefore, the 10 fractions of the distributed

over

fresh root distillation

not

were

two

days,

with 5

samples

each. It did

only

take

longer

to distill 100 ml water, the oil was also extracted more

slowly

from the fresh roots

(dried

and

fresh)

than from the

dry

roots

(Figure 6.4,

6.5).
Since

only

minor amounts of oil

were

obtained

by rinsing

the cooler after

distillation of
roots.

dry roots,

the cooler

was

initially

also not rinsed after

some

distilling

fresh

However, after distillation of the fresh roots,

oil remained in the

cooler, since it smelled strongly like vtiver oil. Therefore, for the experiment
with the dried fresh roots, the cooler distillation
was

again

rinsed with 10 ml MTBE after

(sample app), yielding

adhering

about 33 % of the total extracted oil

(Figure

6.5).

As the oil

to the cooler was lost from the fresh root

distillation, the

dried root and the fresh root distillations could not be

compared.

108

a) segment
0.750t

Comparison between water distillation of fresh and dried fresh vtiver roots. Fresh () and dried fresh vtiver roots (A) were distilled with phosphate buffer (0.5M, pH8) over two days (samples 1 -5/6-10). All samples were analyzed by GC. The yield of material A, B and C were followed during the distillation (a, b, c). app: cooler rinsed with MTBE; R: remaining water or buffer extracted with MTBE; R acid: remaining water or buffer acidified before extraction with MTBE;
Figure
6.5.
roots: distilled roots extracted with MTBE

109

Influence of

glass

surface adhesion of vtiver oil

on

distillation

From the observation with the dried root distillation, distilled oil remained in the water fraction and
a

we

concluded that not all

to the cooler unit.

part adhered

To determine how much oil remained in the 100 ml distilled water and how much adhered to the cooler, the distillation
100 ml water fraction and the cooler
was was

stopped

after removal of every

rinsed with 10 ml MTBE

(sample xa).

For the first two fractions of 100 ml distilled water, up to 60 % of the material A,

up to 40 % of the material B and up to 20 % of the material C adhered to the

cooler. With the

subsequent

three distillation steps, less than 10 % of the oil

distilled in these fractions adhered to the cooler careful

(Figure 6.6).

An additional
a

rinsing

of the cooler after the five distillation steps resulted in

recovery

of about 4 % of the total distilled oil

(sample 5b, Figure 6.6).

distilled in the first few distillation steps,

Since the

major part

of the oil

was

further distillations

were

reduced to 3 fractions of 100 ml distilled water. To

extract the maximal amount of distilled

oil, the cooler

was

rinsed twice with extracted 3 times

10 ml MTBE

(samples

xa

and

xb)

and the distilled water

was

with 10 ml MTBE

(samples

x, x' and
were

x").
found in the first extraction of the distilled

Most of the oil components

water and in the first

rinsing

of the cooler. However, the

following

two water

extraction steps and the second

rinsing

of the cooler still contained sufficient oil

(Figure 6.8)

to warrant their collection.

Reduction of distillation steps

For small amounts of roots, it is

important

to extract the material in as little

liquid
GC
was

as

possible,

else the concentration will be below the detection limit of the

-

(Chapter 5).
similar

The ratio of the material Ba

not

Bf in the three distillation

steps

(data
the oil

shown).

A reduction in distillation

steps would therefore steps

to 2 or 1

not

change

composition. By reducing
or

3 distillation 80
-

steps,
2/3
or

the oil content would be reduced to 94 %

1/3 of the solvent

90 %

respectively

in

only

(Figure 6.8).

110

a) segment
0.30t

<

uzjfr^
en

E
en

HIIIII
CO
un un

-a o

o o

CO

a:

b) segment
7.50
-i

r^n?^>-oa-^=S=SF=

c) segment C
10.00 8.75

o o
>-

7.50 6.25 5.00 3.75 2.50 1.25 0

"5

's?
E

b
E
en

q=q=rpqqq[^999'
1-

(0

CM

ffl l\]

CO m

^t

CO ^r

un

CO
un

_Q un

ce

"a 0

o o

a:

rinsing of the glass material on the amount of vtiver oil by water distillation. Dry vtiver roots were distilled with phosphate buffer (0.5M, pH8) (samples 1 5). To obtain all oil distilled within 100 ml water, the cooler was additionally rinsed with MTBE (sample xa). All samples were analyzed by GC. The yield of material A, B and C was followed during the distillation (a, b, c). The distillation was performed twice (D, ). 5b: the cooler was rinsed a second time with MTBE; R: remaining water or buffer extracted with MTBE; R acid: remaining water or buffer acidified before Figure
extracted
-

6.6. Influence of

extraction with MTBE; roots: distilled roots extracted with MTBE

111

Solvent extraction

Usually,

benzene is used to extract vtiver roots

(Anonymous 1976;

de

Guzman and
this solvent

Oyen 1999;

Weiss

1997a),

but for

ecological

and health reasons,

was

not tested. To find the best solvent to extract the

complete

vtiver oil from the roots, solvents with different

polarity

were

tested

(hexane,

MTBE, ethyl

acetate and

ethanol).

Since

we

had observed that the

separation pre-wetted
the of

of the solvent and roots after the extraction is easier if the roots with water, about 20 %

were

(v/v)

water was added to the roots before

adding

different solvents. To test if this additional water influences the the extract,
one

composition adding

batch of roots

was

extracted with MTBE without

two paper

water

(MTBE (dry)).

After filtration

through

filters, the MTBE(dry)

extract still

contained small

particles

and had to be filtered

through

0.2

urn

filter. The GC material A

analysis

showed that the

MTBE(dry)

extract contained

slightly

more

than the MTBE extract with

pre-wetted roots,

but less material B and C

(Figure

6.7).

These difference between

MTBE(dry)

and MTBE extracts could be due to the 0.2

urn

losses

during

the additional filtration

on

through

filter

or

due to

beneficial effect of the water

the extraction of B and C

compounds.

Table 6.2. Oil

composition

of the solvent extracts,

as

determined
b

by

GC

Ratio of material X normalized to material B

segmenta
A

hexane 0.014 0.008 1.000 0.047 0.234 0.069 0.182 0.117 0.350 2.410

MTBE(dry)
0.038 0.029 1.000 0.054 0.223 0.068 0.193 0.112 0.349 2.543

MTBE

ethyl

acetate

ethanol 0.019 0.010 1.000 0.065 0.180 0.056 0.208 0.115 0.372 3.713

0.023 0.015 1.000 0.048 0.241 0.066 0.182 0.109 0.355 2.317

0.036 0.018 1.000 0.061 0.217 0.066 0.181 0.107 0.343 2.564

Aa
B

Ba Bb Be Bd Be
Bf

C
a

Division of the segments

see

figure

6.2

:To

directly compare the oil composition of the different distillates the material were normalized to the material B

A, B, C, Aa and Ba

to Bf

The solvents hexane, MTBE and

ethyl

acetate extracted the same material B

and C, whereas ethanol extracted less. Ethanol and hexane extracted fewer

hydrocarbons (segment A)

than the other solvents

(Figure 6.7,

Table

6.2).

For

112

a) segment

Figure

6.7. Solvent

extraction of vtiver roots with different solvents. Before the

dry

vtiver

roots were extracted with

different solvents

(hexane (D),

MTBE

O), ethyl
ethanol
were

acetate

(, (A) and

(O))

the roots

wetted with water % v/v

(-20
b) segment
6.25 t

solvent),

except for MTBE(dry)

B

(),
by

to which roots no

water was added. The extracts were

GC. The

analyzed yield of
was

followed

material A, B and C over the 4

extraction steps (a, b, c). Additionally, the dry weights of the samples was determined (d),
1 2

except for ethanol,

because

by evaporating

c) segment C

the ethanol-water mix the essential oil would also evaporate.

d) dry weight

113

hexane, MTBE(dry), MTBE and ethyl

Ba
-

acetate extracts, the ratio of the material

Bf

was

similar to that of the distilled oil. For the ethanol extract, the ratio different

was

slightly

(Tables 6.1, 6.2).

are

Since

only

the volatile components

was

detectable

by

GC

analysis,

the

dry

weight

of the extract

determined to estimate the amount of the non-volatile

in addition to the volatile

not

compounds.

The

dry weight

of the ethanol extract

was

determined,

as

by evaporation

of the ethanol-water mix, the essential oil

would also evaporate. Since hexane, MTBE and

in the
same

ethyl

acetate extracts resulted

material B and C, the differences between the

dry weight

of these

solvent extracts must have been due to the differences in non-volatile

compounds.
Of the tested solvents, hexane is the best for

extracting

the

complete

oil

(material A, (Figure 6.7).

analyzes,

B and

C)

and the smallest amount of non-volatile

compounds
of the GC

Since non-volatile
was

compounds impair

the

quality

hexane

chosen for further solvent extractions.

Comparison

of distillation and solvent extraction

With the

previous experiments

we

optimized

the conditions for solvent

extraction and for distillation. To determine the amount of non-volatile

compounds
distillate volatile

in

hexane extract, the The

dry weight

of

hexane extract and

were

compared.

H20

distillate contained similar amounts of

compounds (material

B and

C)

after 26 fractions

as a

hexane extract did

after 3 extractions. The calculated difference in distillate and the hexane extract
more was

dry weight

between the

H20
that

about 9 mg per gram roots, of the hexane extract

was

meaning

than

one

third of the

dry weight

due to

non

volatile

compounds.
on

To test the influence of the two extraction methods

nents of the

the volatile compo

oil,
a

roots from the same batch were extracted with hexane or

distilled with

P-buffer

(P-dist/roots).
to observe

Half of the hexane extract that

was

distilled

(P-dist/hexane extract)

changes

might

result from the

high

temperature. Distillation of

roots liberated more oil than the extraction with

more

hexane did. After distillation of the hexane extract

oil

was

detected than in

the hexane extract before distillation, but still less than with direct distillation

114

a) segment
0.30 -r

b) segment
8.75 7.50
o

^"m

>]_i^acfadn

6.25 5.00 3.75 2.50 1.25 0

=*__.---A

-i---

"c >
to

en

E E

>^=

<-fii3=P=-f
CO
X CD

iif??
=

H
.

_t=^=i:=fi==
:

CO

_Q

01

CMCMCMCMCMC5C5C5C5C5LL

co

c) segment C
15.0-U3

12.5" 10.0-7.5--

o o
>-

"
E

'en
E

5.0" 2.5-0

L
CO
X CD

^-?-q-|--i-j
CO CM CM

F/gi/re

6.8.

Comparison

influence of the extraction method vtiver oil, vtiver roots (0.5M, pH8) (D, ) and
were

of distillation and hexane extraction. To compare the on the amount and the composition of the

extracted by distillation with phosphate buffer by hexane extraction at room temperature (, O). To test the influence of high temperatures and the efficiency of hexane extraction, hexane extract and the roots after hexane extraction were distilled (P-dist/ hexane extract (A, A); P-dist/extracted roots (O, )). The distilled water fractions were extracted three times (samples x, x', x") and the cooler was rinsed twice (sample xa, xb). All samples were analyzed by GC. The yield of material A, B and C was followed over the three extractions and the three distillation steps (a, b, c). R: remaining water or buffer extracted with MTBE; R acid: remaining water buffer acidified before extraction with MTBE; roots: distilled roots extracted
withMTBE

or

115

(Figure 6.8).

To compare the extraction

efficiency

of the hexane extraction,

roots after hexane extraction were distilled as well

(P-dist/extracted roots).
only
a

However, the distillate of the extracted

oil

roots contained

small amount of

(Figure 6.8),

less than the difference between the root distillate and the

distillate of the hexane extract. This difference is most

probably

due to

peak

integration

errors.

The differences in the oil content of hexane extract and distillates

roots or hexane

(direct
and the

extract)

are

probably
some

result of the heat

(100

140C)

pH.
and

Under these conditions

were

bound molecules

might

have broken down

subsequently

detectable
-

by

GC.

The ratios of the material Ba the hexane extract

were

Bf of the distillate

(of

roots and of

extract)

and

similar but not identical

(Table 6.3).

With distillation, the

material Bf decreased, whereas the material Ba, Bb and Be increased For the distilled extracted roots,
not

slightly.

only

small amounts of oil

were

distilled

(data

shown)

and

no

clear conclusions could be drawn.

Table 6.3.

Comparison

of the oil

composition

extracts and of distillates from a hexane extract, as determined

Ratio of material X normalized to material B

of distillates from roots, of hexane by GC

b

segmenta
A

P-dist/roots 0.022 0.007 1.000 0.061 0.267 0.100 0.171 0.128 0.273 0.185

hexane extract 0.020 0.008 1.000 0.054 0.248 0.075 0.179 0.116 0.328 2.396

P-dist/hexane extract 0.026 0.009 1.000 0.064 0.259 0.079 0.183 0.118 0.298 0.108 0.031 0.007 1.000 0.054 0.261 0.074 0.177 0.127 0.306 0.109

0.035 0.007 1.000 0.058 0.269 0.097 0.170 0.133 0.273 0.064

0.027 0.009 1.000 0.050 0.248 0.074 0.176 0.122 0.330 2.291

Aa
B

Ba Bb Be Bd Be
Bf

C
a

: :

Division of the segments To

see

figure

6.2

directly

were
c

compare the oil composition of the different distillates the material normalized to the material B

A, B, C, Aa and Ba

to Bf

two

repetitions

From the GC

analyses

we

conclude that the

composition

of the volatile oil

components obtained by distillation and by hexane extraction is similar. Only

when separate

peaks

are

compared

in detail,

can

small differences in of vtiver

composition

be found. Therefore, for the

pre-screening

plantlets,

the

116

less labor-intensive hexane extraction the non-volatile components

can

provides

suitable extraction method, if

or

either be removed

do not disturb the

subsequent

GC

analysis.

ACKNOWLEDGEMENTS

The Vtiver

plants

were

provided by

Mr. Heini

Lang,

Jakarta. This work

was

supported by

Givaudan-Roure

Forschung

AG Dbendorf, Switzerland and

no.

by

the Swiss Federal Office for Economic

Policy, project

2561.1 of the

Commission for

Technology

and Innovation.

117

Chapter

7:

Optimization

of small scale extraction,

purification

and concentration methods for

analysis

of the essential oil from vtiver roots

Ruth E.

Leupin,

Charles Ehret, Karl H. Erismann and Bernard Witholt

118

ABSTRACT

To
in

develop

simple

and small scale

procedure

for the

analysis

of vtiver oil
were

multiple

vtiver root

samples,

hexane extraction and water distillation

optimized.

As hexane also extracted non-volatile

compounds,

which

complicated subsequent
fractionated method of
was

gas

chromatography analysis,
or

extracts were further

by

thin

layer chromatography

column

chromatography. permit

Neither distillation

sufficient. To minimize the scale of distillation and

samples

in

parallel,

water distillation was combined with solid

was

phase

extraction. An Amberlite XAD-2 column the steam, thus

used to adsorb the vtiver oil from

replacing

the

cooling system. analysis

of

The choice of the best system for

multiple

small scale
in the final
case

samples

depends

on

the

acceptability

of non-volatile
are

compounds
as

analysis

assay. If non-volatile
hexane extraction is volatile

compounds
a

acceptable,

is the

for TLC,
If
non

useful method to prepare many small

samples.

compounds
distillation

disturb the

analysis,

as

it is the

case

for gas chromato

graphy,

coupled

to the Amberlite column is better. For very small

a

amounts of

material, both methods require

concentration step.

INTRODUCTION

After al.

regeneration
all

of vtiver

plantlets

via tissue cultures

(Chapter 3, Leupin
composition
of the

et

2000),

plantlets Usually,

have to be tested for the amount and vtiver

essential oil.

plants by

are

harvested after 15-22 months and the

oil is extracted from the roots Weiss

steam distillation
even more

(Roth

and Kornmann 1997;

1997a).

With in vitro
can

plantlets,

time is needed before

regenerated plantlets screening

for

be tested. A micro assay that

permits

an

earlier pre-

changes

in the oil
as

composition

is therefore needed.
-

Several methods such

simultaneous distillation

solvent extraction

(Bicchi

etal. 1983; Godefroot etal.

1981), headspace solid-phase (Craveiro

etal.

micro extraction and

(Field

etal.

1996),

microwave extraction and Ciola 1991 ;

1989)

supercritical

fluid extraction

(Blatt

Sugiyama

and Saito It

1988)

have been

described to extract essential oil from small

samples.

was

shown that the

119

composition

of the oil varies

depending

on

the extraction method used

(Boutekedjiret
In

etal. 1997; Pino etal. 1996; Scheffer 1996; Simndi etal.

we

1999).

previous experiments,

have

optimized

and

compared

water distillation

and solvent extraction at

5 g

room

temperature

to extract oil from

larger samples
better,
as

of

dry

roots

(Chapter 6).
compounds

For

large

amounts of roots, distillation is

only

volatile

are

extracted, but the distillation of many samples

raises

practical problems.
can

Solvent extraction has the

advantage

that many small

samples

be extracted in

parallel. However,
cause

since such extracts also contain the

non-volatile

compounds,
gas

these may

problems during

subsequent
it
was

analysis by

chromatography.

In the

previous study (Chapter 6),

shown that hexane, about the volatile the

same

methyl ferf-butyl ether, ethyl

acetate and ethanol extracted amounts of non

amount of volatile

components but different

were

compounds,

of which fewest

extracted with hexane. Nevertheless,

dry weight

of the hexane extract

(23.7
mg

mg

g~1 dry roots)

is still

significantly
that the

higher

than that of the distillate

(14.9

g~1 dry roots), indicating

compounds.

hexane extract does contain non-volatile

In this

chapter

we

describe the

optimization

of the small scale hexane

extraction,

test methods to lower the amount of non-volatile

compounds by
a

thin

layer chromatography,
to distill several

column
in

chromatography
a

or

distillation, and try

method

samples

parallel by
extraction.

combination of solvent extraction,

distillation and solid

phase

MATERIAL AND METHODS

Vtiver roots

The

plants

and the

dry

vtiver roots

were

provided by
were

Mr. Heini

Lang, pieces.

Jakarta. For the extraction Fresh roots

were

experiments,

the roots

cut in 5

mm

harvested from the Vetiveria zizanioides of

our

stock. The
-

plants

were

grown outside

(summer)

and in

greenhouse

at 10

15C

(winter).

120

Vtiver oil

The vtiver oil Bourbon Dbendorf.

was

provided by

Givaudan-Roure

Forschung,

Hexane extraction at

room

temperature

Dry

vtiver roots, cut in 5

10

mm

pieces,

were

wetted for about 1 hour with

water and

subsequently
was

extracted

overnight
extract

at room

temperature with hexane.

hexane
was was

The hexane

removed

(hexane

1)

and

new

added. After removed

extract

another 24 hours and the roots

stirring,

the hexane

(hexane

extract

2)

again

were

extracted for the third time with

new

hexane

(hexane

3).
For

experiments

with hexane extract

were

as

starting material,

5 g vtiver roots,

pre-wetted

with 10 ml water,

extracted with 50 ml hexane to avoid

variation due to To

inhomogeneous

root material.

optimize
or

the extraction of small amounts of oil, different

or

starting

materials

(thick
the

thin roots, cut

ground
600
or or

with

sand),

different amounts of water to wet

dry

roots

(0, 150, 300, (1.5,

3

1200 ul water per 300 mg

roots),

different

amounts of hexane

6 ml hexane per 100 mg

roots)

and different

extraction

periods (sequentially
tested,
as

for 1, 1 and 3

days,

for 2 and 3

days

or

for 5

days)

were

described in the result and discussion section.

were

The acidic the

same

compounds
a

removed

by shaking

out the hexane extract with

volume of

1M

potassium hydroxide

solution

(KOH).

The hexane
extract
-

phase

contained the hexane extract without the acids

(hexane
phase.

KOH),

whereas the acidic

compounds

remained in the water

Concentration

or

purification

with silica columns

Pasteur

pipettes

were

plugged

with cotton and

dry

silica

(Merck, Kieselgel

60, 0.062-0.200 mm)

columns
were

was

added to make

simple

silica columns. Before use, the ether

washed with 1 ml ethanol, 2 ml

were

methyl ferf-butyl
on

(MTBE)
hexane:

and 3 ml hexane. Hexane extracts

loaded

columns

(outflowing

121

H)

and oil

was

eluted in 0.5 ml steps

All

(elution

with different solvents: s, elution

with MTBE: To

M).

samples

were

analyzed by

gas

chromatography (GC).
we

analyze

the concentration effect of these columns,

tested different

amounts of silica

(30, 50,

100 and 160

mg),

different solvents to elute the

acetate and

extract from the column

(MTBE, isopropanol, ethanol, ethyl

or

diethyl
mg

ether)

and different amounts of hexane extract

vtiver oil Bourbon

were

(0.5

ml1 in hexane). When solvents other than MTBE

the column, these
were

used to elute the oil from

used instead of MTBE to wash the column.

Small scale distillation in combination with solid

phase

extraction

Optimization

of the micro-distillation

Pasteur

pipettes

were

used to make Amberlite columns

(Figure 7.1a).
which

Loose
was

cotton was used to retain the column material

(Amberlite XAD-2),
were

added

as a

suspension

in water. Before use, the columns

washed

sequentially

with 3 ml ethanol, 3 ml MTBE, 2 ml hexane, 2 ml ethanol and water

and dried for several hours at 100C.

Figure
Amberlite column

7.1. Miniaturized

distillation apparatus with Amberlite column.

cutoff
-Pasteur

rni
pipette
-

a)

Amberlite column
a

made from

Pasteur

pipette b) distillation apparatus

5 ml P-buffer

Amberlite
loose cotton

with Amberlite column

(0.5M, pH8)
+

extract or roots

Samples (distillate (P-buffer, 0.5M, pH8)

nitrogen (HexN)
silica
was or

or

hexane
a

extract)

were

added to 5 ml
was

phosphate

buffer

in

Pyrex

tube and the hexane

evaporated

with
on

the

compounds
was

in the hexane extract

were

adsorbed

(HexSil)

and the silica

transferred in the P-buffer. The XAD-2 column and put in the

oven

connected to the tube

(Figure 7.1b)

at 120C. After

122

5 hours about 3 to 4 ml of the P-buffer had been

was

evaporated

and the

distillation

stopped.
-

The Amberlite column

was

eluted three times with

2 ml MTBE

(M1

M3),

and the residual buffer

was

extracted with 1 ml MTBE

(R).

All

samples

were

analyzed by

GC.

Comparison
distillation

of hexane extracts and roots

as

starting

material for micro-

Three different

samples

were

distilled: 1 ml hexane extract 1, concentrated

by evaporating
on

the hexane under

nitrogen (HexN);
hexane:

1 ml hexane extract, loaded

50 mg silica

(HexSil) (outflowing
was

H);

and 100 mg of

dry

roots. After

about 3 hours the distillation residual buffer

was

stopped.

For the second distillation, the

new

filled up with water to 5 ml and

was

XAD-2 columns

were

used. After another five hours the distillation

stopped.

The Amberlite

columns of the first and the second distillations

ml MTBE

were

rinsed three times with 2

-

(first
1

distillation: M1 and the roots

were

M3, second distillation: M4

extracted with MTBE

M6).

The residual

buffer

(~

ml)

were

(R).

As controls, roots
was

extracted twice with hexane and the hexane extract

prepared by

as

for the distillation, either

by adding

the extract to the P-buffer,

or

followed

the
on

evaporation

of the hexane with

nitrogen (HexN)

by loading

the hexane

the silica column

were

(HexSil),

but instead of the

or

subsequent

distillation, the compounds

then re-extracted

re-eluted, respectively. All

samples

were

analyzed by

GC.

Thin

layer chromatography (TLC)

For TLC

analysis,

extracts were

separated

on as

silica

gel plates (Silica gel

60

F254, MERCK), using hexane and chloroform

dimension. To separate non-volatile solvents

consecutive solvents in

one

compounds

from the essential oil, different

(hexane, isooctane, isopropanol, diethyl ether, acetonitrile, methanol,

were

methylene chloride, chloroform, toluene) compounds,

acid
were

tested. To detect the different acid-sulfuric Merck

UV

(254 nm),
:

iodine vapor and

anisaldehyde-acetic

(2

ml

20 ul

40

ul) staining

for essential oils

(Cosicia 1984;

1970)

used.

123

Gas

chromatography (GC)

A Hewlett Packard 5890A gas

Chromatograph equipped

with

flame
urn

ionization detector and

DB-WAX column
was

(25

m x

0.32 mm, 0.25

a

film

thickness)
2 ml

was

employed. Hydrogen
were

used

as

carrier gas at mode.

rate of

min1. The samples

injected

in the

splitless

Injector

and The

detector temperatures column

oven

were

maintained at 200C and 300C,

respectively.

temperature

was

programmed
was

for 80C

(after

min)

to 220C at

2.5C min1 and the final temperature

retention times 3390A
were

held for 20 min. Peak

areas

and

measured

by

electronic

integration (Hewlett

Packard

integrator).
injecting
in the

Before

GC, dibutyl phthalate

was

added to all

samples

as

internal standard. To compare the

composition

of the extracts, the GC

chromatogram (Figure 7.2)

was

divided in three main segments

(A, B, C).
mainly
the

Segment

A contained

mainly hydrocarbons, segment

C contained

acidic components and segment B contained residual components

(alcohols,
on

ketones,..) (Chapter 5).

oil

To detect the effects of the extraction methods

the Ba
-

composition

in

more

detail, the segments A and B

were

subdivided

(Aa,

Bf; Figure 7.2). In the following text, X compounds refer

to the

compounds

running

within segment X of the GC

chromatogram.

Figure 7.2. Gas chromatogram of vtiver oil on a DB-Wax column. The chromatogram was divided in segments A, B and C for data analysis. Segment A contains mainly hydrocarbons, segment B contains mainly alcohols, aldehydes and ketones and C contains mostly acids. The segment B was subdivided in Ba Bf. IS: dibutyl phthalate (internal standard)
-

124

Estimation of the amount of oil based

on

GC

analysis

To determine the amount of any

compound

Z from

GC

chromatogram,

the

response factor of that

compound

Z and the internal standard

(IS)

has to be

determined. The FID response is

atoms

roughly proportional

to the number of carbon

present in the compounds, however the response is also affected by

hetero atoms and various functional groups each

(Flanagan 1993). Therefore,

for

compound

the response factor

can

be different and has to be determined

separately.

response

factor

-^

peak area

amount

Using

this response factor, it is

Z from
a

possible

to estimate an unknown amount of

compound

GC

chromatogram.

amount of

compound
K

[ug]
LMaj

peakareaZ
peakarealS

response factor IS
response factorZ

|S

As many vtiver oil components

are

not isolated and the structure not

elucidated, it is

not

possible

to determine the response factor for each

are

single

component. Since vtiver oil components

expected

to be all

sesquiter-

penoids, they
are

should have C15 and

exceptionally

C14. The main differences

the number and the

we assume

position

of the functional groups. To

simplify

the

calculation,
as

that all vtiver oil components have the This results in:

same

response

dibutyl phthatate (C16H2204).

amount of compound

^r

i
=

peak area
r
[J
HnK

Z
r^

[ug]

amount IS

rtInn

peak

area

IS^*

The amount of oil in each segment X standard

was

estimated relative to 1 ul internal

containing

0.025 ug

dibutyl phthalate by summing

up all

approximate

amounts of the

compounds

within segment X.

125

....

iVr

matenal

in

segment a

ug

LHaj

peak areas in segment

nn

peak area S

0.025 Ha

ug

It has to be noted that the calculated amount is of oil, based

on

only

an

approximate
on

amount

the

assumption

described above.

Depending
was

the commercial

vtiver oil tested the amount used

(see chapter 5), (results

not

the calculated amount

up to 1.5 times of

shown).

RESULTS AND DISCUSSION

In this

study,

we

describe

experiments

to miniaturize the extraction of vtiver

oil from

small amount of roots. Given the

room

simple equipment

necessary for
can

hexane extraction of roots at extracted in

temperature, several samples

be

parallel. However,

non-volatile

compounds

which

are

co-extracted,
them should

disturb the gas be found.

chromatographic analysis

and methods to

remove

Water distillation of roots has the

advantage

that

only

volatile

compounds
due to the

are

extracted, but only

few

samples

can

be distilled To

simultaneously
the
in

limitation of the necessary

cooling system.

replace

cooling system

and
a

therefore make the distillation of several small solid

samples

parallel possible,

phase

extraction

was

tested.

Optimization

of small scale hexane extraction

In

previous experiments, by

we

found that

more

oil

was

extracted from the the

vtiver roots

distillation than

by

hexane extraction

(Chapter 6). Therefore,

extraction of the roots with hexane not extraction

only

has to be miniaturized, but the

procedure

must also be further

improved.

Effect of

solubility

Factors which

can

limit

an

extraction

are

the

solubility

of

compounds

in the

extracting

solvent and the diffusion of solvents into and

compounds

out of the

126

plant

material. To test if the

solubility

of the vtiver oil in hexane is

limiting

factor, different

amounts of hexane were used for the extraction. In

were

previous

experiments,

5 g roots
as

extracted in 50 ml hexane. For 100 mg roots, this

ratio is not useful,

1 ml hexane fails to

submerge

the roots and the

magnetic

stirring
were

bar. Therefore, for 100 mg

pre-wetted roots, 1.5,

was

3 and 5 ml hexane

used. The first extraction step

only slightly
more

more

effective with 5 ml
not

hexane

compared

to 3 or 1.5

ml, extracting

acidic

compounds (results
not be the main

shown). Thus,
reason

limited

solubility

of the oil components

can

for their less efficient extraction.

Since after induction of oil formation, the in vitro

amounts of

plantlets

contain very small

oil, it is necessary

to extract the oil in as little solvent as

a

possible.

As

the increased amount of hexane did not result in

100 mg roots in 1.5 ml hexane
was

major improvement,

the ratio

used for further extractions.

Effect of extraction time

To test the influence of the extraction time

on

the extracted amount of oil, 100 for 1, 1 and 3

mg

pre-wetted
or

roots were extracted

sequentially

days,

for 2 and 3

days

for 5

days.
of the extraction time from 1 to 2

Prolongation

days

did not influence the

same

amount of oil extracted.

However, the extraction for 5 days resulted in the

sum

material A and B
or

as

the

of the

subsequent

extractions for 1, 1 and 3

days

2 and 3

days. Independent

of the time intervals, the total

yield

after 5

days

remained within the variation of the

experiment (results

not

shown).

Effect of diffusion distance

To test the influence of the diffusion distances

roots were

on

the extraction

yield,

the

dry

separated
5
mm

in thick

(>

mm)

and thin roots

(<

mm),

before

cutting

them in 2

pieces.

Thin roots contained about 1.5 to 2 times less oil than thick roots

(Figure 7.3).

However, the dry

contain less oil
roots were

roots were stored for 5

6 years and to test whether thin roots

as a

consequence of

evaporation during

this storage time, fresh

harvested, split in only thick and mainly thin

roots and extracted with

127

hexane.

Again,

more

oil

was

extracted from the thick roots

was

(1.6

-1.9

times).

With both

experiments,
we

the oil

not extracted faster from thin roots.

an

Therefore,
influence

concluded that the diameter of the roots did not have

on

the effectiveness of the extraction.

To increase the surface of the root

particles

further and

simultaneously samples
of

destroy possible

diffusion barriers of the intact root surface, 100 mg

were

thick and thin roots

ground

with sand. This treatment did not extract

one

more

oil, but it

was

extracted faster: after

was

extraction step

already
-

about 72

82 %

of the total extracted oil

recovered, whereas only 63

were

72 % of the oil

was as

extracted from control roots, which the

not

ground (Figure 7.3). However,

and
some

grinding represents
was

an

extra

working step

oil

might

be lost

during

this process, it

not used further.

segment A
o o

segment B

segment C

-a en

Figure ground

7.3. Extraction of thin, thick

were

and thick vtiver roots

cut in

ground vtiver roots with hexane. Thin pieces and either directly extracted, or first
or

with sand before

they

were

extracted three times with hexane

extraction,

analyzed

^ second extraction, |~J third extraction). The extracts were by GC. The yield of material A, B and C was determined after each

(H

first

extraction step.

Influence of water

on

extraction of vtiver oil from

dry

roots

Up

to now all extractions were

performed might

with

dry roots,

but

as

after the

induction

experiments

fresh material

also be extracted, it is

important

to

know how water influences the hexane extraction. Therefore, different amounts of water

(0, 50, 100, 200,

400 ul per 100 mg

roots)

were

added to

dry

roots

128

before extraction with hexane. Without water, many small

particles

were a

found
urn

floating

in the hexane, and the extract needed to be filtrated

through

0.2

filter. With 50 ul water, ul water the oil

or

only

few

particles

were

observed and after

adding

100

more, filtration was not necessary. Addition of water also affected

more

composition: by adding

water, less material A

was

extracted,
was

whereas for beneficial

polar components (B

and C

compounds),

the addition of water

(Figure 7.4).
on

To compare the influence of fresh and dried material

an

the extract, roots of extracted

in vivo grown

plant

were one

harvested. One part

was

(1.7 g)
day

was

directly

with 5 ml hexane and

part
was

dried for

one

at room

temperature. The
of dried roots

weight

loss after

one were

day

between 73 and 76 %.

Samples
-

(370

to 450

mg)

re-wetted with 600 ul water

same

(61

57

%)

and extracted with

5 ml hexane. About the

material B and C

were

extracted from dried and

fresh roots. The material A

not

was

slightly (24 %)

lower for the dried roots

can

(results

shown).

We concluded therefore that hexane extraction

be used for

both fresh and

dry

material.

segment A
0.125r

segment B

segment C

|tn

I
o un o o
i-

VA

If m
t
a*
o o
^

m
o o

o o

o o

o un

o o

cm

>*

<m

^r

\i\

water

/100mg dry

roots

pre-wetting the dry roots pre-wetted with 0, 50, 100, 200 and 400 uj_water per 100 mgroots, before they were extracted three times with hexane (^ first extraction, second extraction, |~J third extraction). The extracts were analyzed by GC. The yield of material A, B and C was determined Figure
with different amounts of water. Vtiver roots
were

7.4. Extraction of vtiver roots with hexane after

after each extraction step.

129

Reproducibility

of extractions

To test the influence of the

inhomogeneous

material

(e.g.

thin and thick

roots)

on

reproducibility

of small scale extraction, ten times 100 mg

dry

roots were

pre-wetted

with 200 ul water and extracted

overnight

with 1.5 ml hexane. Each

extract was

analyzed by

GC in

duplicate. compared
and
a

The ten hexane extracts

were

variation of found

maximally
not

8 % for

the material B and C, and of 16 % for material A

was

(results

shown).

Compared
from the

to material B and

C, only small

amounts of material A were extracted

dry

roots used in our

experiments. Therefore,
contents could have a

GC

integration
effect
on

errors

and variations in

hydrocarbon

large

material A.
are

It is thus difficult to determine if observed the

changes

in the material A

due to

inhomogeneous material,

to

integration

errors or

to the extraction

treatments.

Concentration of the hexane extract

The in vitro tissue may, after induction of the oil oil than the in this

production,
a

still contain less

study

used

dry

in vivo roots. As

result,

concentration step

may be necessary. One way to achieve this is to evaporate the hexane with

nitrogen;

another is to reduce the volume

by running

it

over a

silica column.

Evaporation

with

nitrogen

To determine how

evaporation
volatile

with

nitrogen changes

oil

composition,

especially

of the

more

compounds,

500 ul hexane extract 1 and 500 ul

were

of vtiver oil Bourbon

(0.5

mg ml"1 in

hexane)

taken to

dryness
was

under

nitrogen

for 2.5, 5 and 10 minutes. After 2.5 minutes all hexane

were

evapo

rated. The dried extracts

re-dissolved in 500 ul hexane and

analyzed by
for 10

GC. The

evaporation

affected the A

compounds.

After

evaporation

minutes, 30 % of the material A found in the original hexane

extract 1 was
more

recovered. Whereas for the vtiver oil Bourbon, which contained 14 times material A per 500 ul diluted solution than the hexane extract of the

dry roots,

130

only

10 % of this material A
was

was

recovered. The loss in the other

chromatogram original

segments

within the variation of the

analysis (93

110 % of

material B, Table

7.1).

Table 7.1. Influence of the vtiver oil

evaporation
determined

time

on

the hexane extract and the

composition,
h
e
x

as

by

GC
t

si

extract1

oil2

GC

material

segments

recovery after evaporation with N2 for

2.5
mm

material

recovery after evaporation with N2 for

2.5
mm

[Mg per
500

mm

10

mm

[Mg per
500

mm

10

mm

pi]

[%]
87 109 88

[%]
69 108 108

[%]
29 110 94

Ml]

[%]
110 110 99

[%]
59 102 97

[%]
9 93 97

segment

2.35 164.83 233.45

31.85 155.18 11.58

segment B segment C
1 2 3

: :

dry

vtiver roots extracted in

hexane,

vtiver oil Bourbon

(Givaudan-Roure),

100 mg roots ml1 hexane 0.5 mg ml1 in hexane

: see

figure

Concentration

by running

over

silica column

It is known that oils

hydrocarbons
on

can

be isolated from
or

terpenoids
as

or

essential

by chromatography

silica with hexane

pentane

eluents

(Croteau

and Ronald 1983; Kubeczka

extracts with small columns

1985).

We used this fact to concentrate hexane different amounts of silica

-

containing

gel (30, 50, subsequently

were

100 and 160

mg).

0.5 ml hexane extract 1

KOH

was

loaded and

rinsed 5 times with 0.5 ml hexane. Three hexane fractions of 1 ml each collected and

analyzed by

GC. The first hexane fraction contained

mainly only
traces

hydrocarbons (segment A).

of B and C
were more

The next two hexane fractions contained

compounds.

With 30 mg silica

slightly

more

B and C

compounds gel.
As

found in the hexane fractions than with solvent is necessary for

larger

amounts of silica
was

larger columns,

50 mg silica

chosen for

further

experiments.

Elution of the hexane extract components from silica columns

To concentrate the hexane extract, it elute the

was

necessary to

use

less solvent to

compounds

from the silica column than the volume of extract loaded

131

on

the column. Different solvents

(MTBE, isopropanol, ethanol, ethyl

ethyl

acetate

and

diethyl ether)

were

therefore tested. MTBE and

acetate re-extracted

about 100 %, ethanol and ether about 120 % and

isopropanol only
-

90 % of the

original

material B. All solvents tested extracted about 64

77 % of the

original

material C

(Figure 7.5).
complete elution,
recover was

To determine the minimal amount of solvent necessary for the extract

more was

eluted in three steps and

analyzed by isopropanol

GC. To
or

98 %

or

of the total eluted material B, 0.5 ml

or

ethanol

sufficient,

whereas for MTBE

ethyl

acetate 1 ml and for

diethyl

ether 1.5 ml

were

necessary
it has
a

(Figure 7.5).

Since MTBE eluted 100 % of the

which makes it easy to load

original

material B and

low

boiling point,

samples

on a

TLC

plate,

MTBE

was

chosen for routine elution of the oil from silica columns.

segment A

segment B

segment C

Figure

7.5. Elution of

vtiver root hexane extract from

silica column loaded

on

using

different solvents. 2 ml vtiver root hexane extract 2 columns with

was

silica

(50 mg silica). The vtiver oil components were eluted from the column methyl ferf-butyl ether (D), isopropanol (O), ethanol (A), ethyl acetate (O) or diethyl ether (X). The outflowing hexane fraction (H) and the eluted solvent fractions (s1 s3) were analyzed by GC. The total recovery of material A, B and C was compared with that in the original hexane extract 2 ().
-

To further reduce the amount of MTBE needed to elute the oil from the

column,

we

examined the influence of the hexane left after

loading

the hexane
were

extract. Two columns were eluted

directly,

whereas the other two columns

were

first blown
MTBE.

dry

with

pipetting

ball before

they

eluted three times with

132

Removal of hexane from the column with MTBE: after contained 96 % without
one or

improved

the effectiveness of elution

two elution

steps, the eluates of the dry columns

of the total eluted material B, whereas
or

or

99 %

respectively only

prior

hexane removal found back

76 %
not

96 %

respectively

of the total eluted

material B

was

(results

shown).

Adsorption capacity

of silica columns

The

capacity

of 50 mg silica columns

was

determined

by loading

different

hexane extracts and vtiver oil Bourbon in hexane in 0.5 ml steps and

analy
or

zing

the

outflowing

hexane per 0.5 ml fraction

were

(H1-Hx).

After
-

loading

10 ml

of the extracts, the columns

were

eluted with MTBE

(M1

M5).

All fractions

analyzed by

GC.
or

By loading
contain about

10 ml of hexane extract 2
as

3 ml of hexane extract 1, which

same

much material B, it
was

was

shown that the

amount of

material B and C

rinsed

through (Figure 7.6a, b).

This suggests that the

loading capacity
The

of the silica is unrelated to the volume loaded onto the column. of the oil influenced the amount of extract adsorbed
on

composition

the

silica column. After

loading

5 ml hexane extract 1 about 10 % of the material B

were

and 22 % of the material C

rinsed

through

the silica column, whereas after

loading

5 ml vtiver oil Bourbon

(0.53

mg ml"1 in

hexane) only
through

about 2 % of the

material B and 1 % of the material C

were

rinsed

the column

(Figure

7.6a, c). The differences between the composition of hexane extract 1 and the
vtiver oil Bourbon is that the latter contains
more

of the

hydrocarbons (A

com

pounds),

whereas the hexane extract 1 contains

more

acids
on

(C compounds).
the

To

test whether the acidic

compounds they
were

have

negative

effect

ability by

of the

silica to adsorb the oil,

removed from the hexane extract 1

KOH
on

extraction. 5 ml of this hexane extract the column. As

was

(hexane

extract 1

KOH)

were

loaded

the

case

for the vtiver oil Bourbon,

were

only

about 2 % of the

material B and 1.5 % of the material C the has

rinsed

through

the column

during

loading (Figure 7.6d).

a

We concluded therefore that the acid concentration

on

negative

influence

the

adsorption capacity

of the silica column.

133

a)

hexane extract 1

b)

hexane extract 2

c)

vtiver oil

d)

hexane extract 1-KOH

e)

vtiver oil filled

on

symbols:

material X loaded

the silica column

from the

(calculated original extract)

open

symbols: material X remaining on the column, i.e.,

material X loaded
on

the silica

column

(calculated)
fractions

minus

material X detected in the

outflowing
ihhhihi'

of 50 mg silica columns. Different vtiver root hexane extracts of and vtiver oil (Bourbon) in hexane (0.5 mg ml"1) were

Figure

7.6.

Loading capacity

loaded in 0.5 ml steps on silica columns (50 mg). After loading 5 or 10 ml extracts, the columns were eluted with methyl ferf-butyl ether (MTBE). The

outflowing
were

hexane fractions

collected and

(H1 analyzed by
over

and C the

(A, A) was followed subsequent elution.

(M1 M5) GC. The recovery of material A p, D), B (, O) the loading (outflowing hexane fractions) and
-

Hx)

and the eluted MTBE fractions

134

All of the B and C

on a

compounds

in 2.5 mg vtiver oil Bourbon

was

were

adsorbed

50 mg silica column. When 5.3 mg oil

was

loaded, about 7 % of the

About 98 % of the

material B

rinsed

through

the column

during loading.

adsorbed oil

was

eluted from the column with 0.5 ml MTBE

was

(Figure 7.6e).

The

material B of about 5 mg vtiver oil hexane to 0.5 ml MTBE. If

a

therefore concentrated from 10 ml

loss of 5

6 % is

accepted,

the hexane extract 1

could be concentrated about 8 times. If the hexane extract without acid

compounds
even more

behaves
to 18

as

does the vtiver oil Bourbon, it could be concentrated first

(up

times) by

removing

the acidic

compounds.

Comparison

of extract concentration

procedures

Which of the two above described methods is used to concentrate the extract

depends nitrogen,
were

on

which

compounds
were

of the oil

are

of interest.
on

By evaporation

with

compounds

partially lost,

whereas

the silica column

they

not adsorbed and therefore not concentrated. For B

compounds,

it did not

matter which method was used. No C

compounds
on

were

lost

during evaporation eluting

with of

with

nitrogen,

whereas

part remained

the silica column after

0.5 ml MTBE and

high

concentrations of acids reduced the

loading capacity

the silica column. To concentrate the total oil,

evaporation

with

nitrogen

seems

very

promising

for small amounts of extracts, whereas for the silica column method alone

larger

amounts of hexane extracts

a

(Box 1)

or

in combination with

subsequent

evaporation

with

nitrogen

is useful.

Box 1
-

Optimized

silica column concentration method:

load 50 mg silica in Pasteur diameter

<

pipette (or

column with smaller

50 mg silica

gel)

wash column with ethanol, MTBE and hexane

if extract contains
a

lot of acids: shake out with KOH

load the hexane extract: A

in the

compounds

are

mainly

found back

outflowing
hexane

hexane

remove

by blowing

air

through

the column

-elute with <0.5 ml MTBE

135

Removal of non-volatile

compounds

from the hexane extract

One

problem

of solvent extraction is that the volatiles

are

isolated

together
may

with non-volatile rise to

compounds,
such
as

which

during subsequent peaks

or

GC

analyses

give

problems

additional

base line for

shifting.

Therefore it is

necessary to

use some

kind of

sample preparation,
of the

example

TLC

or

column

chromatography,

for

preliminary clean-up

samples (Croteau

and Ronald

1983; Scheffer 1996). We have tested the separation ability of different solvents

by

TLC. None of the tested solvents

separated

the non-volatile

compounds

from

the volatile

compounds (result

not

shown).
it
was

In the concentration

experiments,
on

found that up to 25 % of the acidic

compounds

remained
If

the silica after the first elution with 0.5 ml MTBE the non-volatile

(Figure 7.5).
place

simultaneously

compounds, running

at the same to

and slower than the acidic oil components of the vtiver oil,
on

were

remain

the silica column,

partial cleaning might

be

possible.

To test how

efficient the

precleaning

was, a hexane extract of distilled roots was, after

extraction of the acidic

compounds

with KOH, loaded

on

silica columns and

eluted with different solvents

(MTBE, isopropanol, ethyl

acetate and

diethyl

ether).

TLC

was

used to detect the non-volatile

compounds.

With all solvents

used, the major portion of the non-volatile spots eluted in the first fraction

(results

not

shown).

The

precleaning

effect is thus

only

minor.

Small scale distillation in combination with solid

phase

extraction

The most efficient method to separate volatile and non-volatile

to use distillation. One

compounds cooling

is

disadvantage

is that distillation

requires
in

system, which makes it complicate

cause

to distill several
area

samples

parallel

and may

losses due to the

large

surface

of the

glass
solid

material. A method to

avoid these

problems

is to

replace

the cooler

by

phase

extraction

column, which adsorbs the oil components from the

steam.

136

Adsorption

of vtiver oil

on

Amberlite XAD-2

To avoid

plugging

of the column and

possible

overpressure, the column

material should not be Amberlite XAD-4 it is

packed

too

densely.

Machale

(1997) reported
compounds

that with
con

possible

to extract essential oil

from

densate water, and Menon free and

(1999)

used Amberlite XAD-2 columns to isolate

a

glycosidically

bound volatiles from

water extract of cardamom. We

therefore selected Amberlite, which consists of small To test if the

spheres (20

50

mesh).

apolar

Amberlite XAD-2 adsorbs all distilled vtiver oil compo

nents from cold water, an Amberlite column was fixed behind the cooler of the

distillation apparatus.

Only

about 3 % of the total material B

was

found in the

distilled water fractions after the Amberlite column The next step
was

(results

not

shown).

to test whether the Amberlite XAD-2 also adsorbs the

vtiver oil from steam. Distillation with the Amberlite column before the cooler did not work,
as

the water condensed in the Amberlite column and the

same

over a

pressure caused the Amberlite to be blown out. The

happened

with

Pyrex

tube with Pasteur

pipette

column

(Figure
an oven.

7.1

b)

in

an

oil bath at 140C. tube

as

An alternative is to

place

the system in

The

Pyrex

well

as

the

Amberlite column the risk of

an

are

heated, the

water does not condense in the column and

Amberlite blow out is reduced. To test whether such Amberlite

columns adsorb the volatile with XAD-2 columns

were

compounds
in

from steam, small scale distillations

an oven

performed

at 120C.

Table 7.2. Minimized distillation of vtiver distillate with absorb the oil from the steam

XAD-2 column to

original
GC
distillate
1

combined distillation

solid

phase

extraction

percent

of

original
total 109.4 97.6 78.4

distillate

[%]
total

segments
material

eluate of Amberlite column

extract of

[ug]

3 8.7 0.8 0

residual buffer
7.1

segment A segment B segment C

1 2 3

23

4.78 432.30 28.55

87.3 69.7 67.6

13.3
27.1

116.4 97.6 95.4

0.1 17.0

10.9

distillate of vtiver roots dissolved

in

hexane

: see :

figure 2 large variations

137

The eluate of the Amberlite column contained the for material A, the contained The
sum

major part

of the oil.

Except

of the eluates and the extracts of the residual buffer

only
oil

few percent less than the remain in the steam

original starting
or

distillate

(Table 7.2).

missing

might

not be extractable from the column

material. The material A varied, due to

116
+

evaporation

and small amounts, around

15%

(Table 7.2).
XAD-2.

This

experiment

showed that the

major part

of the oil

adsorbed

on

Effect of Amberlite column

height

on

adsorption

To find

an

optimal
or

amount of
on

Amberlite, the effect of the Amberlite column

height (0.8,

1.8

cm)

the

adsorption

of volatile

compounds

from the

steam was examined. With the 0.8 cm

high

columns up to 60 % of the
were

original

material A and about 10 % of the

1.8 and 3
cm

original

material B

lost, whereas for the

columns up to 40 % of the material B

were

original
not

material A and less than 10 %

of the

original

lost

(results

shown).
of the hexane, it
was

Due to the loss of material A

during evaporation

not

possible

to determine how much of material A was not adsorbed

was

by

the

Amberlite. The elution of the Amberlite columns

not influenced

by
-

the
96 % of

height

of the column material: with all three column

was

heights

about 90
more

the total eluate

re-extracted with the first 2 ml MTBE and

than 99 %
2
cm

within the second 2 ml

(results

not

shown).

Therefore

we

selected

Amberlite column for further

experiments.

Sample preparation

of hexane extracts for distillation

Due to the

explosion danger,

hexane has to be removed before the hexane

to the concentration

extract is distilled in the oven.

Analogous

experiments nitrogen

described above, this

can

be done

by evaporation
on

of the hexane with

(HexN)

or

by adsorption

of the oil

compounds

silica

(HexSil).
major portion
hexane of A

Since

during preparation

of the HexSil concentrate the

compounds
were

of the hexane extract remained in the

nor

outflowing

(H), they
the

neither distilled

lost due to

evaporation

with

nitrogen. Therefore,

138

Figure 7.7. Influence of sample preparation of the hexane extract

on

the distillation behavior. The

was

hexane of the hexane extract removed either with

by evaporation nitrogen (HexN D) or by adsorption of the oil components on a silica column (outflowing hexane: H) and suspending the dry silica in P-buffer (HexSil A).
HexN and HexSil concentrates distilled in
a

were

miniaturized

distillation apparatus with XADcolumns (see Figure 7.1) to retain the vtiver oil components. The XAD-2 columns were eluted three times buffer

(M1
was

M3)

and the residual

extracted

(R).

The

original
buffer

reextracted

hexane extract and HexN, directly from the P-

(HexN contr.), were used as samples were analyzed by GC. The recovery of
controls. All material A, B and C
was

determined after each extraction

step.

segment C
rBl
625
T--

500

"

375

a>

250

125

D-

kZ
T-

H
<M

1
CO

1
__

c5

139

outflowing
more

hexane and the distillate of HexSil concentrate

together

contained

material than the distillate of HexN concentrates

(Figure 7.7).

After about 4 hours distillation, the eluates of the Amberlite columns of the

HexSil distillation contained less material B and C than those of the HexN

distillation, but after extraction of the residual buffer (R), both resulted in the
same

recovery

(Figure 7.7).

This indicates that it takes

longer

to distill the oil

adhering

to the silica than the oil

floating

free in the

phosphate

buffer.

Comparison
distillation

of hexane extracts and roots

as

starting

material for small scale

To compare the effect of the hexane extracts

starting

material

on

the distillation
to the

performance,
were

(HexSil, HexN)
a

and roots added

a

directly

P-buffer,
was

distilled. To obtain

complete distillation, large

scale distillation

second distillation step

more

added.

Analogous by

to the

(Chapter 6),

oil

was

extracted

distillation from the roots than

by

solvent extraction: the first and the second

distillation steps

yielded larger

amounts of oil

(about

1.3

material

B)

than the

hexane extracts 1 and 2,

respectively. nearly
all oil components of HexN
were

After the first distillation step

distilled,
recov

whereas the second distillation step still resulted in 5 -10 % of the total

ered oil for the HexSil distillation and about 20 % of the total recovered oil for the root distillation. Scheffer

(1996) already

described that distillation

removes

the volatiles much faster from solvent extracts than from We found that

plants containing

them.

adsorption

of the hexane extract

on

the silica

similarly prolonged

the distillation time

(Figures 7.7, 7.8).

After the two distillation steps, the HexN and HexSil distillates contained about 90 % material B of the buffer increased this amount

starting

hexane extract 1 and extraction of the rest

1 %. Therefore about 9 % of the
can

maximally by
This

original

material B is
errors

missing.

discrepancy

have several
some some

causes:

integration
be lost

of the GC may

cause

small variations;

of the oil could of the oil may still

during sample preparation

on

before the distillation;

was

be adsorbed of the oil

the Amberlite column and the column;

or

not removable with

MTBE;
a

some

evaporated through

or some

oil

might

still be in

not

extractable form in the rest buffer

the wet silica. The first

option

is

unlikely,

140

segment A
15.0

Figure

7.8. Combined

distillation-solid

phase

extraction of hexane extracts and roots. A vtiver root hexane extract, evaporated with nitrogen (HexN D) or adsorbed distilled in
on

silica

and vtiver roots

a

(HexSil A) (O) were

miniaturized

distillation apparatus with XAD-columns

in two

(see Figure 7.1)

steps. After the first distillation, the buffer was re

plenished
segment B
625
T-

and the Amberlite

column

was

replaced.

After

the distillations, the columns were eluted (first distillation:

M3; second distillation: M4, M5) and the residual

M1
-

buffer and the roots extracted

were

(R). As controls, roots were extracted twice (first

hexane

with hexane
extract

^;

second extract
were

[2]).

The hexane extracts

prepared
instead of
ra

as a

for distillation, but

x-c

x c

>< c

o
l8

cdo

xo

subsequent distillation, the compounds were reextracted (HexN contr.:

first extraction extraction

[T^)

^,
or

second

reeluted

(HexSil
hexane

contr.:

|~J, first_e_lution

outflowing

), respectively. All samples were analyzed by GC. The yield of

second elution material A, B and C per 1 ml hexane extract or per 100 mg dry roots was determined after each extraction step.

i-

<M

CO

141

as

all three

repetitions

resulted in recovery around 90 % of the have

an

original

hexane

extract. All other

options might

influence,

after

evaporation

smaller

amounts of material A were recovered and also after elution of the silica column

less oil

was a

found back,

after three times elution of the Amberlite columns with

MTBE,

few percent of the oil could still be extracted with ethanol

-

(results

not

shown)
directly
was

and

from HexN, which

was

after the

evaporation

of the hexane,

reextracted from the P-buffer,

only

about 85 % of the

original
on

material B

recovered
as

(Figures 7.7, 7.8).

a

part of the oil might still stick

the silica

gel,

the eluate from

wet silica column contained

only

about 40 % of the

original

hexane extract after 3 times

rinsing

with 0.5 ml MTBE

(results

not

shown).
From this distillation

experiment,

we can

conclude that

prior

extraction with

hexane is not necessary. In fact, direct distillation of dried material results in

even

better

yields.

The distillation of the roots takes

longer

than the distillation

of the hexane extract, but

more

already

after the first distillation step

(up

to 5

hours),

oil

was

extracted than after three extractions with hexane.

Adsorption capacity

of XAD-2 columns

The distillation of the roots has proven that

cm

more

oil

can

be retained

by

the 2

Amberlite column than the amount of oil in 1 ml hexane extract. Therefore

different amounts of hexane extract 1 silica and

(1, 1.5, 2, 2.5, 3,

ml)

were

loaded

on

subsequently
of
a

distilled in P-buffer for about 5 hours, to determine the

2
cm

adsorption capacity
The 2
cm

XAD-2 column.

XAD-2 column

was

able to adsorb all tested amounts of oil from

the steam

(Figure 7.9). Only

was

for the smallest amounts, 1 and 1.5 ml hexane

extract, less oil

from all

recovered in the eluate. Most

probably

some

oil

was

lost but for

samples,

for

example by sticking
a

to the Amberlite or to the

glass,

smaller amounts of oil such losses had hexane extract all volatile

bigger

effect. Between 2 and 4 ml of

compounds
tested,
as

were

adsorbed

by

the column.

Higher
12 %

amounts of extract were not

with 4 ml hexane extract 1

were

already

of the total material B and 17 % of the total material C silica column

rinsed

through

the

during loading

of the hexane extract. The residual buffers

material C

[pg]
material B

[pg]
r\j ^j
-> -J-

material A

[|jg]
INJ
o en

-1

w r\ ^j INJ
en o o en o en en o en o en o en o o o o o en o o

w CO
o

CO"
-I 1-

en o o

o o o

en o o

o o o

en o o

o o o

I
H
.

NI

CO

M1
1

.1

a>

M2
1

t-

M3

R
CO ro -si
en o
i i i i i

Lit
CO
o en o

en o o

o o o

en o o

o o o

en o o

o o o

inj -si
en o en o en o o

en o

en o o

o o o

1
i i i

1 ml
zr
i i i i i

I"
CO

_-

-"n
CD
CO

1 ml 1.5 ml
-

CT

1.5 ml

-=^n i
I
i
-

i
i
I I

:
i
I I

:
i
I

CD
CO

]
!
CD
CO

i
i;
3
CD
, , ,

CO

CD

5
CD
Z3

2 ml

I |
i,
o

J
i
: :
DD

J
l
i:

i i

i i

i i

2 ml
"

i
' '

:
2.5 ml
-

CQ

2.5 ml

li
I I I

i
I

i
I

CD

|
-J-

3 ml

3 ml
' '

i
4 ml

4 ml

'|i
-si
en o o en o

->

INJ
o o en o o o

INJ inj -si

en o en o en o o o o o

CO inj
en o

en o

o o

en o

|\J
o

|\J
en

CO
o

-\

4=

M1

"ft

Ft

M2"fr

T^kT
11nil
:
1

t\i A
1

l-j^-l

143

contained about 5 % of the total material B and up to 74 % of the total material

(Figure 7.9).

Comparison
solid

of small scale hexane extraction and combined distillation extraction to

screen

phase

for oil variants

In this

chapter
for
a

hexane extraction and distillation with

phosphate

buffer

were

optimized

small amount of root material and

(100 mg).

For hexane extraction,

roots were

pre-wetted

subsequently

extracted three time with hexane. For buffer

distillation, the

roots were distilled in

phosphate
on

(0.5M, pH8)

in

an oven

at

120C, with

an

Amberlite XAD-2 column

the tube to adsorb the oil from the

steam. The column was rinsed three times with MTBE.

For both methods, the

major part

of the oil

was

extracted from the roots in the

first extraction step and from the Amberlite column in the first elution step. Additional material
was

extracted in

subsequent

extraction
more

or

elution steps, but

the concentration of the total extract decreased, and removed to detect the oil components oil. The ratio of the material Ba
or
-

solvent needs to be

by GC, especially
about the
same

for small amounts of after the first, second it is

Bf

was

third extraction
one

or

elution step,

respectively. Therefore,

possible

to use

only

extraction

or

elution step. Due to variations in the recovery of the first

eluate

compared

to the total eluate after three elutions or

extractions, the

Adsorption capacity of a 2 cm XAD-2 column for small scale phase extraction. Different amounts of hexane extract (1 (), 1.5 (), 2 (), 2.5 (D), 3 (O) and 4 ml (A)) were loaded on silica columns (outflowing hexane: H). The silica was distilled in a miniaturized distillation apparatus with a 2 cm XAD-column (see Figure 7.1) to adsorb the Figure
7.9.

distillation combined with solid

vtiver oil from the steam. After the distillation, the XAD-2 columns were eluted with methyl ferf-butyl ether (M1 M3) and the residual buffer was extracted (R).
-

All

samples were analyzed by GC. The recovery of material A, B and C was determined after each extraction step. a, c, d, f, g, i) sum of the material X of the outflowing hexane during loading of

the silica column, the eluate of the XAD-2 columns after distillation and the residual buffer extracts

h) material X of extract used analyses of 1 ml hexane extract

b,
e,

as

starting material,

calculated from GC

144

quantitative analysis
will be detectable.

will not be exact, but

qualitative changes

of the vtiver oil

With both methods, the concentration of the very small amounts of induced oil in the eluate

(2 ml)

or

extract

(1.5 ml)

will still be too low for detection

by

GC.

Therefore, before the analysis of the

be added. Hexane extraction of the roots is wetted roots have

extract

by GC,

concentration step has to

more

convenient to

perform
over

as

the prewhereas

only

to be

put in hexane and extracted

night,

for the distillation, columns have to be

prepared

and eluted after the distillation.

extract with

However, the removal of the non-volatile compounds of the hexane

the tested methods distillation distilled volatile
-

was

either not effective or,

was

as

in the

case

of the combined

solid

phase extraction,

not necessary, because roots can be

a as

directly.

Therefore hexane extraction is do not disturb the

useful method it is for TLC.

as

only

if

non

compounds
if
more

analysis,

Finally,

sensitive

analysis methods,
a

such

GC, should be used,

distillation with the Amberlite column is

very useful tool.

ACKNOWLEDGEMENTS

The Vtiver

plants

were

provided by

Mr. Heini

Lang,

Jakarta. This work

was

supported by

Givaudan-Roure

Forschung

AG Dbendorf, Switzerland and

no.

by

the Swiss Federal Office for Economic

Policy, project

2561.1 of the

Commission for

Technology

and Innovation.

145

Chapter
material

8: Initial

study

on

induction of vtiver oil

production

and accumulation in tissue culture:

preparation

Ruth E.

Leupin,

Charles Ehret, Karl H. Erismann and Bernard Witholt

146

ABSTRACT

To pre-screen

regenerated production might

vtiver

plantlets

for

changes

in oil

composition

in

tissue cultures, oil

and accumulation must be induced. Since the

state of differentiation

influence the induction of essential oil

biosynthesis,

in vitro

plantlets,

root cultures and calli should be tested.

Nevertheless, the oil

tissue for extraction

content of in vitro tissue is

expected

to be low and

enough
-

and

analysis

is therefore necessary. After about 4

6 months

growth,
the

about 100

mg

(dw)

roots can be obtained from in vitro

plantlets. By varying plants

plant

growth regulator composition

were

in the medium,

with different root types

obtained, which could be interesting for oil induction experiments. All trials

to obtain

continuously growing

root cultures were unsuccessful.

However,

continuously growing
oil

root cultures

might

not be necessary for the induction of

biosynthesis

or

precursor

feeding.

The calli from root

are a

tips

grown

on

modified

MS medium

supplemented production

with NAA

very

interesting
some

tissue for the

induction of oil

or

accumulation, since

of them smell of vtiver

oil. However, here also it takes at least 2 months to obtain extraction.

large

calli for oil

INTRODUCTION

Vtiver oil is

valuable

raw

material in
or

perfumery.

New vtiver variants

containing
are

more

of the essential oil

another ratio of the different

compounds
traditional

therefore of interest. New variants could be obtained either

et al.

by

breeding (Gupta
or

1983; Lai et al. 1998; Sethi 1982; Sethi and Gupta 1980)
et al.

via tissue cultures

(Chapter 3, Leupin

2000), potentially

altered with

additional chemical

or

physical mutagenesis.
are

To determine whether the

regenerated plantlets
with the
at an

oil variants, the oil has to be the essential oil

can

analyzed

and

compared

original

oil.

By inducing screening

biosynthesis
for

and accumulation
in the vtiver
or

early stage,

the

be done

specific

changes

oil. Since undifferentiated cultures

only rarely
are

accumulate

monoterpenoids

sesquiterpenoids

in

quantities

that

comparable

with those present in the

are

parent plants (Charlwood and Charlwood 1991), in vitro cultures of vtiver

147

expected

to contain very small amounts of

oil, implying that the oil production

material has to be available to

or

accumulation has to be induced, and

extract and

enough

analyze

the oil with the miniaturized

procedures

described in

chapters

5 and 7.
we

In this work,

studied the usefulness of different tissue cultures such

as

in

vitro

plantlets,
or

root cultures and callus cultures for the induction of vtiver oil

production analyze

accumulation and the

feasibility

to

produce enough

tissue to

the oil content.

MATERIAL AND METHODS

In vitro

plantlets

To

study

the influence of the

were

growth

medium

on

the root

production
see

of in vitro

plantlets, they

cultured

on

VRMO, VPM, VRM8 (composition

chapter

2)

or

VBM

(modified

MS medium

(see Chapter 2) supplemented

The

with 1 mg I"1
-

a-naphthalene

acetic acid

(NAA)).

plantlets

were

transferred every 6

weeks, after reducing the leaf length and removing the roots,
All in vitro

to fresh medium.

plantlets

were

cultured at 23C with

12 hours

photoperiod.

Root cultures

To obtain
root

growing

root culture which can be

subcultured, 5-10
on

mm

long

tips

of

plantlets

grown for several subcultures

new

VBM,
on

were

cut 2 weeks

after the last transfer to These media

were or

VBM and &

were

cultured medium

different

liquid

media. and

1/2

Murashige

Skoog

(1/2 MS) (Murashige

Skoog 1962)

VWM

(0.25

g I"1 KCl, 0.144 g I"1

7

Ca(N03)2

H20,

0.5 g I"1

NH4N03,

0.25 g I"1

MgS04
4 mg

H20,

2 g

I"1 NaCI, 0.835 g I"1 KH2P04, 0.688 g I"1

0.5 mg I"1

Na2HP04
sucrose,

H20,

I"1

FeCI3

H20,

thiamine-HCI, 3.0 %

pH 7) supplemented

with the
or

growth regulators 2,4-dichlorophenoxy

acetic acid

(0-0.1

mg I"1

2,4-D)

NAA

(0-1
was

mg

I"1)

and casein

hydrolysate (0,

0.05, 0.5, 5 g
culture.

I"1).

The

growth

of the roots

measured after 1 month in

148

To obtain

continuously growing cultures, they

either the

were

subcultured

by

transferring

complete

roots or root

tips (5 mm)

to new medium.

Root cal M

on

solid medium

To induce calli from roots, 5

or

mm

long

root

tips

from

plantlets

grown

on

VBM

VRMO

were

cultured

on or

solid modified MS medium

containing

various

concentrations of NAA

2,4-D (0.5, 1, 2.5 mg

I"1).

RESULTS AND DISCUSSION

The in vitro accumulation of

monoterpenoids

and

sesquiterpenoids differentiating

was

reported
shoots,

in callus,

suspension

cultures and callus with

roots or

as

well

as

in shoots and root organs and in

fully

differentiated tissue

(Charlwood

and Charlwood

1991).

To induce the essential oil in tissue cultures

in vitro

of vtiver, different callus cultures

or

starting materials, including

plantlets,

root

cultures,

liquid

cultures should be considered.

In vitro

plantlets

By using
Precursors

in vitro
or

plantlets,

the

same

tissues

are

available

as

in in vivo

plants.

parts of the oil could be produced in any tissue and be

trans

ported

to the roots. To obtain sufficient roots for the

analysis,

media

containing

different
some

growth regulators only

after

were a

tested. On vtiver

propagation

medium VPM,

roots grew

long

time without subculture. On VRM8 the roots

did not

develop side-roots,

whereas without any

growth regulators (VRMO),

the

roots have side-roots. On VBM medium calli were first roots with side-roots were

induced, and later thick

on

produced.

After several subcultures

were

VBM, the

callus

production

was

reduced and the roots

induced faster after

subculture. could

By reducing

the NAA content in the medium, the callus induction

already

be reduced in the first culture step.

Since VRMO, VRM8 and VBM each resulted in roots with different

pheno-

types (Figure 8.1 ), it

was

of interest to test each of these root types in oil

149

production experiments.
months until 100 mg

However with all three media, it took

more

than 6

dry

roots were obtained. To increase the amount of roots

versus were

formed, the effect of solid

For that purpose, concentrations

liquid

medium
on

on

root

growth

was

examined.

plantlets

cultured and
on

VRMO

containing

different agar

(0.3, 0.65,

0.9

%)

liquid VRMO, using

was

wire-screens to

support the shoots. After

4

months,

no

obvious trend

detectable.
more

Only

after
-

months, plantlets
were

on

the wire-screens

produced slightly

roots, but 4

months

still necessary to obtain 100 mg roots

(dw)

per

plantlet (data

not

shown).

One

possible advantage

of

liquid

medium is that

subsequently
more

added
in

compounds liquid

for the induction of vtiver oil

biosynthesis

distribute

easily

than in solid medium.

To test the influence of the age of the which

on were

plantlets
or

on

root

production, plantlets,
on

previously

subcultured for 0, 1, 5 of roots

12 times

VRM8,

were

grown

VRMO. The

resulting dry weights

were

were

similar for all

plantlets, except
medium.

for

plantlets

which

directly

transferred from the

propagation
but
over

These with

produced
which

less root material at the

were

beginning,

time the difference

plants

subcultured at least once, dwindled and after 8 months

not not

all differences

disappeared (data
was

shown).
possible
to obtain 100 mg

With all tested methods, it less than 4

-

dry
are

roots within too small.

6 months and for

long

cultures the 400 ml beakers

were

Therefore 1.5 I

glass jars

were

tested, and single plants

cultured in

jars

on

solid VRMO medium. Some of the

plantlets

died and the

surviving plantlets only

150

produced
-

fewer roots in 6 months than in the beakers. After 12 months

300 mg roots

(dw)

were

obtained per

plant. Hence,

the

growth

conditions need

further

improvement.

Since

given

treatment of a

plantlet might
used for

have

an

influence

on

subsequent
can

oil induction, the used for induction

a

plantlets

that The

are

one

induction

experiment

not be

later

repetition.

plantlets
as

must therefore be

propagated

before

experiments, especially

the oil induction treatment could harm the

plant.

150

VRM8

VRMO

VBM

Figure 8.1. Root production of in vitro plantlets on different media. Culture of plantlets on media supplemented with different amounts of growth regulators (VRM8: modified MS medium supplemented with 1 mg 11 kinetin and 0.1 mg 11 indole acetic acid (a, d, g); VRMO: modified MS medium with no growth regulator (b, e, h); VBM: modified MS medium supplemented with 1 mg 11 anaphthalene acetic acid (c, f, i)) resulted in roots with different phenotypes. The plants were harvested after 4 (a, b, c), 6 (d, e, f) and 8 months (g, h, i). bars 5
=

cm.

151

Root cultures

The essential oil is

in the rest of the

mainly

found in the roots, and Ruskin

although

traces may be found root cultures

plant (Vietmeyer starting

1993). Therefore,
experiments.

would be

useful
a

material for oil induction

To obtain cultured medium

on

growing

root culture which can be

subcultured,
was

root

tips

were

different

liquid

media. The best

growth

obtained with VWM which not

supplemented
the

with 50 mg I"1 casein

hydrolysate (VWM-C),

only improved

growth

but also the number of side-roots per root. On

average, the roots grew to grow any

the roots
more was

only

20

mm

and became thin. These roots did not

after

subculturing
not

their root

tips (5 mm).
was

The variation between

to obtain a

large (results
root

shown)

and it

not

possible

continuously growing

culture, producing enough

roots to

analyze

the oil

content, with any of the tested media. However, for oil induction it might not be
necessary to have

continuously growing roots;

it

might

be

enough

to have

living
their

roots. Since roots which were transferred to new medium without

reducing
not

sizes still grew

little and continued to

produce

new

side-roots

(data

shown),

we

concluded that

they

are

still alive after

to cut the

culturing

for 4 weeks in
roots of in vitro

VWM-C. It

might

therefore be

possible

complete liquid

plantlets

and culture them

during

the oil induction in

VWM-C. The
are

advantage by

of such root cultures is that the

regenerated plants
can

not influenced
as soon as

the induction

experiment

and the

experiment

be

repeated

enough

roots are available

again.

Hairy

root cultures

Another

approach

to obtain

continuously growing
in

root cultures is to infect the root cultures.

plant

with

Agrobacterium rhizogenes, resulting

is
a

hairy

Agrobacterium
and

soil bacterium, that

causes

gall (Agrobacterium tumefaciens)

of the

hairy

root formation

(Agrobacterium rhizogenes) by transferring parts

or

bacterial Ti

(tumor inducing)
plant
cells

Ri

(root inducing) plasmid

DNA into the nuclear

root cultures

genome of

(Conner

and Dommisse
can

1992). Hairy

commonly

exhibit very

high growth rates,

and

be cultured in the absence of

plant growth regulators

generally

retain the

capability

of the

parental

tissue

152

to accumulate

secondary products (Charlwood 1993;

Charlwood and
have been
mono

Charlwood

1991). Monocotyledonous plants, particularly cereals,

considered outside the host range for A. tumefaciens, because most

cotyledons
and Hood

do not form tumors

as a

result of A. tumefaciens inoculation

(Smith
A.

1995). However,

gene transfer and

consequently

infection

by

tumefaciens have been proven for many Triticum aestivum, Hordeum formation in

monocotyledons, including

Zea mays,
tumor

vulgare

and
a

Oryza

sativa. The

missing
or

monocotyledons
hormone

is due to

lack of

transcription,

to different

endogenous

physiology compared

with

dicotyledons (Smith

and Hood

1995). However,

if the lack of tumor

production

after infection with A. of

tumefaciens is due to the different

endogenous growth regulator physiology "hairy"

root cultures from vtiver after

monocotyledons,
infection

the chance to obtain

by

A.

rhizogenes might

be small.

Callus culture

on

solid

or

in

liquid

media

Callus cultures

on

solid media

Another

possibility

for

screening
on

the
or

regenerated plantlets liquid media,

is to

use

tissue

cultures, like callus cultures

induction
root

solid

for the essential oil slices

experiments.

To induce calli, leaf slices,

crown

(Chapter 2)
root

and
are

tips

can

be used, but since the

as

plant
root

should not be

destroyed,
on

tips

ideally

used

expiants. Therefore,

tips
or

were

cultured

solid media

containing

various concentrations of NAA calli

were

2,4-D (0.5, 1, 2.5 mg

calli from
are

I"1).

With both
0.5

growth regulators,
or

induced. On

some

plates containing
also

1 mg

I"1 NAA, root tips

were

observed. These calli

or

interesting,

since

the accumulation of

monoterpenoids improved by

sesquiterpenoids degree
of

of undifferentiated
or even

cultures could often be

some

cytodifferentiation,

morphological

differentiation

(Banthorpe 1988;

Charlwood and Charlwood 1991 ;

-

Charlwood et al.
calli

1989). However,

it took at least 1

2 months to obtain

larger

(data

not

shown).
are

Nevertheless, these cultures

root

very

interesting,

because

some

calli from

tips

cultured
a

on

modified MS medium

supplemented

with 0.5 and 1 mg I"1

NAA

produced

smell similar to that of vtiver oil. A very concentrated

methyl

153

terf-butyl

ether extract of these

on a

smelly

root calli and of their medium showed

similar spots

thin

layer chromatogram (silica gel plates, developed

as

with

hexane and chloroform

consecutive solvents and stained with These

anisaldehydesame

acetic acid-sulfuric acid;

Chapter 5).

compounds

ran

with the

Rf

as

the acid components and vtiver oil

(very faintly)

at the lowest "non-acid"

spot of the
calli
was

(Figure 8.2). Unfortunately,

the induction of

smelling

not

reproducible. However,
in later

the results of these

experiments

should not be

ignored

stage oil induction experiments.

Figure

8.2. Thin

layer chromatogram

of extracted

tips were containing 0.5 (sample 1) or 1 mg 11 a naphthalene acetic acid (sample 2). The calli and the corresponding media were separately extracted with methyl ferf-butyl ether. The concentrated extracts were analyzed by thin layer chromatography (developed with hexane and
induced
on

root calli and their media. Calli from root

medium

chloroform with

as

consecutive solvents and stained and

anisaldehyde-acetic acid-sulphuric acid) compared with vtiver oil (25 ug, sample V).

Liquid

cultures

Previously,

we

obtained well

growing liquid
of compact

cultures that

were

tested for Mucciarelli and

plantlet regeneration, consisting Leupin,

in

clumps (Chapter 4;
were

press).

The

starting

material for these cultures

are

compact calli

from leaf and

crown

slices, which

in

principle

not available when the

plant

should stay alive. The induction rate of these

more

liquid

cultures

was

low and it took induction

than 6 months until the cultures

were

homogenous. Therefore,
the

of these

liquid

cultures is not useful to

screen

regenerated plantlets.
to

However, such liquid cultures might be interesting

reactors. For this purpose, the

produce

the vtiver oil in

growth rate,

biomass level and

yield

of vtiver oil

154

in the in vitro material has to be sufficient to

on

compete with oil produced in vivo

plantations.
As the
occurrence

of somaclonal variation increases with the duration of the

disorganized phase

and the extent of the

disorganization (Karp 1994), suspension during

cultures for pre-

it has to

be taken in account that

by using

callus

or

screening,

detected oil

changes might

arise

the culture and may not be

intrinsic to the

plant.

CONCLUSIONS

Different methods

are now

available to obtain in vitro vtiver tissue for

experiments
In vitro

to induce the oil

biosynthesis

or

accumulation.

plantlets produce

about 100 mg roots

(dw)
the

in 4

6 months, with

different

phenotypes

of the roots,

depending

on

plant growth regulator directly

for oil induction

composition
or

in the medium. The

plantlets

could be used

the

complete

roots could be cut off and cultured in

liquid

medium for oil

induction. Another very

promising place

tissue is callus from root

tips.

To test if

differentiation has to take

for the

production

and accumulation of vtiver calli and calli with

oil, it is necessary

to

distinguish

between

unorganized

regenerated Using

root

tips. study,
oil induction

the material described in this

experiments produce
after

can

be

started. These

experiments plants

will show whether it is feasible to

can

vtiver oil

in vitro, and whether

be screened in

an

early stage

regeneration.

155

Chapter

9: Conclusions and outlook

156

The aim of this

project

was

to obtain vtiver variants with a

higher

vtiver oil
we

yield

or a

different oil

composition
to

via tissue culture. To reach this

goal,

developed procedures
we

regenerate plantlets via tissue cultures. Additionally

optimized

methods to extract the oil from many small

samples

and detect

qualitative

and

quantitative changes.

1. TOOLS TO OBTAIN VETIVER VARIANTS

1.1. In vitro cultivation of vtiver

During

this

study

an

efficient

plant regeneration system

via callus culture

was

developed
the other vitro

for the used

non-flowering

vtiver variant from Java. In contrast to

crown

regeneration
were on

methods described for vtiver,

as

and leaf slices of in

were

plantlets

used

starting

material.
crown

Regenerated plantlets
on

found

within 18 weeks The

up to 55 % of the

slices and

up to 60 % leaf slices.

regeneration

via

liquid

cultures

was

less successful than via callus, since it

took 6-14 months before compact structures before the cultures

were

developed

and

even

longer

homogeneous

and at that time

plantlets

did not

regenerate anymore. Therefore, regeneration via callus is the

method to obtain somaclonal variants.

more

promising

It is not

possible

to

predict

whether

our

regeneration regeneration

method via callus would methods did not result in

also work for other vtiver variants. Other

regenerated plantlets
would be

from

our

in vitro

non-flowering

vtiver variant from Java. It is related

same

interesting

to know whether the lack of

regeneration ability
in vitro

to the medium or the

starting

material

(in

vivo

or

expiants).

At the

time it would be necessary to test whether useful for other vtiver variants.

our

regeneration

method is also

1.2.

Obtaining

vtiver variants

First, regenerated plantlets should be screened for somaclonal variants with

the desired trait. If the

frequency

of variation is too low, it

can

be increased

by

157

inducing plants

mutations

by

irradiation

or

mutagenic

chemicals. To avoid chimeric irradiation of leaf slices, sub

a

due to mutation of

pre-existing meristems,

sequent callus induction and plant regeneration would be

However, the correlation between irradiation dose
must still be determined.

useful

procedure.
rate

rate and

regeneration

To obtain variants via since

genetic engineering,

extensive studies

are

necessary,

nothing

is known about the molecular

biology

of vtiver and genes that

limit the

biosynthesis

of the vtiver oil components must be identified.

2. IDENTIFICATION OF VARIANTS

To determine whether the be

regenerated plantlets original

are

oil variants, the oil has to

analyzed

and

compared plant

with the

oil. Since it takes about 15-22 vtiver oil

months until the

contains the

complete

(Roth

and Kornmann time is

1997; Weiss 1997a), and starting from in vitro plantlets

even more

needed,

pre-screening

of the

plantlets

in

an

earlier stage would be advan

on

tageous. DNA based pre-screenings have been developed, based

random

amplified polymorphic

DNA

(RAPD),

restriction

fragment length polymorphism

microsatellite repeat

(RFLP), amplified fragment length polymorphism (ALFP),

polymorphism (also polymorphic
known
as

simple

sequence
et al.

repeat)

and cleaved

amplified

sequence

(CAPS) (Jones

1997; Rafalski and Tingey 1993;

is known about the

Ridout and Donini between vtiver oil

1999). Unfortunately, nothing

production
for

relationship
result it is for the

and such DNA based of

changes.
and

As

necessary to

screen

changes

plant phenotype

especially

production

of

more or

altered oils.

2.1.

Analysis

methods

Different

analysis

methods have been

compared

for their
as a

ability

to detect

qualitative
small

and

quantitative changes

of the vtiver oil,

pre-screen of many
nor

samples

from in vitro cultures. Neither

were

olfactory

detection

the inhibition

of bacterial

growth gain

able to
was

replace

the time

consuming

gas chromato

graphy.

The

of time

too small

compared

to the loss of information.

158

With thin

layer chromatography, qualitative compounds

more

and

quantitative changes

and

even

the non-volatile

of the extract

were

detectable. However, GC

analysis provides

detailed information and

requires only

0.5 ug oil per

analysis. Therefore, samples,

TLC is

preferred

for

preliminary analysis

of many
a

whereas GC

analysis provides

more

detailed information for

smaller

set of selected

samples.

2.2. Extraction methods

To extract the oil from the roots, water distillation and solvent extraction

were

compared

for their effectiveness in

extracting

all vtiver oil and for the

possibility

to miniaturize the

procedures

for many small

samples. by

The

long

distillation time necessary for vtiver roots could be reduced buffer

using

phosphate
of the

(0.5M)

at

pH

8 instead of water.
an

Additionally,

the
to

replacement

cooling system by
in

Amberlite column made it

at 120C.

possible

distill many small

samples

parallel

in

an oven

All the tested solvents

(hexane, methyl ferf-butyl ether, ethyl

same

acetate and

ethanol)

extracted about the

amount of volatile

components but different

were

amounts of non-volatile

compounds,

of which fewest

extracted with

hexane. Due to the

simple equipment requirements,

hexane extraction could be

miniaturized to extract 100 mg roots in 1.5 ml hexane.

Although
gas

the extraction method has

an

influence

on

the oil

composition,

the

chromatograms

of the hexane extract and the distillate

were

similar. The

choice of the extraction method

depends

therefore

largely

on

the

experiment.

Hexane extraction is less labor intensive than combined water distillation with solid

phase

extraction. However, hexane also extracts non-volatile

compounds,

which

complicated subsequent

gas

chromatography analysis. problem

a

For both extraction methods,

one

is the dilution of the

samples.

If

only

trace amounts of the oil are

available,

concentration step has to be

included, since GC analysis requires

concentrate the total

at least 0.5 ug oil per

analysis.

To

oil, evaporation with nitrogen is very promising for small eluates, whereas for larger
or

amounts of hexane extracts or MTBE

amounts of
a

hexane extracts the silica column method alone

in combination with

subsequent evaporation

with

nitrogen

is useful.

159

2.3. Amount of tissue necessary for small scale extraction and

subsequent analysis

The

yield

of vtiver oil from in vivo vtiver roots varies between 0.1-3.3 % oil, the vtiver variant

depending

on

(Akhila
the oil

et al. 1981 ;

Anonymous 1976;

Roth about

and Kornmann

1997).

To

analyze

by

gas

chromatography (GC)

0.5 ug oil is necessary per

injection (Chapter 5). However,

one

the minimized

extraction does not extract the total oil in better

extraction step

(Chapter 7)
dry

and for

handling

we

need 100 ul extract. Therefore, about 5 mg

roots are

necessary for roots

than 0.1 % oil, cultures

containing

1 %

oil, and for plant material containing less

material is needed. Since undifferentiated
or

more

than 50 mg

dry

only rarely
are

accumulate

monoterpenoids

sesquiterpenoids

in quan

tities that

comparable

with those present in the parent

are

plants (Charlwood
to contain very

and Charlwood

1991),

in vitro cultures of vtiver

expected
or

small amounts of oil,

implying

that the oil

production

accumulation has to be

induced, and enough material has

with the miniaturized

to be available to extract and

analyze

the oil

procedures.

3. INDUCTION OF OIL BIOSYNTHESIS AND ACCUMULATION

The

ability

to pre-screen

regenerated plantlets depends

on

the

possibility
or

to

induce the oil

production

and to accumulate the oil in the

a

plantlets

the in vitro

cultures. Therefore the next step will be to find and /

or

way to induce the

biosynthesis

to accumulate the vtiver oil in in vitro cultures.

To induce the essential oil know

biosynthesis

in the

plant,
not

it would be useful to for

why

the

plant produces

it. Essential oils and

are

absolutely required
are

viability, though especially

of

mono-

sesquiterpenoids

important help

mediators

plant-plant, plant-insect, plant

and

plant-pathogen

interactions and

to

adapt

the

to different environmental conditions

(Charlwood 1993;

Kribii et al.

1999; Newman and Chappell 1999).

However, nothing is known about the biosynthesis pathway

of essential oil
will

or

the

regulation

biosynthesis

in vtiver. This

implies

that the induction of the oil

mainly

be

matter of trial and error.

Nevertheless, the induction might be

160

feasible, because

some

calli from root

tips

on

modified MS medium

supple
The

mented with 0.5 and 1 mg I"1 NAA did smell of vtiver oil induction of

(Chapter 8).
so

smelling calli, however,

was

not

reproducible,

that

a more

systematic approach
or

has to be followed to induce

sesquiterpenoid biosynthesis

to increase the amount of oil

compounds

in vtiver tissue cultures.

3.1. Medium

composition

and environmental conditions

The usual cultures is to

sources

approach

to induce

secondary product

accumulation in carbon and

plant

cell

manipulate

the culture medium

by varying

nitrogen

and their concentrations, the

or

phosphate

concentration and the range of

as

plant growth regulators,

to

change

environmental conditions such

et al.

temperature, light and gas composition (Scragg 1997; Stafford

1986).

Especially interesting,

different concentrations and ratios of

as

growth regulators

are as

they

can

influence the root type of the in vitro

plantlets,

described above. For callus cultures, these

manipulations

may result in

changes

in

cytodifferentiation production
of

or

morphological
essential oil
or

differentiation which in turn may oil with

a

result in the

more

composition

that differs

from that of undifferentiated cultures

(Charlwood

and Charlwood 1991 ;

Charlwood etal.

1989)

3.2. Elicitor treatment

By applying

stress to the

plant cultures, they

is

may react with

one

secondary

metabolite formation. mechanisms

Phytoalexin production

of the

plant-defense
consist of

already recognized against pathogens. Phytoalexins

flavonoid/isoflavonoids, terpenoids and polyacetylenes (Weissenborn etal.

1995).

Their

production

is

triggered

either and

by pathogen

attack

or

by

biotic and

abiotic elicitors include cells

or

(Chvez-Moctezuma

Lozoya-Gloria 1996). viruses), applied

Biotic elicitors

plant pathogens (bacteria, fungi

fractions thereof
or

or

in the form of

living
as

(glucan polymers, glycoproteins, organic

cell-wall of the

acids such

arachidonic acid,

fungal

material), degradative

enzymes and

resulting

cell wall

fragments
and

plant (DiCosmo

and Misawa 1985; Threlfall

and Whitehead

1991),

signal

molecules involved in defense reactions such

161

as

methyl jasmonate (Singh

etal.

1998)

or

salicylic

acid

(Ram
UV

etal.

1997).

Abiotic elicitors include stress

inducing

conditions such

as

light, pH
etal. 1988;
on

changes,

osmotic stress,

wounding

and

heavy

metal ions

(Holden

Threlfall and Whitehead

1988).

The effect of any elicitor is

dependent

the

specificity

of the elicitor, elicitor concentration, the duration of treatment and the of the culture

growth stage

(Holden
only

etal.

1988).

Treatment of

plants

or

tissue but

cultures with elicitors leads not also other, non-antimicrobial

to the accumulation of
can

phytoalexins,
etal.

compounds

be formed

(Holden

1988).

The vtiver

plant

shows resistance to infections etal.

by

root-knot nematodes

(Meloidogyne spp.) (West (Vietmeyer

and Ruskin

1999),

the roots

are

used to

repel

insects and

1993),

the oil inhibits bacteria and

fungi (Chaumont

Bardey 1989;

Dikshit and Husain 1984;

Gangrade

etal. 1991 ;

Gangrade

etal.

1990; Hammer etal. 1999; Maruzzella and Sicurella 1960) (Chapter 5) and
some

bacteria

are

observed in the The oil

or

same

root cells as the oil

(Viano

etal. 1991a;
a

Viano etal.

1991b).

at least some

compounds

could therefore have of oil is takes

defensive function in vivo. The

question

is whether the

biosynthesis

constitutive, inducible

or

both. The inducible

phytoalexin biosynthesis

place

over a

short, well-defined period of time (Wolters and Eilert 1983) and the
are

phytoalexins
Whitehead

normally

not detectable in

healthy plants (Threlfall

and

1988).
approaches
to

Additional

improve terpenoid biosynthesis by

precursor

are

the induction of the

polyploidy by
carbon flux

colchicine treatment, the

feeding

or

by diverting

through

isoprenoid pathway
the
same

towards the lower

isoprenoids by

inhibiting

other

pathways using

precursors/ intermediates (Banthorpe

1988; Charlwood and Charlwood 1991

; Charlwood etal.

1989).

3.3. Colchicine treatment

Lavania

(1988,

1991

obtained

tetraploid

vtiver

plant

with

significant

improvement
colchicine.

of the oil

productivity, by treating germinating

the

vtiver seeds with

Additionally,

tetraploid plants

resulted in vtiver oils with

enhanced levels of khusinol and reduced levels of shift in

-vetivone, which caused

optical

rotation

(Lavania 1988;

Lavania

1991).

162

Since the treatment resulted in

selection for selection

mixaploid plantlets (2n

20 to

40),

diploid

and

tetraploid plantlets

had to be done. For the

plants,

the

was

done via

subsequent sequential separation

In
our

of tillers from the

or

mixaploid plant (Lavania 1988).

case,

using

callus

suspension
while

cultures, it would be practically impossible

to select for

tetraploid cells,

mixaploid
as

cultures

are

not useful for

testing

the

parental plants

for oil variations

the

ploidy

influences not

only

the oil content but also the oil

composition.

3.4. Precursor

feeding

The oil level of the The

plant

cultures could be increased

by

precursor
can

feeding.
in

terpenoid building block, isoprene diphosphate (IPP),

via two different

be

synthesized

plants
from

biosynthesis pathways:

the mevalonate

pathway starting

acetyl-CoA

via mevalonate

(MVA)

to

IPP, which takes place in the cyto

plasm,
via

and the DOXP

pathway

from pyruvate and

to

glyceraldehyde-3-phosphate

1-deoxy-D-xylulose-5-phosphate (DOXP)

IPP, which is found in the

chloroplasts (Lichtenthaler 1999). Examples

of

sesquiterpenoid biosynthesis
can

are as

described, which show that IPP synthesized via both pathways

be used

building

blocks for

sesquiterpenoid biosynthesis.
via the mevalonate since isoforms of

The IPP thesis of

biosynthesis

pathway

is involved in the

biosyn

sesquiterpenoids,

(S)-3-hydroxy-3-methylglutaryl-

CoA reductase CoAto

(HMGR:
are

enzyme
induced

catalyzing during
the

the irreversible conversion of HMG-

mevalonate)

biosynthesis

of

sesquiterpenoid
radio
or

phytoalexins (Stermer isotopes

of acetate
or

et al.

1994). Incorporation experiments using

efficiently

mevalonate resulted in

labeled sterols

sesquiterpenoids
In contrast, in

in the

cytoplasm (Rohmer 1999).

secretory cells isolated from glandular trichomes of pepper

mint, it

was

shown with precursor

feeding,

that the

cytoplasmic pathway

is

blocked at HMG-CoA reductase and that the IPP utilized for both monoter-

penoid

and

sesquiterpenoid biosynthesis
et al.

was

biosynthesized exclusively

in

plastids (Ramos-Valdivia

1997).
pathways
should also not be

The combination of IPP from both IPP

excluded, since for chamomile sesquiterpenoids

blocks
were

two of the

isoprene building
whereas the third

predominantly

formed via the DOXP

pathway,

163

unit

was

of mixed

origin, being

derived from both mevalonate and the DOXP

pathway (Adam

and

Zapp 1998).
acetate and mevalonate for the mevalonate

Therefore, precursors like

pathway

and pyruvate,

deoxy-D-xylulose

and

methyl deoxy-D-xylulose

(Lichtenthaler 1999)
and pyruvate
are

for the DOXP

pathway

should be examined. Since acetate

also involved in several other

biosynthesis pathways,

the would

intermediate mevalonate, be
more

deoxy-D-xylulose

and

methyl deoxy-D-xylulose
can

specific

for

terpenoid biosynthesis.

Other precursors

also be

tested, but it has

the

to be considered that the precursors have to be taken up

by

plant

material.

3.5.

Diverting

the carbon flux

By diverting
more

the carbon flux the

through

the

terpenoid biosynthesis pathway

and

or

exactly through

sesquiterpenoid biosynthesis pathway

the amount of vtiver oil
are

by

inhibiting competing pathways,

might

be increased. metabolism of

However, acetyl-CoA and pyruvate

involved in the and

primary fatty

plants (e.g. glycolysis, TCA-cycle, photosynthesis

as

acid

synthesis)

and

these

pathways

are

essential for

viability,

inhibition

might

be lethal to the

cultures. Within the

terpenoid biosynthesis, sterol-,

with the

carotenoid- and other

terpenoid

synthesis might compete building

blocks
or

sesquiterpenoid biosynthesis
In
some cases

for the IPP redirect the Thus after

for

farnesyl diphosphate.

plants

precursor into the needed

a

pathway by inhibiting competing pathways.

some

pathogen

attack

or an

elicitor treatment with arachidonic acid, and increase the

plants
to

inhibit the sterol

biosynthesis

sesquiterpenoid biosynthesis

improve

the

phytoalexin production (McCaskill

Weissenborn etal.

and Croteau 1998; Newman and the essential oil

or

Chappell 1999;

1995).

In

plant cultures, by using

accumulation has been increased in

some cases

carotenoid

sterol

synthesis
cultures,

inhibitors. However, in

Pelargonium

tomentosum shoot

proliferation

progressive
was

dedifferentiation

resulting
on

in

non-accumulating

aggregate cultures
etal.

observed, dependent
be

the inhibitor used the vtiver oil

(Charlwood

1989). Thus,
the sterol

it

might

possible

to

improve

biosynthesis by

inhibiting

biosynthesis.

164

4. END PRODUCT TOXICITY

To be able to
must not

analyze

the essential oil in the culture, the oil

biosynthesis

only

be induced but the oil should also accumulate. Since it has been

demonstrated that many

monoterpenoids

and

sesquiterpenoids

are

toxic to

plant cells, end-product toxicity

in undifferentiated cultures
a

may be the

regulating

factor in oil accumulation

(Banthorpe 1988;

Charlwood et al.
mean a

1989). However,
pathway

lack of

product

accumulation does not

always

lack of

enzyme

expression.
of

Some cultures that did not accumulate detectable

or

quantities

monoterpenoids

sesquiterpenoids,

nevertheless

appeared

to

possess the full

accumulate

enzymatic machinery (Banthorpe 1988;

the rate of its

Charlwood

1993).

To

product,

synthesis

has to be it

larger

than the rate of

product

catabolism. In several

suspension cultures,
was

was

shown that the

conversion of the

monoterpenoids

higher

than the rate of their

synthesis

(Charlwood 1993)
To obtain

and thus the cultures do not

a

toxify

themselves.

terpenoid accumulation,

possibility
or

is to induce sufficient

differentiation to allow formation of storage

excretion sites, like secretory and trichomes, without

cells, resin

or

oil ducts, hairs,

glandular epithelial cells,

reaching plantlet regeneration (Banthorpe 1988).

An alternative

strategy that may minimize end-product toxicity and enhance

productivity
of

of the

terpenoid
and

accumulation would involve the continuous removal from the culture. In the
case

monoterpenoids

sesquiterpenoids

of

an

excretion of the essential oil to the medium, the accumulation of the

in undifferentiated cultures could be increased

terpenoids

by using

lipophilic

second

phase (e.g. Miglyol)

or

by adding adsorbing

resin

(e.g. XAD) (Banthorpe 1988;

products
the toxic could be avoided,

Scragg 1997).
labile

Loss

by

volatilization of the excreted

products

could be stabilized and

by removing

compounds

the

viability

of the cultures could be maintained

(Banthorpe 1988).

165

5. TOWARDS A PRACTICAL PROTOCOL FOR THE

PRODUCTION OF VETIVER OIL VARIANTS

Since the culture condition and the culture type

can

influence the
et al.

composition

and content of the essential oil

(Charlwood
a

1989; Scragg

1997),

the induced oil of the in vitro cultures may have

different
are

composition
differences in when

than that

produced by plants produced by

same

grown in vivo. However, if there

the vtiver oil these

are

the

original plant

and the

regenerated plantlets

treated the

way, it is

possible

that there will also be differences

are

after 15-22 months. There is the risk that oil variants

some

not detected because

compounds might changes

are a

not be induced with the selected treatment, or that matter of the treatment and not of
a

observed is

genetic

variation. It

important
a

therefore for

useful

pre-screening method,

that the induced oil

has

composition
on

similar to that of the oil extracted from

plants

cultivated for 2

years
take

soil. Furthermore, the oil

production

and accumulation in vitro should

to prove that the obtained

place

in less time than needed in vivo.

are

Finally,

compounds

oil

compounds,

it is necessary to

analyze

them

by

GC-MS.

After

developing
are

methods to induce vtiver oil The selected

synthesis, regenerated
oil variants must
are

plantlets

pre-screened.
on

putative

subsequently
can

be cultured for 2 years then be

soil,

to confirm that

they

variants. Variants

analyzed genetically
a

or

biochemically

for the mutated trait,

ultimately

perhaps permitting plantlets developed

search for variants

by analysis

of DNA modifications in

in vitro.

166

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Curriculum vitae

Oct. 19, 1967

Born in Zrich, Switzerland

1974

1980

Primary

school Ksnacht, Zrich

1980

1983

Secondary

school Ksnacht, Zrich

1983

1987

Mathematisch-Naturwissenschaftliches

Gymnasium (MNG),

Rmibhl, Zrich:
Matura

Typus

1987

1992

Eidgenssische Diploma

Technische Hochschule Zrich

(ETHZ):

Biology study (plant science)

of Natural Sciences

1992

1993

Phytotech Labor,

Bern

(Prof.

K.H.

Erismann):
project

Collaborator, working

on

the vtiver

1993

2001

Eidgenssische

Technische Hochschule Zrich

(ETHZ):

PhD-student at the Institute of

Biotechnology