FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fragr. J. 2001; 16: 319–324

DOI: 10.1002/ffj.1002

Characteristics of an isomenthone-rich somaclonal

mutant isolated in a geraniol-rich rose-scented geranium
accession of Pelargonium graveolens
Ritika Gupta,1 G. R. Mallavarapu,2 S. Banerjee1 and Sushil Kumar1Ł
1 Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow 226015, India
2 CIMAP, Field Station, GKVK PO, Bangalore-560 065, India
Received 11 October 2000
Revised 19 February 2001
Accepted 15 March 2001

ABSTRACT: A somaclonal essential oil mutant (IRPG) was identified among the regenerants induced in callus
cultures initiated with the leaf explants of geranium Pelargonium graveolens cv. Kunti. While the shoot essential
oil of the wild-type parent was rich in geraniol, the oil of the IRPG had isomenthone as the major monoterpenoid
component. The IRPG oil had about 71% isomenthone, 6% citronellol and 3% geraniol, as compared to the
parental variety oil, in which isomenthone, citronellol and geraniol contents were about 8%, 13% and 40%,
respectively. Copyright  2001 John Wiley & Sons, Ltd.
KEY WORDS: geranium; essential oil variant; isomenthone-rich oil; mutant essential oil; geranium somaclone;
Pelargonium graveolens mutant

Introduction Bipuli (intermediate to the Reunion Island and Egyptian

Geranium oil is one of the expensive essential oils used
in the perfumery, flavour and cosmetic industries.1 It
is obtained by hydrodistillation of the aerial parts of
rose-scented geranium, Pelargonium graveolens.2 There
are several cultivars of geranium that are commer-
cially grown for the production of the essential oil.
The important cultivars of geranium are the Reunion
Island type, the Egyptian or North African type, and the
Chinese type. The oil of the Reunion Island type con-
tains citronellol and geraniol (1 : 1) and citronellyl for-
mate, guaia-6,9-diene and isomenthone in high amounts.
The Chinese type oil contains high amounts of cit-
ronellol and citronellyl formate and a low concentration
of geraniol. The Egyptian type oil contains citronellol
and geraniol (1 : 1) and citronellyl formate, isomenthone
and 10-epi-
-eudesmol as major constituents. The dif-
ference between the Bourbon and Egyptian type oils
is that the former contains good amounts of sesquiter-
pene guaia-6,9-diene and is practically devoid of 10-
epi-
-eudesmol, whereas the Egyptian type contains
fairly good amounts of 10 epi-
-eudesmol and low
amounts of guaia-6,9-diene.3 – 9 In India three cultivars
of geranium are available that are known by the names
*Correspondence to: S. Kumar, Central Institute of Medicinal and
Aromatic Plants (CIMAP), Lucknow 226015, India.
E-mail: E-mail: cimap@satyam.net.in
Contract/grant sponsor: Department of Biotechnology, Government of
India.
types), Hemanti (similar to the Chinese type) and a third
type, Kunti, whose oil is rich in geraniol (40–50%) and
poor in citronellol (1–10%) compared to Reunion Island
type.10,11 During the course of our present study on
genetic improvement in geranium, we have isolated a
somaclone from the cultivar Kunti, whose oil was found
to be rich in isomenthone. The morphological differences
associated with the essential oil quality differences and
genetical differences observed between the somaclone
and the parental types are reported in this paper.
Experimental
Plant Material
The rose-scented geranium, P. graveolens cv. Kunti, was
the source of explants for the present study. Healthy
potted 5–6 month-old glasshouse grown plants served as
the explant resource. In preparation of their inoculation,
the explants, i.e the young leaves, were sequentially
washed under running tapwater for 30 min, dipped in
HgCl2 solution with constant agitation and rinsed three
times with sterile double-distilled water.
Culture Medium and Conditions for Generating
Regenerants
Murashige and Skoog’s (MS)12 medium containing 3%
sucrose and 100 mg/l myoinositol was used as the basal

Copyright  2001 John Wiley & Sons, Ltd.

320 R. GUPTA ET AL.

medium. The MS medium was supplemented with a
combination of kinetin (2.5–5.0 mg/l) and naphthalene
acetic acid (NAA) (0.1–1.0 mg/l) at variable concentra-
tions (Table 1). The pH of the medium was adjusted to
5.8 and autoclaved under 104 kPa at 121 ° C for 15 min.
The sterilized leaf explants were placed horizontally with
the abaxial side down on the medium. Each treatment
consisted of 10 explants, replicated three times. Cul-
tures were maintained at 25 š 1 ° C temperature, 60%
relative humidity and 35 µE/m2 /s flux density. Observa-
tions were recorded at weekly intervals. After 4 weeks of
culture, the observations on percentage callus induction
from leaf explants, percentage callus segments forming
shoots and average number of shoots/cm2 of callus were
recorded and have been summarized in Table 2.
The induced calli were subsequently subcultured on
the same medium for further proliferation, as well as on
MS medium supplemented with kinetin (Kn), NAA and
adenine disulphate (ADS) for inducing shoot regenera-
tion from the calli. The in vitro regenerated shoots from
callus were separated and transferred to hormone-free
Table 1. In vitro response of leaf explants to different
phytohormonal concentrations and combinations in
the rose-scented geranium Pelargonium graveolens cv.
Kunti
MS medium for elongation. In vitro regenerated shoots
of 2–3 cm size with 3–4 fully expanded leaves were
transferred to half-strength MS medium containing
50 mg/l myo-inositol, 1.5% sucrose and 1.0 mg/l indole
butyric acid (IBA) for rooting.
Acclimatization and Transfer of Regenerated
Plants to the Field
Well-rooted plantlets with 8–10 fully expanded leaves
were removed from the culture medium and the roots
were washed gently under running tap water to remove
the traces of medium. The plantlets were transferred into
pots containing coarse sand and kept for 4 weeks in a
glasshouse at 26 š 1 ° C, 80% relative humidity, and then
transplanted to pots containing sand, soil and farmyard
manure in 1 : 1 : 1 ratio. The calliclones and controls
were transferred to the field in three replications during
the third week of October 1998, using a randomized
block design (RBD) for agronomic evaluation. Data were
recorded at the time of crop harvest for agronomical
traits, viz. height, canopy size, number of branches, leaf
area, leaf: stem ratio, petiole length, oil content and
oil composition for all the somaclones for two years
(1998–1999 and 1999–2000).

Type of
Phytohormone and its Explants response and Extraction of Oils
concentration (µg/ml) that number of
responded shoots
Kna NAAb ADSc (%) formed/explant Six month-old plants of the calliclones were harvested
and hydrodistilled in a Clevenger-type apparatus for 2 h
5 1.0 0 72 0d
5 0.5 0 28 2.0 š 0.4e to obtain the essential oils. The oil samples were dried
5 0.1 0 64 12.0 š 1.0f over anhydrous Na2 SO4 and stored at 4 ° C in a refrigera-
5 0.1 3 80 30.4 š 2.0f tor until analysed. Among the calliclones, a variant was
a
Kinetin. identified whose essential oil differed from that of the
b
Naphthalene acetic acid. parent in being rich in isomenthone. Detailed comparison
c
Adenine disulphate. of the variant somaclone (mutant) and the parental type
d
Callus proliferation.
e Callus proliferation and subsequent shoot regeneration. showed that the isomenthone-rich clone was a chemo-
f
Callus-free shoot regeneration. typically and genetically distinguishable mutant.

Table 2. Expression of the essential oil yield related characters in the plants of
the (Pelargonium graveolens cv. Kunti) and its isomenthone-rich somaclonal
variant field-grown in winter–summer seasons of 1998–1999 and 1999–2000 in
subtropical agroclimate of Lucknow

Expression (mean š SE) in:

Isomenthone-rich
Sl. no. Character cv. Kunti somaclone
1. Plant height (cm) 35 š 4 30 š 2
2. Canopy size m2  0.6 š 0.2 0.4 š 0.1
3. Herb yield (kg/plant) 2.0 š 0.1 1.8 š 0.1
4. Number of branches/plant 22 š 1 18 š 1
5. Number of leaves/plant 1363 š 177 1200 š 208
6. Petiole length (cm) 9.0 š 1.1 5.5 š 0.4
7. Leaf lamina area cm2  108 š 5 50 š 9
8. Leaf: stem ratio 1.2 š 0.1 1.0 š 0.1
9. Essential oil content in shoot (%) 0.30 š 0.01 0.15 š 0.01
10. Essential oil yield (g/plant) 6.0 š 0.5 2.7 š 0.1

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 319–324
 ISOMENTHONE-RICH SOMACLONAL MUTANT OF PELARGONIUM GRAVEOLANS
Gas Chromatography (GC)
GC analysis of the oils was performed on Perkin-Elmer
Gas Chromatograph 8500 equipped with FID, using two
fused silica capillary columns BP-1 coated with dimethyl
siloxane (30 m ð 0.25 mm, 0.25 µm film thickness) and
BP-20 coated with Carbowax 20 M (25 m ð 0.25 mm,
0.25 µm film thickness). Carrier gas, nitrogen, at 10 psi
inlet pressure. Temperature programming, 60–220 ° C at
5 ° C/min for BP-1 column and 60–200 ° C at 5 ° C/min
for BP-20 column. Split ratio, 1 : 80.
Gas Chromatography–Mass Spectrometry
(GS–MS)
GS–MS was performed on a Schimadzu QP-2000 instru-
ment using ULBON. HR-1 fused silica column (50 m ð
0.25 mm, 0.25 µm film thickness). Temperature pro-
gramme, 100–250 ° C at 10 ° C/min. Carrier gas, helium
at 2 ml/min. MS conditions, EI mode, 70 eV and ion
source temperature, 250 ° C.
Identification of the Compounds
Compounds were identified by comparing the retention
indices (relative to C8–C21 alkanes) with those reported
in the literature,13 – 16 by peak enrichment on co-injection
with standards wherever possible, and by comparison
of mass spectra of the peak with those of compounds
reported in literature.3,15,16 Relative amounts of individ-
ual components were based on peak areas on the BP-1
column without FID response factor correction.
Molecular Analysis of Selected Clone
DNA was isolated from 1 g young leaves of the
selected clone and that of the control parent using
a method described by Khanuja et al. (1999)17 and
digested with EcoR1 restriction endonucleases. Poly-
merase chain reaction (PCR) was carried out using
20 decanucleotide primers (MAP 01–MAP 20) series
(M/S Bangalore Genie, India) in 25 µl reaction mix-
ture containing 20–40 ng plant genomic DNA, 125 µM
buffer, 0.2 U Taq DNA polymerase enzyme, 100 µM
each of the dNTPs, and 5 pmol primer. Genomic DNA
was amplified in DNA Engine (M. J. Research, USA)
thermal cycler programmed for 45 cycles of 1 min
at 94 ° C, 1 min at 36 ° C and 2 min at 72 ° C. The
cycle was concluded with final extension at 72 ° C for
5 min. The amplification products were electrophoresed
in 1.5% (w/v) agarose gel, visualized by ethidium bro-
mide 0.5 µg/ml staining. The pictures of the gel were
scanned for the presence of polymorphic fragments
Copyright  2001 John Wiley & Sons, Ltd.
321
which were scored for the presence C or absence
 of bands. The data so generated were used for
calculating the index of genetic similarity, using Nei
and Li’s matching coefficient.18 The similarity indices
were calculated by using the formula (number of similar
bands between the two accessions/total number of bands
in two accessions) 2.
Results and Discussion
The present study demonstrated that the leaf explants
of rose-scented geranium Pelargonium graveolens cv.
Kunti were highly responsive towards in vitro callus-
ing, as has been reported earlier in floricultural geranium
species and in rose-scented geranium P. graveolens cv.
Hemanti.19 – 20 The medium containing 5 mg/l Kn and
1 mg/l NAA showed the best callusing response, where
72% of the leaf explants formed callus, within 20 days
of culture initiation. About 65% organogenesis in callus
could be achieved by reducing the NAA concentration
to 0.1 mg/l, where 12 š 1 shoots appeared from 1 cm2
callus (Table 1). Addition of ADS to the same medium
at a concentration of 3 mg/l greatly improved the regen-
eration to about 80% and the number of shoots formed
to 30 š 2 for 1 cm2 callus.
The in vitro-regenerated shoots were separated from
callus and subcultured on hormone-free MS medium for
elongation, where they attained a size of 1.5–2.5 cm
in 10 days. They were then transferred to half-strength
MS medium containing 1 mg/l IBA. Thirty in vitro-
grown plants were transferred to the glasshouse for
hardening into pots containing coarse sand for 2 weeks
and thereafter transplanted into pots containing sand, soil
and farmyard manure, where 83% survival was noted.
Subsequently, all the healthy-looking calliclones, along
with the control parent, were transferred to the field
during October 1998, to serve as resource plants to
obtain cuttings. The surviving clones were field-planted
in randomized blocks, replicated three times. The field
trial was repeated again in the winter–summer cropping
season of 1999–2000. Data was recorded for various
agronomical traits. The crops were allowed to grow
for 6 months and then the aerial parts were harvested
for determination of the yield and quality of oil. GC
analysis of the oil of all the clones produced during 2
consecutive years led to the isolation of an isomenthone-
rich somaclone. The composition of the oil of this
isomenthone-rich clone was studied in detail by GC and
GC–MS analyses.
Composition of the Oil of the Isomenthone-rich
Geranium Somaclone
The essential oil of the isomenthone-rich geranium
strain obtained from tissue culture of the parent clone
Flavour Fragr. J. 2001; 16: 319–324
322 R. GUPTA ET AL.

Kunti had a minty odour. The oil was analysed by
GC and GC–MS. Its composition was compared with
the oil of parent clone Kunti. Seventy-five constituents
of the oil of the variant were identified by Kováts
retention indices and mass spectra. The identified com-
pounds are presented in Table 3, along with the com-
pounds of the parent oil for comparison. The oil of
the isomenthone-rich variant (IRPG) was found to con-
tain, besides the main constituent isomenthone (71.0%),
limonene (1.4%), linalool (1.2%), menthone (1.8%),
citronellol (6.1%), geraniol (3.1%), citronellyl formate
(1.4%) and 10-epi-
-eudesmol (1.7%) in substantial
amounts. The oil of the parent clone Kunti had the
main constituents linalool (5.1%), isomenthone (8.2%),
citronellol (13.0%), geraniol (31.9%), geranial (3.0%),
citronellyl acetate (1.0%), decanoic acid (2.4%), guaia-
6,9-diene (1.4%), geranyl propionate (2.3%), geranyl
butyrate (3.2%), 2-phenyl ethyl tiglate (1.3%), 10-epi-

-eudesmol (5.2%), ß-eudesmol (2%), citronellyl tiglate
(4.2%) and geranyl tiglate (2.8%). A comparison of the
compositions of the two oils showed that the oil of IRPG
had higher concentrations of limonene, menthone, iso-
menthone and citronellyl formate than the oil of the
parent clone, whereas the oil of parent was found to have
higher amounts of linalool, geranial, citronellol, geraniol,
citronellyl acetate, decanoic acid, iso-undecanoic acid,
guaia 6,9-diene, geranyl propionate, geranyl butyrate, 2-
phenylethyl tiglate, furopelargone B, 10-epi-
-eudesmol,
ß-eudesmol, citronellyl tiglate, geranyl isohexanoate,
geranyl hexanoate and geranyl heptanoate than the oil

Table 3. Chemical compositions of the essential oils of Pelargonium graveolens cv. Kunti and
its isomenthone-rich somaclonal variant

Content in essential oil of:

Sl. Retention Index cv Kunti Somaclonal variant
no. Compound BP-1 (%) of cv. Kunti (%)
1. ˛-Pinene 936 ND 0.3
2. Sabinene 970 tr 0.2
3. Myrcene 986 0.1 0.4
4. p-Cymene 1014 ND 0.8
5. Limonene 1023 tr 1.4
6. (Z)-ˇ-Ocimene 1030 ND 0.3
7. (E)-ˇ-Ocimene 1042 0.1 0.1
8. cis-Linalool oxide (furan) 1063 0.2 0.2
9. trans-Linalool oxide (furan) 1076 0.2 tr
10. Linalool 1088 5.1 1.2
11. cis-Rose oxide 1098 0.2 0.2
12. trans-Rose oxide 1116 0.1 tr
13. Menthone 1136 0.2 1.8
14. Isomenthone 1148 8.2 71.0
15. Borneol C neomenthol 1154 ND 0.3
16. Menthol 1163 ND 0.2
17. Terpinen-4-ol 1165 tr 0.1
18. neo-Isomenthol 1173 0.6 0.5
19. ˛-Terpineol 1177 0.3 0.4
20. Isomenthol 1184 ND 0.1
21. Citronellol 1215 13.0 6.1
22. Piperitone 1232 0.1 0.6
23. Geraniol 1241 31.9 3.1
24. Geranial 1248 3.0 0.7
25. Citronellyl formate 1260 0.1 1.4
26. Bornyl acetate 1272 ND 0.2
27. Geranyl formate 1282 0.3 0.5
28. Citronellic acid 1305 0.2 0.1
29. Phenyl ethyl propionate 1315 0.9 0.1
30. Citronellyl acetate 1334 1.0 0.1
31. ˛-Cubebene 1352 0.2 0.5
32. Geranyl acetate 1365 0.6 0.1
33. Decanoic acid 1363 2.4 0.1
34. ˛-Copaene 1380 0.3 0.1
35. ˇ-Bourbonene 1387 0.4 0.2
36. Iso-undecanoic acid 1399 0.6 0.1
37. ˇ-Caryophyllene 1422 0.5 0.2
38. Citronellyl propionate 1430 0.1 0.1
39. Guaia-6,9-diene 1442 1.4 0.7
40. Geranyl propionate 1448 2.3 0.1
41. ˛-Humulene 1456 0.1 0.1
42. allo-Aromadendrene 1463 0.1 tr
43.
-Muurolene 1473 0.1 ND
44. Germacrene D 1480 0.1 0.2

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 ISOMENTHONE-RICH SOMACLONAL MUTANT OF PELARGONIUM GRAVEOLANS 323

Table 3. (Continued)

Content in essential oil of:

Sl. Retention Index cv Kunti Somaclonal variant
no. Compound BP-1 (%) of cv. Kunti (%)
45. ˛-Selinene 1486 0.1 tr
46. ˛-Muurolene 1494 0.2 0.2
47. Citronellyl butyrate 1506 0.4 ND
48. cis-Calamenene 1514 0.1 tr
49. υ-Cadinene 1518 0.1 0.1
50. Geranyl butyrate 1534 3.2 0.2
51. (E)-Nerolidol 1550 0.4 0.1
52. 2-Phenylethyl tiglate 1558 1.3 0.1
53. Furopelargone B 1566 0.8 0.2
54. Spathulenol 1571 0.2 0.1
55. Caryophyllene oxide 1578 0.3 0.1
56. Geranyl isovalerate 1588 0.1 tr
57. Citronellyl valerate 1599 0.3 0.1
58. 10-epi-
-Eudesmol 1615 5.2 1.7
59. Geranyl valerate 1625 0.1 0.1
60. ˇ-Eudesmol 1631 2.0 0.1
61. ˛-Eudesmol 1637 0.1 tr
62. Citronellyl tiglate 1647 2.4 0.1
63. Citronellyl isohexanoate 1663 0.2 tr
64. Geranyl tiglate 1672 2.8 ND
65. Geranyl isohexanoate 1681 0.8 0.3
66. Citronellyl hexanoate 1700 0.1 0.1
67. Geranyl hexanoate 1726 0.4 tr
68. Citronellyl isoheptanoate 1758 0.2 tr
69. Geranyl isoheptanoate 1784 0.1 tr
70. Citronellyl heptanoate 1796 0.1 ND
71. Geranyl heptanoate 1826 0.6 tr
72. Citronellyl isooctanoate 1865 0.1 ND
73. Geranyl isooctanoate 1882 0.1 0.1
74. Citronellyl octanoate 1894 0.2 ND
75. Geranyl octanoate 1928 0.3 0.1
76. Unidentified compounds (approx) 1.7 1.4
ND, not detected, tr, traces.

of IRPG. The compounds borneol and bornyl acetate,
which were detected in the oil of IRPG, were not found
in the oil of the parent clone. On the other hand,
-
muurolene, geranyl tiglate, citronellyl heptanoate, cit-
ronellyl butyrate and geranyl tiglate, which were present
in the oil of the parent clone, were not detected in the oil
of IRPG. Although the citronellic acid and geranic acid
were reported earlier in oils of P. vitifolium,5 decanoic
acid and iso-undecanoic acid found present in the oils
of the parent and mutant in this study have not been
reported in any Pelargonium oils so far.
This is the first report of the isolation of a isomenth-
one-rich strain of geranium from tissue culture using the
cultivar Kunti of P. graveolens as parent. Earlier, iso-
menthone was found as the major compound in the oils
of P. australe, P. graveolens, P. radens, hybrids of P.
graveolens and P. radens,4 P. tomentosum,21,22 the cul-
tivar Menthe (hybrid of P. capitatum and P. radens)22
and the clones 53 and 79 obtained from the leaf cut-
tings of the Indian cultivar Bourbon.23,24 Although all
these oils contained isomenthone as the major com-
pound, there are significant differences in their compo-
sition. The cultivar Menthe and the clone 79 contained,
besides isomenthone, considerable amounts of ˛-pinene
(8.5–11.0%) in their oils, whereas the oil of P. tomen-
tosum was found to contain a fairly high concentration
of menthone (26.0–33.0%) in addition to isomenthone
(45.8–62.0%).21 Isomenthone-rich variants were previ-
ously reported from the cultivar Bipuli (Bourbon type).
Isomenthone is biosynthesized in the plant from ger-
anyl pyrophosphate through a series of reactions such
as cyclization, oxidation and reduction.22 It is likely that
conversion of geraniol and citronellol to isomenthone in
the plants of Kunti is controlled by the enzymes present
in the plant, resulting in the formation of large amounts
of isomenthone and small amounts of menthone, men-
thol, neoisomenthol, etc.
The distinctness of the IRPG somaclone was con-
firmed by the molecular analysis of the somaclone and
the parent. The RAPD profiles, performed by using
20 decanucleotide DNA primers (MAP01–MAP20),
showed that the somaclone was different from the wild-
type parent in certain features only. The primer MAP11
showed least similarity, primers MAP4, MAP5 and
MAP16 showed an intermediate degree of similarity
and primer MAP9 showed close relatedness, while the
remaining primers showed an exact similarity of the
somaclone to the parent (Table 4). It was evident from

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 319–324
324 R. GUPTA ET AL.

Table 4. Similarity index (homology) between the iso- geranyl tiglate and citronellyl octanoate, were absent
menthone-rich somaclonal variant and control parent from the oil of IRPG clone, unlike in the oil of the par-
in rose-scented geranium Pelargonium graveolens cv.
Kunti ent Kunti cultivar. The profile of the IRPG somaclonal
mutant recovered in the study differed from all the
SI. no. Primer Base sequence Similarity index previously known isomenthone-rich scented geranium
1. MAP-01 GTGCAATGAG 1.00 genotypes.
2. MAP-02 AGGATACGTG 1.00
3. MAP-03 AAATCGGAGC NR Acknowledgements—Grateful thanks are due to S.P.S. Khanuja for
4. MAP-04 AAGATAGCGG 0.66 help in the DNA-fingerprinting, S. Ramesh in GC analysis, and the
5. MAP-05 GGATCTGAAC 0.66 Department of Biotechnology, Government of India, for providing
6. MAP-06 TTGTCTCAGG 0.66 partial financial support for the GeneBank activity at the Institute.
7. MAP-07 GTCCTACTCG NR
8. MAP-08 GTCCTTAGCG NR
9. MAP-09 TGCGCGGATC 0.75
10. MAP-10 AACGTACGCG 1.00 References
11. MAP-11 GCACGCCGGA 0.50
12. MAP-12 CACCCTGCGC 1.00 1. Gulati BC. Ind. Oil Soap J., 1960; 26(2), 35.
13. MAP-13 CATCCCGAAC NR 2. Charlwood BV, Charlwood KA. Pelargonium spp. (Geranium):
14. MAP-14 GGACTCCACG NR in vitro culture and the production of aromatic compounds. In
15. MAP-15 AAGATAGCGG 0.60 Biotechnology in Agriculture and Forestry. Vol. 15, Medicinal and
16. MAP-16 CTATCGCCGC 1.00 Aromatic Plants III , Bajaj YPS (ed.). Springer-Verlag: Berlin,
17. MAP-17 CGGGATCCGC 1.00 Heidelberg, 1991; 339.
18. MAP-18 GCGAATTCCG NR 3. Teisseire P. In Capillary Gas Chromatography in Essential Oil
19. MAP-19 CCCTGCAGGC NR Analysis, Sandra P, Bicchi C. (eds). Huethig: Heidelberg, 1987.
20. MAP-20 CCAAGCTTGC NR 4. Van der Walt JJA, Demarne F. S. Afr. J. Bot. 1988; 56: 617.
5. Demarne F-E, Van der Walt JJ. J. Essent. Oil Res. 1992; 4: 345.
NR, nil response. 6. Demarne F-E, Van der Walt JJ. J. Essent. Oil Res. 1993; 5: 233.
7. Lawrence BM. Progress in essential oils. Perfum. Flav. 1984; 9:
87.
the similarity indices paired test that the isomenthone- 8. Vernin G, Metzger J, Fraisse D, Scharff C. Parf. Cosm. Arômes
rich somaclone (IRPG) and the wild-type parent were 1983; 52: 51–61.
genetically distinguishable (Table 4). The observations 9. Fraisse D, Sharf C, Vernin G, Metzger J. IX International Essen-
tial Oil Congress, Singapore, 1983. Book 3, pp. 100–120.
on similarity indices also confirmed the relationship of 10. Kaul PN, Rajeswara Rao BR, Bhattacharya AK, Singh CP,
the somaclone with the wild-type parent. Singh K. PAFAI J 1995; 17(4): 21.
11. Mallavarapu GR, Prakasa Rao EVS, Ramesh S, Narayana MR. J.
Essent. Oil Res. 1993; 5: 433.
12. Murashige T, Skoog F. Physiol. Plant. 1962; 15: 473.
Conclusion 13. Jennings W, Shibamoto T. Qualitative Analysis of Flavor and
Fragrance Volatiles by Glass Capillary Column/Gas Chromatog-
raphy. Academic Press: New York, 1980.
The leaf explants of rose-scented geranium P. graveolens 14. Davies NW. J. Chromatogr. 1990; 503: 1.
cv. Kunti regenerated shoots via callus formation on MS 15. Adams RP. Identification of Essential Oil Components by Gas
medium containing kinetin, NAA and ADS at 5, 0.1–1 Chromatography and Mass Spectrometry. Allured: Carol Stream,
IL, 1995.
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Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 319–324