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61
CHAPTER 4
FIELD EVALUATION OF PATCHOULI GERMPLASM
4.1. Introduction
The leaves of the patchouli plant are dotted with oil containing glands on both their
adaxial and abaxial epidermis. Internal mesophyll glands are also reported to carry
patchouli oil (Maeda and Miyake 1997). Since the oil has a base note character, it is
popular as a natural fixative for heavy perfumes (Hasegawa et al. 2006). Therefore, it
is used both in traditional and contemporary women’s and men’s fragrance (Anonis
2006). Although the aroma of undiluted patchouli oil is disliked by many, it is
considered very pleasant when used in small quantities.
Despite the commercial importance of patchouli oil and many attempts to elucidate
its chemistry, relatively little is known about the principles which are responsible for
the characteristic strong odor of this oil (Guenther 1974). True patchouli oil is
reported to contain 1% terpenes, 50% sesquiterpenes and 30 to 40% patchouli and
related alcohols (Lawrence 1981). The oil contains approximately 97% of compounds
which have no influence on the aroma. Out of these 40 to 45% belong to the
sesquiterpene group and the balance seems to consist of patchouli alcohol. The oil
contains small amount of benzaldehyde, eugenol, cinnamic aldehyde, an alcohol
with rose like fragrance, a ketone with an orris root like odour, two bases possessing
a strong benumbing odor, azulene, a sesquitrepene alchohol, βpatchoulene, gamma
gauaiente, α- bulonesene, α terpene, cadinene, benzaldehyde and patchouli
alchohol ( Farooqui and SreeRamu 2001). It is also reported to contain βpinene,
62
camphene, α quaiene and β bulnesene. Patchoulenol is reported to be the true
colour carrier of the patchouli oil. Patchouli pyridine, epiguai-pyridine, dhelwangin
and a tri-cyclic sesquiterpene seychellene are the alkaloids reported from P.
patchouli and P. heyneanus (Rastogi et al. 1969).
Several factors like environmental regime, processing technique, chemical analysis,
condition of the raw material etc. are reported to influence oil quality. Forty one
compounds were separated from the essential oil of Indonesian patchouli using GC
(MS). Out of this 28 were identified and four new compounds namely gurjarene
(2.2%), germacrene D (0.2%), aciphyllene (3.4%) and 7-epi selinene (0.2%) were
isolated (Sellier and Bure 2004).
Sources also disagree over how to obtain the best quality oil. Some claim that the
highest quality oil is usually obtained from fresh leaves distilled close to the
plantation while others claim that bailing the dried leaves and allowing them to
ferment a little is best (Leung and Foster 1996).
However, olfactory evaluation remains the most prevalent method of oil quality
assessment in the market even today. Small scale distillers sometimes make use of
colorimetry to measure and ensure consistent color clarity. Patchouli oil is usually
dark brown in color. But, good quality distilled material from modern distilleries and
redistilled oil is pale yellow in color. Natural light colored oil gives 12% to 27% color
transmission or clarity at 525nm of light. Very light colored oil gives 28 to 43% color
transmission. Analytical methods like gas chromatography assure good quality
control. The identification of non-isolated constituents of essential oils can be
considered feasible when MS and GC retention indices are strictly identical to those
63
of reference samples present in databases using the same methodology (Singh
et al.1995). The GC method of quantitative determination of patchouli alcohol
provides a reliable method for standardization of the raw drug (Kang et al. 1998).
Standard fingerprints of Pogostemon cablin collected from different regions of China
were developed using GC-MS (Hu et al. 2006; Zheng et al. 2006). Three types of
fingerprints namely the patchoulol type; the pogostone type and an interim type
were reported from the study. Variety identification of Pogostemon cablin could be
made using digitalization mode of GC-MS characteristic fingerprint chromatogram
(Wei 2007). Application of capillary gas chromatographic fingerprint was also used in
quality control of Pogostemon cablin (Meng et al. 2006). There are many
specifications such as EOA, ISO and BSI standards for patchouli oil since most
perfume blending companies have their own requirement of color, odor and
solubility for extract.
Though many studies are available on the chemical characteristics of patchouli oil, a
systematic study correlating growth parameters, oil yield and oil quality is lacking in
the crop.
4.2. Materials and Methods
4.2.1. Preparation of the field
The patchouli accessions were evaluated under field conditions at the Department
of Botany, C.M.S. College, Kottayam. The experimental plot was ploughed and well-
decomposed manure was applied three weeks in advance of the actual planting
64
time. The experiment was laid out in a Randomised Block Design plot (Fig.7) with six
replications and planting was carried out on 10th April 2005.
4.2.2. Plant Material
The six accessions of patchouli short listed based on RAPD characterisation were
carried forward for the field evaluation study. Six replications constituting eight
plants each per accession was considered for the study. Therefore, 48 stem cuttings
of equal size from each of the six accessions were treated with 1000ppm IAA for
rooting and planted out. The rooted stem cuttings were planted at a spacing of
60 cm, irrigated and weeded as deemed necessary.
4.2.3. Data Collection
Characterisation of the field-grown patchouli plants were carried out after three
months of planting in the field. The same method was repeated in the sixth, ninth
and twelfth months as well. Three parameters namely physical, yield and oil quality
with four, two and thirteen variables respectively, were considered for the present
study.
a. Physical Parameters
i. Height of the plant
The height of plants was measured as in 2.2.3.2.a.
ii. Internode length
The length of the fourth Internodes was measured as in 2.2.3.2.b.
65
iii. Length of the petiole
Length of the petiole of the fourth pair of leaf was measured as in 2.2.3.2.c.
iv. Leaf area
The leaves at the fourth node were drawn on a graph paper and the small squares
occupied by the leaf surface were counted. The number thus obtained was
multiplied by the area of the small squares i.e. 1sq.mm. The leaf area was then
expressed in sq. cm.
b. Yield Parameters
i. Herbage yield
The number of leaves per plant was measured as in 2.2.3.2.d.
ii. Patchouli oil yield
Harvest of patchouli leaves
Tops of patchouli plants consisting of three to five pair of leaves were cut using
scissors or sharp knives early in the mornings before sunrise, at an interval of three
to five months.
Curing of patchouli leaves
After harvesting, the leaves and stocks were spread in thin layers on concrete floors
and shade dried. During the drying process, the leaves were regularly turned over by
hand to promote even drying and prevent fermentation. This process takes about
66
three to four days. Proper drying is of great importance for the quality of both leaves
and oil.
Distillation of patchouli oil
The dried herbage was subjected to steam distillation using a Clevenger apparatus
for a period of six to eight hours.
Storage of patchouli oil
All free water in the oil was completely removed after distillation. The remaining
traces of water was removed by adding anhydrous sodium sulphate at the rate of 20
to 30 grams per liter, stirred, left for 4 to 5 hours and filtered. Pure oil thus obtained
was stored in dark colored bottles and kept in a cool dry place. The oil thus obtained
was expressed as percentage dry weight of the leaf tissue.
c. Chemical Parameters of Patchouli oil
i. GC (MS) Characterisation of patchouli oil
A Clarus 500 Perkin Elmer Gas Chromatogram/Mass Spectrometer, featured with an
elite 5MS capillary column of 30m length and 0.25mm diameter was used for the oil
profile analysis. The oven temperature was maintained at 200ºC and the experiment
time was set to 23 min. The flow rate of the carrier gas helium was set at 40ml/min.
Methanol was used as the solvent for dilution. Double the amount of methanol was
used to dilute the oil extracted from the patchouli cultivar MEG 1 since the oil failed
to form peaks when injected in the same concentration as that of the other oils. One
67
µl each of the oil samples was loaded at the injection port and the chromatograms
were generated on Poly Ethylene Glycol (PEG) columns. The chromatograms
generated thus, indicated time on x-axis and percentage intensity on y-axis. The two
numbers on the chromatogram represented mass and retention time. The atomic
mass spectrum of the major chromatographic peaks was taken with the help of mass
spectrometer featured with an electron ionization mode detector. The source
temperature was set at 250ºC and the inlet line temperature was maintained at
220ºC. The mass spectrum generated thus, represented mass/charge (m/z) on the x-
axis and percentage intensity on the y-axis. A Turbo mass 2005 software was used
for the GC(MS) characterization of patchouli oil.
ii. Interpretation of GC results (UW Gen. Chem. Pages 1995-1996)
A substance’s affinity for the stationary phase is expressed through the retention
time. Substances with long retention times gave broad peaks in the chromatogram.
The ratio of the size of peaks gave the ratio of the relative amounts of substances in
the sample. The size of peaks was found by calculating the areas under the peaks.
The areas of the peaks were calculated using the formula
Area = h X W ½
where,
h = height of peak
W ½ = width at half height of the peak.
Using the peak areas, the percentage of each compound in the sample was
calculated as follows
68
Percentage of compound A = Area of peak A X 100
Total area of all the peaks
d. Selection of Elites (Balakrishnan et al. 2000)
With the aim of selecting patchouli elites from the germplasm of six accessions,
selection indices were computed for the four growth parameters i.e. height of the
plant, inter node length, petiole length and leaf area at the end of three months, six
months and nine months. Selection index was computed using the formula
SI = ∑ [1 + Pi (Xi – Av)]
where, SI = Selection Index
Pi = Probablity
Xi = Mean value of the ‘i’ th genotype
Av = Average
Here,
4
( i ) = ∑ [ 1+ pj (Xij – Avij)]
j=1
j=1……….6
69
where,
(i) = Index
Pj = Probability for j th character (assumed equal probability for each character)
i.e. here pi = 0.25
Xij = Value of ‘i’th genotype for the ‘j’th character
Avj = Average value of ‘j’ th character
4 .3 Results
Six patchouli accessions were selected for the field evaluation based on the RAPD
characterisation studies. All the six accessions were planted out in the field in a
Randomized Block Design (RBD) plot (Fig.7). The growth and yield parameters of
patchouli accessions were recorded at an interval of three months for a period of
twelve months (Fig. 8). An analysis of variance (ANOVA) was performed for the six
patchouli accessions using the SPSS software to test the variation between the
accessions. The data recorded at twelve months was not considered for the ANOVA
since KAR 1, i.e. the Johore variety did not survive after the ninth month growth
period.
Growth parameters
The average values of the plant height, inter node length, petiole length and leaf
area for the six patchouli accessions were computed at the end of three, six and nine
month growth periods. An analysis of the results show that the highest growth rate
for all the four parameters was recorded in MEG 1 and the lowest in KAR 1 at the
70
71
72
end of the third (Table 11), sixth (Table 12) and ninth (Table 13) month growth
periods.
Contrary to the above, it was observed that the average number of leaves per plant
was highest in the accession KER 1 followed by MEG 1 and the lowest in KAR 1. The
mean values of all the growth parameters between the accessions was observed to
be significant at the end of the first growth period i.e. 3 months and not significant
at the end of the sixth and ninth month periods (Table 14).
But, computing the average values of the total leaf area per plant across the six
accessions confirm that MEG 1 exhibits the highest leaf area per plant in contrast to
KAR 1 that has the lowest leaf area per plant during all the growth periods (Table
15).
The computation of selection index (Balakrishnan et al. 2000) for the four growth
parameters across the three growth periods showed that the selection index value
was highest in MEG 1 followed by CHE 1, DEL 1, KER 1, KAR 2 and KAR 1 in their
order of decrease (Table 16).
In addition to growth parameters, physical, morphological and micromorphological
characters were also recorded for the six patchouli accessions as in chapter one
(Table 17, Fig.9). An analysis of these parameters confirms that, there exists a
variation between the patchouli cultivars constituting the germplasm.
73
Table 11
The average values of growth parameters across six patchouli accessions at the end of three months
Accession
Height (cm)
Internode (cm)
Petiole (cm)
Leaf area (sq.cm)
KER 1
73.83
3.33
1.52
31.08
KAR 1
36.67
2.17
1.27
19.72
CHE 1
73.33
4.37
1.55
60.73
MEG 1
102.50
7.35
4.32
99.87
DEL 1
46.0
3.73
1.30
49.22
KAR 2
37.50
3.75
1.72
30.37
VR
**
**
**
**
CD (%)
2.14
0.46
0.20
1.78
VR – Variance Ratio
** Significant variance
74
Table 12
The average values of growth parameters across six patchouli accessions at the end of six months
Accession
Height (cm)
Internode (cm)
Petiole (cm)
Leaf area (sq.cm)
KER 1
84.3
3.85
1.75
43.42
KAR 1
55.67
2.60
1.53
24.58
CHE 1
78.17
4.82
1.78
65.40
MEG 1
130.17
7.82
4.62
107.80
DEL 1
76.83
4.12
1.60
53.33
KAR 2
75.5
4.17
1.92
33.52
VR
**
**
**
**
CD (%)
3.22
0.45
0.23
2.40
** Significant variance
75
Table 13
The average values of growth parameters across six patchouli accessions at the end of nine months
Accession
Height (cm)
Inter-node (cm)
Petiole (cm)
Leaf area (sq. cm)
KER 1
92.33
4.38
2.0
51.55
KAR 1
59.83
3.06
1.53
28.33
CHE 1
82.5
5.25
1.95
71.18
MEG 1
153.33
8.33
4.88
116.18
DEL 1
87.67
4.51
1.9
57.48
KAR 2
85.5
4.56
1.92
37.1
VR
**
**
**
**
CD (%)
3.12
0.44
0.22
2.09
** Significant variance
76
Table 14
The average values of the number of leaves per plant across six patchouli accessions at three different growth periods.
Accession
3 months
6 months
9 months
KER 1
141.5
154.3
165.5
KAR 1
52.3
56.5
62.5
CHE 1
62.0
67.3
74.7
MEG 1
82.8
128.1
156.5
DEL 1
90.5
114.6
130.2
KAR 2
91.0
110.1
123.5
VR
**
Not significant
Not significant
CD (%)
4.99
** Significant variance
77
Table 15
The averages of total leaf area per plant across the six patchouli accessions at three different growth periods.
Accession
3 months (in sq.m.)
6 months (in sq. m.)
9 months (in sq.m.)
KER 1
43.97
67.01
85.32
KAR 1
10.32
13.89
17.71
CHE 1
32.65
44.03
53.15
MEG 1
82.72
138.09
181.82
DEL 1
44.54
61.15
74.82
KAR 2
27.64
36.91
45.82
Table 16
Selection indices of the six patchouli accessions
Accession At 3 months At 6 months At 9 months
KER 1 2.39 1.11 1.26
KAR 1 -10.10 -11.13 -13.12
CHE 1 9.94 5.32 3.92
MEG 1 28.46 30.38 34.38
DEL 1 0.01 1.75 1.59
KAR 2 -6.72 -3.44 -4.03
78
Table 17 Characterisation of the six patchouli accessions
Accession CSI Leaf margin Leaf texture Trichome
density
Trichome
size(µm)
Vein Termination
Number
KER 1
0.55
Serrate
Pilose
6
450
4
KAR 1
0.75
Serrate
Tomentose
13
188
7
CHE 1
0.11
Crenate
Glaucus
2
150
4
MEG 1
0.00
Serrate
Glabrous
3
250
11
DEL 1
0.70
Serrate
Rugose
2
180
5
KAR 2
0.25
Crenate
Herbaceous
1
200
4
Table 18 The average oil yield across six patchouli accessions at four different growth periods.
Accession
3 months (%)
6 months (%)
9 months (%)
12 months (%)
KER 1
1.2
1.4
1.8
2.0
KAR 1
1.5
2.0
2.0
NIL
CHE 1
1.0
1.1
1.3
1.3
MEG 1
1.0
1.5
2.0
2.3
DEL 1
1.5
1.9
2.0
2.0
KAR 2
1.3
1.6
1.8
1.3
79
80
Patchouli oil yield
The patchouli oil yield was assessed across four growth periods i.e. at the end of
three months, six months, nine months and twelve months. It was observed that in
KER 1 and MEG 1, the oil yield increased gradually over the four growth periods
whereas in CHE 1 and DEL 1, the increase in oil yield was observed only during the
first, second and third growth periods. The yield at the end of the fourth growth
period remained same as that of the third growth period.
In contrast to the above, the accessions KAR 2 and KAR 1 exhibited a gradual
increase in the oil yield over the three, six and nine month growth periods. But, a
steep decline in oil yield was observed at the end of the fourth growth period i.e. at
twelve months (Table 18).
Oil quality parameters
Essential oils extracted from the patchouli accessions KER 1, KAR 1, CHE 1, DEL 1 and
KAR 2 exhibited a golden brown color whereas MEG 1 the cultivar collected from the
wild showed a dark brownish yellow color (Fig. 11a). All the six oils were
characterised using Gas Chromatography. Analysis of the Gas Chromatograms
showed that a total of thirteen peaks were generated across the six patchouli
accessions at the retention times 4.6, 9.1, 10.6, 11.0, 12.0, 12.3, 12.7, 12.9, 13.4,
13.9, 14.1, 14.4 and 14.7 seconds. Each of these peaks represented a compound
constituting the oil. The percentage composition of oil constituents was computed
from the Gas Chromatograms for all the six patchouli accessions (Fig.10, Table 19).
81
Table 19
Percentage composition of patchouli oils across the six accessions
Ret. Time peak
KER 1
KAR 1
CHE 1
MEG 1
DEL 1
KAR 2
4.6
17.8
9.1
15.4
10.6
4.2
5.6
1.7
1.5
2.2
2.7
11.0
15.8
3.7
1.8
1.8
5.0
12.0
5.6
12.3
2.5
11.5
24.5
12.7
36.4
12.9
21.4
12.3
7.3
6.2
11.1
39.0
13.4
7.4
28.0
8.2
13.9
2.8
4.3
30.4
8.1
4.1
14.1
55.8
58.6
19.3
45.8
35.0
21.9
14.4
7.0
14.7
6.0
82
83
84
It was observed that three compounds were present across all the six patchouli
accessions, though in varying quantities. These compounds separated out at the
retention times 10.6 sec., 12.9 sec. and 14.1 sec. (Table19, Fig.10). Identification of
components was performed on the basis of the fragmentation pattern of the mass
spectra generated (Fig.12a and 12b). The mass spectral analysis of these GC peaks
followed by Wiley library search showed that the peaks at 10.6 and 12.9 retention
times had a molecular mass of 204 and represented the compounds β patchoulene
and α patchoulene whereas the peak at 14.1 retention time had a molecular mass of
212 that represented the compound patchouli alcohol (Fig.12). A comparison of GC
retention indices of the oils obtained from the six patchouli accessions helped in
establishing relationships within the germplasm. The quality of patchouli oil is
determined in the market by the amount of patchouli alcohol present in it. In view of
this, it is observed that the patchouli accession KAR 1 contains the highest amount
of patchouli alcohol i.e. 58.6% followed by KER 1 , MEG 1, DEL 1, KAR 2 and CHE 1
(Fig.11b). However, it is noted that the composition of a-patchoulene and b-
patchoulene also play an important role in quality assessment of patchouli oil along
with patchouli alcohol (Fig. 11c.and Fig.13).
Patchouli accessions were ranked based on the percentage oil yield and percentage
of patchouli alcohol (an indicator of patchouli oil quality) using the rank-sum method
(Kang 1991). It was observed that the accession KAR 1 was ranked one, followed by
KER 1, MEG 1, DEL 1, KAR 2 and CHE 1 in the order of their oil yield and quality
(Table 20).
85
Table 20
Ranking of the six patchouli accessions based on oil yield and oil quality
Accession
Oil yield (in %)
Patchouli Alcohol (in %)
Rank
3 months 6 months 9 months
KER 1
1.2
1.4
1.8
55.8
II
KAR 1
1.5
2.0
2.0
58.6
I
CHE 1
1.0
1.1
1.3
19.3
VI
MEG 1
1.0
1.5
2.0
45.8
III
DEL 1
1.5
1.9
2.0
35.0
IV
KAR 2
1.3
1.6
1.8
21.9
V
86
87
88
4.4 Discussion
Growth parameters
The results of the field evaluation study clearly indicate that the patchouli accession
MEG 1 stands out as the most vigorous and KAR 1 the least vigorous in comparison
to the other accessions with reference to their growth parameters i.e. height of
plant, inter node length, petiole length, leaf area and total leaf area per plant. But,
the herbage yield per plant was observed to be highest in KER 1 followed by the
MEG 1 accession. It was also observed that the KAR 1 accession of patchouli did not
survive after nine months proving that it has a low regeneration capacity and shorter
life span when compared to the other accessions. Though KAR 1 showed a gradual
increase in oil yield over the three, six and nine month growth periods, a steep
decline in herbage and oil yield was observed at the end of the fourth growth period
i.e. twelve months. This may be correlated with the woodiness of the stem leading
to a low leaf yield (Sarma et al. 1995). The earlier field study reports (Samuel 2002)
also support this view point. The Chlorophyll Stability Indices (CSI) of the patchouli
accessions lend additional support to this observation since CSI is an indicator of the
tolerance and survival chances of a plant (Dhopte 2002).
The Chlorophyll Stability Index (CSI), leaf margin, leaf texture, trichome density,
trichome size and vein termination numbers in all the six patchouli accessions are
observed to be features that help in distinguishing one accession from the other
(Table 17). The report (Sugimura et al. 2006) that cultivars are distinguishable from
one another by their characteristic leaf morphology and trichome density supports
89
this finding. Various natural and controllable factors like the crop growth under sun
and shade (Prakash Rao et al. 1997), soil heterogeinity (Sugimura et al. 2006), quality
of planting material (Sharma 1999) and cultivation practices (Sarma and Kanjilal
2000) are observed to affect the yield and quality of patchouli oil. It is speculated
that a variation in the characteristic features between patchouli cultivars arose as
adaptations to diverse climatic conditions. An increase in trichome number and
trichome size as in KER 1 and KAR 1 (Penang and Johore patchouli) can be correlated
to a hot humid climate with minor variations. A decrease in trichome numbers and
trichome size coupled with the appearance of a waxy coating and wrinkled nature of
the leaves as observed in CHE 1 and DEL 1 accessions (Java and Medicinal patchouli)
are features that can be correlated to a hot and dry climate with minor variations.
However, fewer trichomes, moderate trichome size and an increase in vein
termination numbers like in MEG 1 (Wild patchouli) are features that can be
correlated to a hot and humid but water stressed condition. A decrease in number
of trichomes, size of trichomes and the vein termination numbers like in KAR 2
(Mysore patchouli) are features that can be related to a cool and rain-fed climatic
condition (Table 1 & 17, Fig.9). The results prove that the patchouli plants possess a
higher ecological amplitude (Bhatia 1998) which is the reason for its ecosystem
diversity. Reports also suggest that secretory structures like glandular trichomes and
glandular hairs can serve as genetic markers for oil quality as in many essential oil
producing plants (Croteau et al. 1981 and Massino et al. 1986)
90
Patchouli oil yield
The oil yield analysis across the six patchouli accessions is observed to support the
growth rate results. The oil yield increase in the accessions KER 1 and MEG 1 at the
end of third, sixth, ninth and twelfth months is in conformity with the growth rate of
the said accessions in the field. The results also suggest that the accessions KER 1
and MEG 1 have a longer life span when compared to the others. Contrary to the
above, the oil yield in the accessions KAR 2 and KAR 1 decreased at the end of the
twelve month growth period indicating that these two accessions have a short life
span. Though the accessions CHE 1 and DEL 1 showed a gradual increase in the oil
yield at the end of the third, sixth and ninth months, the yield remained stable at the
end of the fourth growth period i.e. at twelve months (Table 18). Therefore CHE 1
and DEL 1 can be considered as accessions with a moderate life span.
Oil quality parameters
The result of chemical characterization of the essential oils extracted from KER 1,
KAR 1, CHE 1, MEG 1, DEL 1 and KAR 2 showed that three compounds namely
β patchoulene, α patchoulene and patchouli alcohol were present in all the six
accessions of patchouli as mentioned in previous reports (Sellier et al. 2004 and Hu
et al. 2006). Therefore, these compounds can function as chemical markers and aid
in the identification of true patchouli oil. Patchouli essential oil with 50.66% to
54.3% patchouli alcohol and 4.27% α patchoulene are reported to be good quality oil
and readily accepted in the market (Singh et al. 2002). Reports also suggest that
patchouli alcohol readily loses water to form the sesquiterpene patchoulene
91
(Guenther 1974). This supports the fact that external factors like environmental
regime have an influence on the patchouli oil character. The existence of a set of
sesquiterpenes in patchouli oil that is influenced by environmental conditions
(Banthorpe et al. 1972 and Tsai ying-chieh et al. 2007) lend further support to the
presence of other chemical constituents in the oil. However, the gas chromatograms
generated from the oils of KER 1 and KAR 1 exhibited an identical pattern (Meng
Shao-jin et al. 2006) proving that KER 1 and KAR 1 belong to the same genetic stock
(Fig.10). This is also in conformity with the RAPD profiles (Figs. 3, 4 and 5) and the
results of the cluster analysis (Fig.6). Therefore, it can be undoubtedly established
that the patchouli accessions KER 1 and KAR 1 are closely related to each other (Wei
gang 2007) and hence constitute the primary gene pool. The results also
recommend KER 1(Penang patchouli) as an ideal substitute for KAR 1( Johore
patchouli).
In case of patchouli oil extracted from MEG 1, double the amount of methanol was
added in the oil to dilute it since the oil failed to form peaks when injected in the
same concentration as that of the oils extracted from the other accessions (4.2.3.c.i).
However, the GC profile of MEG 1 was observed to match with the GC profiles of the
other five accessions. It was also observed to possess all the three chemotyping
components b-patchoulene, α-patchoulene and patchouli alcohol as found in the
other accessions of patchouli i.e. KER 1, KAR 1, CHE 1, DEL 1 and KAR 2 (Table 19).
This indicates that MEG 1, the wild variety of patchouli could be a natural polyploid.
The difference in the color of patchouli oil extracted from MEG 1 cultivar (Fig.11a)
and the vigorous growth exhibited by the plant (Table 11, 12, 13 and 16) lends
92
support to this inference. Further studies in this direction were beyond the scope of
the present work.
The rank-sum method (Kang 1991) was adopted to correlate and rank patchouli
cultivars in order of their oil yield and quality. KAR 1 ranked first, followed by KER 1,
MEG 1, DEL 1, KAR 2 and CHE 1. It was observed that, though KER 1 and KAR 1
ranked top in their oil yield and quality (Table 20), they showed the least growth rate
(Table 16).These characters confirm the report that, though a perennial crop,
patchouli needed renewal after two to three years of its cultivation (Sarma et al.
1995). More recent reports suggest that the patchouli crop requires renewal after
every one to two years (Samuel 2002). However, the present study shows that KAR 1
(Johore cultivar) needs a renewal after every nine months i.e. after two to three
harvests (Table 18). This is attributed to the strong lignification, low leaf production
and decreased regeneration capacity in the Johore patchouli owing to the adoption
of repeated propagation by stem cuttings (Bhasker and Vasanthakumar 2000).
The amount of patchouli alcohol, the major constituent of patchouli oil is the factor
that decides the quality of patchouli oil. The more the composition of patchouli
alcohol in patchouli oil, the better it is considered in the national and international
market (Singh et al. 2002 and Zhao et al. 2005). KAR 1 therefore stands out as the
accession producing the best quality oil followed by KER 1 and MEG 1 (Table 19,
Fig.11b).
93
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