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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