Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol29.Issue3.Mar2001:

Biochemical Systematics and Ecology 29 (2001) 267–285

Variation in volatile compounds from tansy
(Tanacetum vulgare L.) related to genetic and
morphological differences of genotypes
Marjo Keskitaloa,b,*, Eija Pehua, James E. Simonb
a

b

Department of Plant Production, P.O. Box 27, University of Helsinki, FIN-00014, Finland
Department of Horticulture, Center for New Crops and Plant Products, Purdue University, West Lafayette,
IN 47906-1165, USA
Received 18 January 1999; accepted 15 February 2000

Abstract
Air-dried flower heads of 20 Finnish tansy genotypes were extracted with petroleum ether
and analyzed using GC–MS. A total of 55 volatile compounds were detected, and 53 were
identified. Of the tansy genotypes studied, 15 were well defined and five were mixed
chemotypes. Complete linkage analysis differentiated the populations into six clusters. The
most frequently found monoterpene was camphor with or without several satellite compounds

such as camphene, 1,8-cineole, pinocamphone, chrysanthenyl acetate, bornyl acetate and
isobornyl acetate. In 13 genotypes, camphor concentration exceeded 18.5% and in seven
genotypes, camphor was less than 7.2%. Other chemotypes rich in trans thujone, artemisia
ketone, 1,8-cineole, or davadone-D were also identified. Davadone-D and a mixed chemotype,
containing tricyclene and myrcene, were identified from a Finnish tansy for the first time.
Geographically, most chemotypes containing camphor originated from Central Finland,
whereas chemotypes without camphor such as artemisia ketone, davadone D and myrcene–
tricyclene originated from South or Southwest Finland. Morphologically, the 20 tansy
chemotypes based on the groups formed from complete linkage cluster analysis, were
compared. The group containing the highest concentration of camphor chemotypes had the
tallest shoots. The groups consisting from chemotypes containing davadone-D or artemisia
ketone, which originated from Southwest Finland, produced the highest number of flower
heads, had the tallest corymb, and were last to flower. Also, the group consisting from
chemotypes with a high concentration of camphor and originated from South Finland started
to flower late. The correlation between the genetic distance matrices based on RAPD patterns

*Corresponding author. Present address: Plant Production Research, Agricultural Research Center of
Finland, FIN-31600 Jokioinen, Finland. Fax: +358-3-4188-2437.
E-mail address: marjo.keskitalo@mtt.fi (M. Keskitalo).
0305-1978/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 5 6 - 9

268

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

reported previously (Keskitalo et al., 1998. Theor. Appl. Genet. 96, 1141–1150.) and the
chemical distance matrices of the present study of the same tansy genotypes was highly
significant (0.41, P50:0001). # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Asteraceae; Davadone-D; Essential oils; Intraspecific variation; Monoterpenes; Multivariate
analysis; Petroleum-ether extraction; Tanacetum vulgare L.

1. Introduction
Tansy is an aromatic perennial plant, widely spread in the northern hemisphere
(Heywood, 1976; Hussey, 1974; Hultén, 1968). Occasionally, it has been cultured in
gardens (Mitich, 1992; Heywood, 1976) and used in salads, omeletts, cakes and
spices (Grieve, 1984; Hussey, 1974). As a herbal remedy, tansy has traditionally been
used in balsams, cosmetics, dyes, insecticides, medicines, and preservatives (Grieve,
1984; Hussey, 1974; Millspaugh, 1974) and as anthelmintic, for migrane, neuralgia,
rheumatism and loss of appetite (Blumenthal, 1998), though the effectiveness for

these uses have not been documented. Recent studies have also shown that the
essential oil or extract of tansy exhibits anti-inflammatory (Brown et al., 1997;
Mordujovich-Buschiazzo et al., 1996), antibactericidal (Neszmélyi et al., 1992;
Héthelyi et al., 1991; Holopainen and Kauppinen, 1989; Stefanovic et al., 1988),
antifungicidal, (Neszmélyi et al., 1992; Héthelyi et al., 1991), and a repellent against
insects (Hough-Golstein and Hahn, 1992; Nottingham et al., 1991; Suomi et al.,
1986; Panasiuk, 1984; Schearer, 1984). The activity against microbes and insects was
dependent on the chemical composition of the essential oil (Héthelyi et al., 1991;
Holopainen and Kauppinen, 1989; Panasiuk, 1984; Schearer, 1984). Components in
the essential oil are also of potential interest as aroma chemicals in perfumery
(Lawrence, 1992).
The composition of tansy oils varies markedly and several chemotypes from
different geographical origins have already been classified. Tansy from Tierra
del Fuego, Argentina (Gallino, 1988), and from England and USA (Ekundayo,
1979) were found to contain mainly b-thujone. Tansy oil obtained from two
Canadian-grown plants contained b-thujone (d-isothujone) (Collin et al., 1993;
von Rudolf and Underhill, 1965), a-thujone (l-thujone) (von Rudolf and Underhill,
1965), camphor-1,8-cineole-borneol, chrysanthenone, or dihydrocarvone (Collin
et al., 1993) as the major oil components. In contrast, chemotypes such as
artemisia alcohol, camphenol, davadone, lyratol, lyratyl acetate and 4-thujen2a-yl (Héthelyi et al., 1981; Tétényi et al., 1975), and trans-chrysanthenyl

acetate (Neszmélyi et al., 1992) have been detected in tansy grown in Hungary.
Naturally occurring tansy from northeastern Netherlands contained artemisia
ketone, chrysanthenol/chrysanthenyl acetate, lyratol/lyratyl acetate and b-thujone
(Hendriks et al., 1990). Chrysanthenyl acetate and camphor/b-thujone chemotypes
were identified in tansy grown in Belgium (De Pooter et al., 1989). In contrast,
only one chemotype, chrysanthenyl acetate, was detected from tansy grown in

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

269

Piedmont, Italy (Nano et al., 1979). Several authors have studied the
intraspecific variation of essential oil in Finnish tansy. Sorsa et al. (1968) found
that a-pinene, b-pinene, 1,8-cineole, g-terpinene, artemisia ketone, thujone,
camphor, umbellulone and borneol were the most common chemotypes. They also
observed that camphor was the main component in northern, and thujone in
southern Finland (Sorsa et al., 1968). Later, isopinocamphone (Forsén and von
Schantz, 1971), trans-chrysanthenyl acetate (Forsén, 1974), sabinene, bornyl acetate,
and germacrene D (Holopainen, 1989) chemotypes were detected from tansy grown
in Finland.

Tansy also contains other secondary products such as less volatile sesquiterpenes
(Chandra et al., 1987b) and several nonvolatile compounds. The latter group consists
monoterpene glucosides (Banthorpe and Mann, 1971); sesquiterpene lactones (Sanz
and Marco, 1991; Ognyanov and Todorova, 1983) such as parthenolide (Hendriks
and Bos, 1990); sesquiterpene diketone (Ognyanov et al., 1983); triterpenes
(Wilkomirski and Kucharska, 1992); and flavonoids (Chandra et al., 1987a;
Ognyanov and Todorova, 1983).
In addition to different tansy chemotypes of which the concentration of one
compound exceeds 40% (as a definition for well-defined chemotypes; Holopainen,
1989), more than 100 volatile mono- and sesquiterpenes have been identified from
tansy by the authors including those compounds reported in this paper. Many
factors are known to influence oil composition, including source of extractable plant
tissue (leaf/flower), ontogeny of plant at sampling (Hendriks et al., 1990;
Holopainen, 1989), seasonal changes (Németh et al., 1994; von Schantz et al.,
1966; von Rudolf and Underhill, 1965), and even the extraction method (Collin
et al., 1993; Holopainen, 1989) may cause variation in the composition of volatile
oil.
We are interested in to enhance the insecticidal properties of tansy. Thus, we have
developed micropropagation, regeneration and protoplast fusion methods
(Keskitalo et al., 1999, 1995) for the improvement of secondary metabolism by

genetic engineering. We also studied genetic variation of 20 tansy genotypes collected
from different regions of Finland by RAPD-PCR. Genetically, the genotypes
clustered into two major groups, which were further divided into smaller groups,
and the clustering was correlated to the geographical origin of the genotypes.
Morphologically, the two clusters differed in their flowering (Keskitalo et al., 1998).
To ascertain whether there is a connection between the chemical and genetic
differences of tansy, we extracted the volatile compounds from the same tansy
genotypes and analyzed these compounds with GC–MS. The goals of this study
were two fold: (1) to identify the volatile terpenes extracted from air-dried
tansy flower heads; and (2) to compare the variation to the geographical origin
of the genotype to link the genetic and morphological variation among the
tansy genotypes. Assessment of the chemical variation of tansy would allow
us to identify unique chemotypes with potential industrial value and bioassay
the activity of the genotypes with specific terpene composition. With such
information the most desirable chemotypes can be selected for genetic engineering
studies.

270

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

2. Materials and methods
2.1. Plant materials
The geographical locations of 20 tansy genotypes (Tv 12Tv 20) collected and used
in this study are listed in Table 1. The genotypes were transplanted to an orchard of
the Department of Plant Production, University of Helsinki (608100 N), Finland in
1991 (Keskitalo et al., 1998). The vouchers have been deposited at Botanical
Museum of University of Helsinki (H). In the orchard, the plants were grown 1.5 m
apart in two rows without fertilization. Morphological observations such as height
of the stem, number of nodes per stem, number of flower heads per stem, length of
corymb and date at the beginning of flowering were carried out as described
(Keskitalo et al., 1998). The flower heads were excised at the onset of flowering, air
dried at 388C, and stored in room temperature in the dark until extracted.
2.2. Isolation of volatile compounds
Two grams of air-dried flower heads crushed in a mortar, were transferred to
centrifuge tubes containing 5 ml methanol, shaken for 1 h, centrifuged and the
supernatant was collected to another tube. The flower heads were extracted with
methanol 3 times. About 1–2 ml of saturated NaCl and 7 ml petroleum-ether (36–60)
Table 1
Geographical origins (National Landsurvey Institute, 1996) of the 20 tansy genotypes

Tansy genotype
Collector #
Keskitalo 21440
Tv
Tv
Tv
Tv
Tv
Tv
Tv
Tv
Tv
Tv
Tv
Tv
Tv
Tv
Tv
Tv
Tv

Tv
Tv
Tv

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

17
18
19
20

Region

Latitude (N)

Longitude (E)

Voucher at (H)

Iisalmi
Eno
Koli
Kangasniemi
Helsinki
Halikko
Lohjanharju

Tampere
Sääksmäki
Porvoo
Sipoo
Vihti
Hämeenlinna
Lemu
Tervakoski
Lieto
Hanko
Alavus
Hailuoto
Köyliö

638330 N
628470 N
638060 N
618590 N
608100 N
608230 N
608300 N
618290 N
618110 N
608230 N
608220 N
608250 N
608590 N
608330 N
608480 N
608300 N
598490 N
628350 N
658000 N
618060 N

278120 E
308080 E
298480 E
268390 E
248570 E
238030 E
248240 E
238480 E
248030 E
258400 E
258150 E
248180 E
248270 E
218590 E
248380 E
22827’E
238000 E
2383700 E
248430 E
228200 E

H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H

1698296
1698297
1698298
1698299
1698300
1698301
1698302
1698303
1698304
1698305
1698306
1698307
1698308
1698309
1698356
1698355
1698354
1698353
1698352
1698351

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

271

were added to the methanol extraction (15 ml), after which the solution separated
into two phases. The top layer containing petroleum-ether was pipetted to an
Erlenmeyer flask, and dried with anhydrous sodium sulfate in +48C in the dark for
1–3 days. The addition of NaCl and petroleum-ether was done twice. The dried
supernatant was filtered and evaporated to 1 ml and stored in 2 ml vials in +48C in
the dark until the GC analysis. The extraction from the flower heads of each tansy
genotype was carried out 4 times. The different extraction times are referred as A–D.
2.3. Chemical analysis of the volatile compounds
Prior to the gas-chromatography (GC) analysis, 1 ml of an internal standard
(tetradecane; stock 50 mg/1 ml petroleum-ether) was added to a volume of 100 ml of
the extractions of A–C. The fourth sample (D) was run without the internal
standard. A sample of 1 ml was injected into the GC-Varian (Walnut Creek, CA)
3700 gas chromatograph, fitted with a flame-ionization detector and HewlettPackard model 3396 series II integrator (Palo Alto, CA). A fused silica capillary
column (30 m0.25 mm i.d., 0.20 mm film thickness, SPB-5; Supelco, Bellefonte, PA)
was used. Helium was used as the carrier gas, and oven temperature was held
isothermal at 808C for 5 min and then programmed to increase 38C/min to 2408C.
The injector and detector temperatures were maintained at 180 and 2508C,
respectively. GC/MS analyses were conducted using a Finnigan (San Jose, CA)
GC (model 9610) and mass spectrometer (model 4000) system equipped with a
capillary column under the same GC conditions and interfused to a Data General
Nova/4 data processing system. The MS conditions included ionization voltage,
70 eV; emission current, 40 mA; scan rate, 1 scan/s; mass range, 40–500 Da; and ion
source temperature, 1608C. The volatile components of the three separate
extractions (A–C) in each tansy line were identified by comparing their relative
retention times, retention indices (RI) and mass spectra with those of authentic
samples, and by matching the mass spectra of each compound with different MS
compound libraries for best fit (Adams, 1995; Finnigan et al., 1978; Stenhagen
et al., 1974) and to the Kovats’ indices of authentic samples (Adams, 1995). The
retention indices of our samples were calculated using n-alkanes (C8–C18) as a
reference (Table 2).
2.4. Statistical analysis
The volatile components of the tansy flower heads were scored as being present (1)
or absent (0) in an extracted oil as determined by GC–MS. The pair-wise similarity
and distance matrices of volatile compounds of the 20 tansy genotypes were
calculated using the SAS program (SAS Institute, 1984) modified by Levy et al.
(1991), similarly as we reported using RAPD-data (Keskitalo et al., 1998). The
similarity and distance matrixes were calculated according the formula of Nei and Li
(1979): [Sxy ¼ 2Nxy : ðNx þ Ny Þ]; where Nxy is the number of terpenes common for
the accessions x and y; and Nx and Ny are the numbers of terpenes of the accessions
x and y, respectively. The dendrogram was synthetized by complete linkage cluster

272

Table 2
Volatile oil concentration (%) and the mean of retention indices (RI) of volatile compounds extracted from air-dried flower heads of 20 tansy genotypes and identified by GC-MS. The results are a
mean of three different extractions (A–C) carried out from each tansy genotype. The groups of tansy genotypes are formed according to the complete linkage cluster analysis based on the presence or
absence of the identified compounda
Groups of tansy genotypes (Tv)
Group 2

Compound

RI

Tv 7

Tv 8

1 Tricyclene
2. a-Thujene
3. a-Pinene
4. Camphene
5. Sabinene
6. Artemiseole
7. b-Pinene
8. Myrcene
9. Yomogi alcohol
10. r-Cymene

912
932
940
957
980
982
986
1002
1002
1029

*

26.84 16.96 0.83

11.
12.
13.
14.
15.

1033
1038
1063
1063
1079

16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.

Limonene
1,8-Cineole
Artemisia ketone
g-Terpinene
cis Sabinene
hydrate
Terpinolene
Artemisia alcohol
cis Thujone
trans Sabinene
hydrate
Chrysanthenone
trans Thujone
Camphor
Chrysanthenol
Pinocarvone
Umbellulone
Terpinen-4-ol
Artemisyl acetate
Borneol
cis Pinocamphone
a-Terpineol
Myrtenol
Verbenone
Nerol
trans Chrysanthenyl
acetate

0.46
2.44

Tv 10 Tv 20

0.08
0.10

0.04

0.06
0.08 0.85
39.39 20.92 1.13
1.43

1.22

2.04

5.14

0.75
1.56

1.95
6.61

0.31

1.39

0.88
0.06

1.53
0.13

1097
1098
1116
1119

1.56

1121

0.12

0.93

0.09

1129
1154
1164
1168
1170
1175
1176
1177
1185
1199
1204
1219
1235
1249

0.43
1.40

0.07
7.15
0.69

0.91
1.35
3.07

0.04
4.71
67.32

0.27
4.27

Tv 3

Group 4

0.06
2.00

15.26 2.45

Tv 5

Tv 6

Tv 12

Tv 9

Tv 11 Tv 13 Tv 15

Tv 17 Tv 19

Tv 14 Tv 16

2.15
0.34
0.67
7.27
0.28

0.16
0.17
2.27
5.15
2.95

0.05
0.39
2.90
7.76
0.85

0.27
0.28
2.07
8.66
0.03

0.04
0.56
4.44
12.60
0.29

0.11
0.30
0.61
4.57
0.40

0.09
0.73
4.69
11.62
1.61

0.33
0.53
0.57
5.06
0.72

0.10

0.36

5.78
2.24
0.64

0.25
0.45
2.71
7.83
1.12

2.01

1.03

1.46

2.57

0.60

2.91

0.40

1.27

0.80

1.46

0.04

1.57

2.74
0.68

1.69
0.43

0.04
0.32
0.64
2.14

0.36
5.72
2.42
1.62

0.54
0.70
6.03
0.03

0.05

2.64

0.61

3.42

0.16
0.78
0.61
0.22

0.15
3.93

1.61

2.91

0.06
3.21

1.36 0.16
11.43 0.15

1.77
0.41

0.54
0.02

1.24

1.18

0.41

0.78
0.12
0.86

0.82

0.65

0.24

0.22

1.32

1.96

1.02

4.00

2.60

0.34 2.53
47.12 8.21

0.38
9.92

1.20
4.79

0.31
0.75

0.78

0.21

0.76

0.18

0.14

0.69

0.14

0.07

6.62
0.45
0.34

0.58

2.05

1.39

1.71

0.81
20.23 7.55
0.54
0.70 0.40
0.55 0.19

0.37
13.73 4.68
0.86
0.08

0.64

0.18

1.24

0.39

0.19

0.42

1.59 1.80
55.08 81.36

0.67
9.31

0.28
3.78

5.98

0.42

0.03
81.87 2.12 0.25
3.70 19.43 69.37 72.16 70.71 29.43 50.07 73.02

0.06 0.04
0.04
50.78 68.79 48.15 65.23

0.15

0.03
0.66

4.94

2.65

2.76

1.01

0.56

0.12

0.65

1.73

3.45

3.01

5.81

0.11

1.39

0.60

4.17

0.16

0.03
0.65

1.16
0.57

0.10

1.22

3.87
0.60

0.23
0.07

0.07

0.30

0.87

Group 6

Tv 18 Tv 2

0.04

3.49
0.17

Group 5

Tv 4

0.83

12.18
4.35

Tv 1

Group 3

0.21

1.11

0.07

4.27

3.95

0.39

3.73
5.01

0.81
0.04
1.01

0.94

0.03
0.62

0.07
18.54 35.71
1.12
4.38
2.46
1.58
3.87 5.08
19.10

0.36
0.02

1.02

1.06
1.53
0.99
0.34
0.33 0.48
10.69
0.21

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

Group 1

Groups of tansy genotypes (Tv)
Group 1
RI

35. Carvone
36. cis Chrysanthenyl
acetate
37. Unknown,
MW 154
38. Bornyl acetate
39. Isobornyl acetate
40. Carvacrol
41.
42.
43.
44.
45.
46.
47.
48.
49.

1306
1336
1387
1390
1426
1485
1501
1525
1553

Thymol
d-Elemene
b-Cubebene
b-Elemene
(E) Caryophyllene
Germacrene D
Bicycloelemene
g-Cadinene
Artedouglasia
oxide C
50. Nerolidol
51. Germacrene
alcohol
52. Spathulenol
53. Caryophyllene
oxide
54. Davadone D
55. Unknown,
MW 222

Tv 7

Group 2

Tv 8

Tv 10 Tv 20

1250
1271

0.89

4.39

1286

0.07

1291
1298
1304

0.67

Tv 1

11.97

0.54

1.36
2.54

0.15
2.05
0.75
1.32

0.43

5.16

Tv 18

0.97

0.65

Tv 2

Tv 5

Tv 6

Tv 12

Group 4

Group 5

Group 6

Tv 9

Tv 17 Tv 19

Tv 14 Tv 16

Tv 11 Tv 13 Tv 15
0.81

0.90

1.56

0.19
0.77
0.66

1.43
2.18

0.81
1.42

0.05

0.81

0.11

11.55 0.04

17.80

1.16

0.31

1.35
0.06
0.06

1.37

2.64

5.72 16.72
12.04 7.48
2.67
0.86
0.65

0.07
0.07
0.96
2.66
0.06
1.87

0.13
0.23
1.14

0.58
1.27

0.86

0.52
0.04
0.82

0.14
1.32
0.79

0.07
0.53

2.92

0.11
0.88
5.08
0.60
0.39

0.86
0.43
1.08
0.05

3.11

1.27

0.03
0.43
0.03

1.38
0.59
0.99

1.31
1.47

0.66
0.26

0.44

0.74

0.40
0.09
1.32
1.12

1.13

0.28
0.86
0.12
1.03

1.18

1565

2.22

0.37

1577

0.04

0.20

1591
1594
1597
1623

Group 3
Tv 4

24.88

0.58

2.25
0.90
1.58

Tv 3

0.22
0.55
65.51
0.39

1.74

0.26

0.07
0.24
0.17

0.55

0.19

0.13

0.41

0.25

0.16

1.81

0.13

0.33

0.15

0.09

0.23

Percentage of total
peak area
identified

85.54 95.85 95.61 98.05

98.70 98.55 97.76 98.89

97.00 98.64 98.91 99.37

99.29 98.68 99.33 99.11

97.79 96.84

97.94 97.39

Total amount (mg)
of terpenes
isolated from 2.0 g
flower heads

0.93

2.84

1.03

1.94

2.22

4.76

a

1.61

1.11

5.46

2.36

3.29

2.29

2.80

1.50

2.73

3.45

5.07

5.65

4.35

3.20

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

Compound

Blank areas = not detected

273

274

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

analysis, which illustrates the chemical distance as the normalized maximum distance
between clusters. The normalized maximum distance is a normalized value (distance
between x and y/mean distance of the population) of the average genetic distance.
Morphological data of tansy genotypes (Keskitalo et al., 1998) were arranged
according to the grouping of complete linkage cluster analysis and the differences
between the groups were calculated with SAS (SAS Institute, 1984) CONTRAST
program. To compare the distance matrices of RAPD-PCR study of our previous
study (Keskitalo et al., 1998) and the distance matrices of this present study
(Table 3), Pearson correlation coefficient was calculated with SAS.

3. Results
3.1. Volatile compounds identified with GC–MS
A total of 55 aromatic volatile compounds were detected from the petroleum-ether
extraction of dried flower heads of tansy. The volatile compounds covered on
average 97.46% (S.D. 2.69) of the total peak area recovered from GC. The concentration of 47 of the 55 compounds varied highly significantly between the tansy
genotypes (P50:0001) and only the genotypic variation in artemiseole, trans sabinene hydrate, nerol, nerolidol, spathulenol, caryophyllene oxide and two unidentified compound was not significant. On average, the total amount of volatile compounds extracted from 2.0 g of tansy flower heads was 2.90 mg (S.D. 1.63) (Table 2).
Fifteen of the 20 genotypes had main component (usually camphor, artemisia ketone,
trans thujone, or davadone D), which consisted at least 40% of the total peak area,
and five genotypes contained at least two terpenes as the main components.
3.2. Complete linkage cluster analysis
The smallest chemical distance observed was 0.116 between the chemotypes Tv 5
and Tv 6 and the largest distance between Tv 7 and Tv 19 (Table 3). The mean
chemical distance was 0.374 among the entire tansy population. Complete linkage
cluster analysis separated the population to six groups. The largest and smallest
normalized maximum distance was 1.94 (0.726/0.374=1.94) and 0.31 (0.116/
0.371=0.31) between group 1 and the cluster of the other groups, and between Tv
5 and Tv 6, respectively (Fig. 1).
3.3. Volatile compounds related to the geographical origin, RAPD-PCR pattern and
morphology
Six of the seven chemotypes, which did not contain camphor as the main
component, originated from Southern Finland (Tv 7, 8, 10, 14, 16, 20), and only the
chemotype containing thujone (Tv 1) was from Central Finland (Fig. 2). Eight of the
13 chemotypes with camphor concentration exceeding 18.5% originated from
Central Finland (Tv 2, 3, 4, 9, 13, 15, 18, 19) and five from Southern Finland (Tv 5, 6,

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

275

Fig. 1. The clustering based on the absence or presence of 55 volatile compounds identified by GC–MS of
the 20 tansy genotypes used in this study. The chemical distance between genotypes were calculated as
described by Nei and Li (1979) using the complete linkage cluster analysis. The bar shows the normalized
maximum distance between clusters.

11, 12, 17). Artemisia ketone (Tv 14, 16, 20) was found only from genotypes
originating from Southwestern Finland. Also, geographically the six groups formed
from complete linkage cluster analysis could be separated from each other. Only the
groups containing a high concentration of davadone D, artemisia ketone or
myrcene-tricyclene (Tv 7, 8, 10, 20 and Tv 14, 16) were found from South-Finland.
The groups containing a high concentration of camphor were found from South and
Central Finland, but the number of chemotypes containing a high concentration of
camphor was higher in Central Finland (Fig. 2).
In a previous paper, we reported the genetic distance matrices based on RAPDPCR patterns (Keskitalo et al., 1998). The groupings of tansy population according
to our previous genetic study (Keskitalo et al., 1998) and this study on tansy volatile
compounds are illustrated in Fig. 2. Because the distance matrices of these both
studies were calculated by the same method, we calculated the correlation between
matrices of genetic data and chemical data. The Pearson correlation coefficient,
0.407 (P50:0001), revealed a 16.565% analogy between genetic and chemical
differences based on absence or presence of compound in the 20 tansy genotypes
studied.
The tansy genotypes were arranged according to the six groups formed from
complete linkage cluster analysis and the variation of morphology was compared
between the groups using the SAS CONTRAST procedure. The group containing
the highest percentage of camphor and 1,8-cineole (Tv 9, 11, 13, 15) had the tallest
shoots (108.2 cm) and differed significantly from the others (P50:0001) whereas the

276

Tv 1
Tv 2
Tv 3
Tv 4
Tv 5
Tv 6
Tv 7
Tv 8
Tv 9
Tv 10
Tv 11
Tv 12
Tv 13
Tv 14
Tv 15
Tv 16
Tv 17
Tv 18
Tv 19
Tv 20

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

0.000
0.351
0.156
0.217
0.200
0.190
0.514
0.348
0.292
0.347
0.333
0.282
0.261
0.463
0.265
0.422
0.362
0.256
0.364
0.526

0.000
0.368
0.384
0.211
0.200
0.600
0.487
0.366
0.476
0.415
0.250
0.333
0.412
0.333
0.421
0.500
0.278
0.405
0.484

0.000
0.234
0.217
0.163
0.579
0.362
0.265
0.360
0.388
0.300
0.319
0.524
0.280
0.435
0.375
0.273
0.333
0.538

0.000
0.234
0.227
0.487
0.417
0.200
0.333
0.200
0.317
0.333
0.535
0.255
0.362
0.388
0.156
0.348
0.600

0.000
0.116
0.632
0.362
0.184
0.400
0.265
0.150
0.149
0.429
0.200
0.304
0.375
0.182
0.333
0.538

0.000
0.600
0.318
0.217
0.319
0.304
0.135
0.182
0.385
0.234
0.349
0.378
0.171
0.238
0.444

0.000
0.590
0.512
0.524
0.561
0.688
0.641
0.588
0.619
0.526
0.600
0.556
0.730
0.613

0.000
0.360
0.255
0.480
0.415
0.375
0.488
0.451
0.489
0.388
0.378
0.435
0.400

0.000
0.283
0.269
0.265
0.200
0.422
0.245
0.224
0.373
0.277
0.417
0.571

0.000
0.358
0.409
0.373
0.478
0.407
0.480
0.385
0.375
0.469
0.442

0.000
0.256
0.280
0.511
0.245
0.388
0.412
0.277
0.417
0.619

0.000
0.220
0.444
0.318
0.400
0.476
0.263
0.385
0.636

0.000
0.349
0.216
0.319
0.224
0.289
0.348
0.550

0.000
0.391
0.286
0.409
0.450
0.512
0.543

0.000
0.320
0.308
0.208
0.265
0.488

0.000
0.500
0.318
0.511
0.538

0.000
0.391 0.000
0.362 0.256 0.000
0.561 0.514 0.474 0.000

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

Table 3
Chemical distance of the 20 tansy genotypes based on the absence or presence of volatile compound using the method of Nei and Li (1979)

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

277

group containing mixed chemotypes (Tv 17, 19) had the shortest shoots (72.7 cm).
The group having the shortest shoots (Tv 17, 19) and tallests shoots (Tv 9, 11, 13, 15)
had the same number of nodes (20.9) in the stems, the number which was the highest
and differed significantly (P50:0001) from the number of nodes in the other groups.
The number of flower heads in the groups, which contained davadone D/myrcene–
tricyclene/artemisia ketone (Tv 7, 8, 10, 20; 50.6), artemisia ketone (Tv 14, 16; 56.5)
or the highest concentration of camphor (Tv 9, 11, 13, 15: 50.9) was the highest, and
the results differed significantly (P50:005) from the other groups. The mixed group
containing pinocamphone-camphor-1,8-cineole-bornyl acetate (Tv 17, 19) had the
lowest number of flower heads (20.9) per stem and differed significantly (P50:001)
from the other groups. Flowering was latest in the groups containing the lowest
concentration camphor (Tv 7, 8, 10, 20 and Tv 14, 16) or a high concentration of
camphor (Tv 2, 5, 6, 12), and the date at the beginning of the flowering differed
significantly (P50:05) from the other three groups (Table 4).
Tansy chemotypes could be joined to two clusters based on camphor concentration. The cluster consisting of chemotypes which camphor concentration was
between 0.1 and 7.2% (Tv 1, 7, 8, 10, 14, 16, 20) had less nodes per stem (average
16.6; P50:001), more flower heads (average 50.4; P50:001) and a taller corymb
(average 13.9 cm; P50:01), than the other cluster consisting of chemotypes which
camphor concentration was more than 18.5% (Tv 2, 3, 4, 5, 6, 9, 11, 12, 13, 15, 17,
18, 19). The number of nodes, number of flower heads and the height of the corymb
of the latter cluster were on average 18.8, 41.2 and 11.1 cm, respectively (data not
shown).
4. Discussion
In total, 55 volatile compounds were detected from air-dried tansy flower heads of
which 53 were identified. The most frequently found compounds exceeding 10% at
least in one chemotype were tricyclene, camphene, myrcene, 1,8-cineole, artemisia
ketone, trans thujone, camphor, umbellulone, artemisyl acetate, pinocamphone,
myrtenol, chrysanthenyl acetate, bornyl acetate, and davadone D, which have
already reported in tansy (Collin et al., 1993; Neszmélyi et al., 1992; Hendriks et al.,
1990; De Pooter et al., 1989; Gallino, 1988; Holopainen, 1989; Héthelyi et al., 1981;
Ekundayo, 1979; Nano et al., 1979; Tétényi et al., 1975; Forsén, 1975, 1974; Forsén
and von Schantz, 1971; Sorsa et al., 1968; von Rudolf and Underhill, 1965), or from
related species such as artemisyl acetate from Artemisia spp. (Worku and Rubiolo,
1996; Epstein and Gaudioso, 1984). An artemisia ketone isomer observed previously
in tansy oil (Hendriks et al., 1990) shows a retention index linear with our
observation of artemisyl acetate. Most minor compounds reported here have been
reported in tansy, but seven minor compounds are reported here for the first time.
All seven have been detected from related species in Asteraceae family. These include
artemiseole from Artemisia arbuscula (Epstein and Gaudioso, 1984), isobornyl
acetate from Artemisia vulgaris (Hwang et al., 1985), artedouglasia oxide
from Artemisia laciniata (Weyerstahl et al., 1997), nerolidol from Tanacetum
cinerariifolium, Artemisia lacinata, and Tanacetum polycephalum (Saggar et al., 1997;

278

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

Fig. 2. (a) The Geographical origins of the 20 tansy genotypes used in this study. The groups 1 and 2 are
defined on the basis of a complete linkage cluster analysis of RAPD-PCR data (Keskitalo et al., 1998). (b)
The groups of 1–6 are defined on the basis of a complete linkage cluster analysis of the presence or absence
of volatile compounds analyzed in this study.

Weyerstahl et al., 1997; Rustaiyan et al., 1990, respectively), germacrene alcohol
from Echinacea purpurea (Bauer et al., 1988), spathulenol from Achillea millefolium
and Achilla laciniata (Afsharypuor et al., 1996a; Weyerstahl et al., 1997), and
caryophyllene oxide from Tanacetum annuum, Achillea wilhelmsii and Achillea
laciniata (Barrero et al., 1992; Afsharypuor et al., 1996b; Weyerstahl et al., 1997,
respectively). Interestingly, davadone D which has previously been found only in

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

279

Fig. 2 (continued.)

Hungarian-grown (Németh et al., 1994; Héthelyi et al., 1991, 1981), was detected
here for the first time in tansy grown in Finland.
Among the 20 tansy genotypes, 15 had a major compound, the concentration of
which exceeding 40%, while the oil of five genotypes was composed of several minor
compounds. Based upon the definition of ‘chemotypes’ by previous authors
(Hendriks et al., 1990; Holopainen, 1989), 15 tansy genotypes are ‘well-defined’
chemotypes, and the remaining five are ‘mixed’ chemotypes. Holopainen (1989)
observed that about 20% of tansy chemotypes resulting from their crossing experiments were also mixed chemotypes. In our study, camphor was the most common

280

Main compound(s) of
chemotypes belonging
to the groupa
1. Davadone D;
Myrcene/tricyclene/artemisyl acetate;
Artemisia ketone;

Tvb

7;
8,10;
20;

2. Camphor;
4,18;
Thujone;
1;
Chrysanthenyl acetate/camphor/1,8-cineole; 3;

Camphorc

Height of
the shoot

No of
nodes per

No of flower
per heads

Height of
the corymb

Flowering
started

%

S.D.

cm

S.D.

shoot

S.D.

shoot

S.D.

cm

S.D.

Date

65.5;
2.49
26.8–17.0/39.4–20.9/4.4–15.3;
67.3;

3.17

91.8

8.8

17.2

1.9

50.6

6.2

12.8

4.0

29th July 3.9

69.4–72.2;
81.9;
24.9/19.4/11.4;

41.2

34.8

93.7

9.2

16.9

1.1

40.0

15.8

11.3

4.3

22nd July 2.9

Concentration or
range (%) of
the compound

S.D.

3. Camphor;
1,8-Cineole/camphor;

2, 6,12; 50.1–73.0;
5;
47.1/29.4;

55.8

20.4

84.4

18.5

17.3

3.2

40.2

8.6

10.0

1.8

30th July 4.2

4. Camphor;
Camphor/1,8-cineole;

9,11,15; 50.8–68.9;
13;
48.2/20.2;

58.2

10.3

108.2

12.0

20.9

2.8

50.9

5.7

12.5

2.6

24th July 4.4

5. Pinocamphone/camphor/1,8-cineole;
Camphor/bornyl acetate;

17;
19;

19.1/18.5/13.7;
35.7/16.7;

27.1

12.1

72.7

17.3

20.9

8.2

24.0

9.0

8.7

5.3

26th July 4.9

6. Artemisia ketone;

14,16;

55.1–81.4;

3.2

3.9

89.6

17.2

15.0

0.6

56.5

33.0

18.8

8.4

30th July 0.5

a

Main compound(s) consisting at least 10% of the total peak area of each chemotype.

b

Tansy chemotype(s) consisting the compound.

c

Concentration of camphor in the group.

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

Table 4
Morphological attributes of tansy genotypes clustered into six groups according to the complete linkage cluster analysis based on the presence or absence of the identified compound

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

281

chemotype, and this is in agreement with others (Hendriks et al., 1990; Holopainen,
1989; Ekundayo, 1979; Sorsa et al., 1968). In mixed camphor chemotypes, camphene, 1,8-cineole, pinocamphene, chrysanthenyl acetate, bornyl acetate, and isobornyl acetate were the most frequently found associated compounds (10–30%). We
also identified three well-defined artemisia ketone chemotypes. Such chemotypes
have been observed previously by Forsén and von Schantz (1971) and Sorsa et al.
(1968) from Finnish tansy, though are common from the Netherlands and Hungary
(Hendriks et al., 1990; Tétényi et al., 1975). A davadone D chemotype, which is
reported now for the first time from tansy grown in Finland, was detected only from
a Hungarian-grown tansy (Németh et al., 1994; Héthelyi et al., 1991, 1981). In
contrast to the previous authors (Holopainen, 1989; Forsén, 1975; Sorsa et al.,
1968) who studied essential oil of Finnish tansy, thujone was not among the most
common monoterpenes in our study. Thujone was found only in small concentrations except for one chemotype where it was the main component. Our observation
was more in accordance with Hendriks et al. (1990), who did not detect high
concentration of thujone together with camphor. The mixed chemotypes containing
tricyclene (16–27%) and myrcene (21–39%) have not been reported previously from
tansy in Finland, although Sorsa et al. (1968) found chemotypes containing either apinene or tricyclene (4–14%) accompanied with a low concentration (0.1–0.3%) of
myrcene.
Geographically, most of the chemotypes containing a significant concentration of
camphor originated from Central Finland, whereas chemotypes containing camphor
as a minor compound originated from South or Southwest Finland. This
observation is in agreement with Sorsa et al. (1968) who found that camphor was
more frequently observed in tansy grown in Northern Finland compared to southern
grown tansy, where thujone was more frequent. Artemisia ketone and davadone
D were detected only from tansy originating from Southwest Finland. Similar
geographical variation in terpene composition and chemotypes of tansy have
been observed within and between other countries (Neszmélyi et al., 1992;
Hendriks et al., 1990; De Pooter et al., 1989; Gallino, 1988; Héthelyi et al., 1981
Ekundayo, 1979; Nano et al., 1979; Tétényi et al., 1975; von Rudolf and Underhill,
1965).
In Finland, the genetic variation between the genotypes originating from different
geographical regions may be the result of naturalization through inhabitation and
agriculture, with the adaptation of tansy to the local climate (Keskitalo et al., 1998).
Tansy has been observed to be one of the most common seed species in the ballast
soil area in Reposaari (Jutila, 1996), which has been an important harbor in
Southwest Finland. Many plant species have spread to Finland by seed embedded in
the ballast soils used in ships (Jutila, 1996). Interestingly, tansy originating from
Southwest Finland contained only artemisia ketone or davadone D as the main components, common compounds observed in tansy from the Netherlands (Hendriks
et al., 1990) and Hungary (Héthelyi et al., 1981). Correlation between the genetic
distance matrices of our previous study (Keskitalo et al., 1998) and the chemical
distance matrices of the present study was 0.407 showing some analogy between the
variation of the two matrices. Unfortunately, only in a few cases has terpene

282

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

variation been studied simultaneously with genetic variation. In agreement with our
study, Juniperus species have been successfully differentiated from each other using
volatile terpenoids analysis and RAPD patterns (Adams et al., 1993).
The group containing camphor and 1,8-cineole, had the tallest shoots while the
mixed chemotypes were the shortest. Németh et al. (1994) observed that chemotypes containing a thujen-acetate had the tallest shoots whereas plants with
1,8-cineole were the shortest. We also observed that the group containing davadone
D were among the groups having a highest number of flower heads per
shoots, which is in agreement with the observation of Németh et al. (1994). We
observed, that groups formed from pure chemotypes produced the highest number
of flower heads whereas groups formed from mixed chemotypes had a low number
of flower heads. Overall, in our study, the chemotypes containing a high
concentration of camphor had less flower heads and initiated flowering earlier than
chemotypes low in camphor. This is in agreement with observations by Németh et al.
(1994).
The underlying causes of the observed chemical variation in tansy is an intriguing
question, and the answer still remains elusive. Genetic variation may be due to
the different geographical origins of tansy (Keskitalo et al., 1998), which
eventually led to differences in the genetic control of essential oil accumulation
(Holopainen, 1989; Lokki et al., 1973). A wide variation in essential oil composition
presumably has ecological advantages in protecting plants against different pests
(Hough-Golstein and Hahn, 1992; Neszmélyi et al., 1992; Héthelyi et al., 1991;
Nottingham et al., 1991). It is also likely, that part of the terpene expression in tansy is
linked to specific environmental or climatic conditions (Sorsa et al., 1968), and to a
lesser extent may be an indicator of other characters such as morphology (Németh
et al., 1994).
The dependency between the geographical origin, genetic, chemical and
morphological variation of tansy show that different factors need to be recognized
when the biodiversity of herbaceous species is to be examined. The analogy between
the relative chemical and genetic differences among the 20 tansy chemo- and
genotypes, respectively, suggest that different terpene compositions resulting from
the differential activation of specific enzymes may be related to the variation in
RAPDs patterns. The association between the main chemical components and
morphology should be considered when selecting the parental chemo- and
phenotypes from tansy populations for future work. The use of morphological
traits as indicators of selected chemotypes would be most useful in breeding and
biochemical studies. Since the bioactivity of the essential oil of tansy depends on the
composition of terpenes (Héthelyi et al., 1991; Holopainen and Kauppinen, 1989;
Panasiuk, 1984; Schearer, 1984), the chemotype with the most effective oil
composition should be selected. According to previous studies of bioactivity of
tansy oil, artemisia ketone (Héthelyi et al., 1981), camphor (Holopainen and
Kauppinen, 1989; Schearer, 1984), chrysanthenyl acetate (Neszmélyi et al., 1992),
1,8-cineole (Schearer, 1984), davadone (Héthelyi et al., 1981), and thujone
(Holopainen and Kauppinen, 1989) chemotypes are among the most interesting
ones for further studies..

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

283

Acknowledgements
The authors thank Professor M. Levy, Department of Biological Sciences, Purdue
University for providing the SAS-supported complete linkage cluster program.
Financial support from the Academy of Finland (grant 7798), Finnish Association
of Academic Agronomists (Agronomiliitto), and the Rotary Foundation of
Southwest Finland (district 1410) is gratefully acknowledged.
References
Adams, R., 1995. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Publishing Corporation, IL USA, 469 p.
Adams, R.P., Demeke, T., Abulfatih, H.A., 1993. RAPD DNA fingerprints and terpenoids: clues to past
migrations of Juniperus in Arabia and East Africa. Theor. Appl. Genet. 87, 22–26.
Afsharypuor, S., Asgary, S., Lockwood, G.B., 1996a. Volatile constituents of Achillea millefolium L. ssp
millefoilum from Iran. Flavour Fragrance J. 11, 265–267.
Afsharypuor, S., Asgary, S., Lockwood, G.B., 1996b. Constituents of the essential oil of Achillea
wilhelmsii from Iran. Planta Med 62, 77–78.
Banthorpe, D.V., Mann, J., 1971. Monoterpene glucosides from petals of Tanacetum vulgare.
Phytochemistry 11, 2589–2591.
Barrero, A.F., Sánchez, J.F., Altarejos, J., Zafra, M.J., 1992. Homoditerpenes from the essential oil of
Tanacetum annuum. Phytochemistry 31, 1727–1730.
Bauer, R., Remiger, P., Wray, V., Wagner, H., 1988. A germacrene alcohol from fresh aerial parts of
Echinacea purpurea. Planta Med. 54, 478–479.
Blumenthal, M. (Translator), 1998. The Complete German Comission E Monographs: Therapeutic Guide
to Herbal Medicines. Tansy Flower and Herb. Unapproved Herbs. American Botanical Council,
Austin, TX, Integrative Medicine Communications, Boston, MA, pp. 379–380.
Brown, A.M.G., Edwards, C.M., Davey, M.R., Power, J.B., Lowe, K.C., 1997. Effects of extracts of
Tanacetum species on human polymorphonuclear leucocyte activity in vitro. Phytothe. Res. 11,
479–484.
Chandra, A., Misra, L.N., Thakur, R.S., 1987a. Germacranolides and an alkyl glucoside from Tanacetum
vulgare. Phytochemistry 26, 1463–1465.
Chandra, A., Misra, L.N., Thakur, R.S., 1987b. Tanavulgarol, an oxygenated sesquiterpene with an
uncommon skeleton from Tanacetum vulgare. Phytochemistry 26, 3077–3078.
Collin, G.J., Deslauriers, H., Pageau, N., Gagnon, M., 1993. Essential oil of tansy (Tanacetum vulgare L.)
of Canadian origin. J. Essent. Oil Res. 5, 629–638.
Ekundayo, O., 1979. Essential oils II. Terpene composition of the leaf oil of Tanacetum vulfgare L. Z.
Pflanzenphysiol. Bd. 92, 215–220.
Epstein, W.W., Gaudioso, L.A., 1984. Volatile oil constituents of sagebrush. Phytochemistry 23,
2257–2262.
Finnigan, Heller, Milne, 1978. 4000 GC/MS; NBS Library 31, 331 entries
Forsén, K., 1974. Begleitstoffe in verschiedenen Chemotypen von Chrysanthemum vulgare Reine transChrysanthenylacetate-type. Farm. Aikak 83, 9–17.
Forsén, K., 1975. Über die Infraspezifische chemische variation bei Chrysanthemum vulgare. Ann. Acad.
Sci. Fenn. Ser. A IV 207, 1–54.
Forsén, K., von Schantz, M., 1971. Neue Hauptbestandteile im ätherischen öl des Rainfarns in Finnland.
Arch. Pharm. 304, 944–952.
Gallino, M., 1988. Essential oil from Tanacetum vulgare growing spontaneously in ‘Tierra del Fuego’
(Argentina). Planta Med. 54, 182.
Grieve, M., 1984. Tansy. In: Leyel, C.F. (Ed.), A Modern Herbal. Penguin Books Ltd, Middlesex, Great
Britain, pp. 789–790.

284

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

Hendriks, H., Bos, R., 1990. The presence of parthenolide in Dutch tansy. Planta Med. 56, 550.
Hendriks, H., van der Elst, D.J.D., van Putten, F.M.S., Bos, R., 1990. The essential oil of Dutch Tansy
(Tanacetum vulgare L.). J. Essent. Oil Res. 2, 155–162.
Heywood, V.H., 1976. Tanacetum L. In: Tutin, T.G., Heywood, V.H., Burges, N.A., Moore, D.M.,
Valentine, D.H., Walters, S.M., Webb, D.A. (Eds.), Flora Europaea, Plantaginaceae to Compositae
(and Rubiaceae), Vol. 4. Cambridge University Press, Cambridge, Great Britain, pp. 169–171.
Héthelyi, É., Tétényi, P., Danos, B., Koczka, I., 1991. Phytochemical and antimicrobial studies on the
essential oils of the Tanacetum vulgare clones by gas chromatography/mass spectrometry. Herba Hung
30, 82–90.
Héthelyi, É., Tétényi, P., Kettens-van den Bosch, J.J., Salemink, C.A., Heerma, W., Versluis, C.,
Kloosterman, J., Sipma, G., 1981. Essential oils of five Tanacetum vulgare genotypes. Phytochemistry
20, 1847–1850.
Holopainen, M. (1989). A study on the essential oil of tansy (Tanacetum vulgare L.). Ph.D. Thesis,
Department of Pharmacy, University of Helsinki, Finland.
Holopainen, M., Kauppinen, V., 1989. Antimicrobial activity of different chemotypes of Finnish tansy.
Planta Med 55, 103.
Hough-Golstein, J., Hahn, S.P., 1992. Antifeedant and oviposition deterrent activity of an aqueous extract
of Tanacetum vulgare L. on two cabbage pest. Environ. Entomol. 21, 837–844.
Hultén, E., 1968. Flora of Alaska and Neighboring Territories. A Manual of the Vascular Plants. Stanford
University Press, Stanford CA, 1008 p.
Hussey, J., 1974. Some useful plants of early New England. Econ. Bot. 28, 311–337.
Hwang, Y.S., Wu, K.H., Kumamoto, J., Axelrod, H., Mulla, M.S., 1985. Isolation and identification of
mosquito repellents in Artemisia vulgaris. J. Chem. Ecol. 11, 1297–1306.
Jutila, H.M., 1996. Seed bank and emergent vascular flora of ballast areas in Reposaari, Finland. Ann.
Bot. Fennici. 33, 165–182.
Keskitalo, M., Angers, P., Earle, E., Pehu, E., 1999. Chemical and genetical characterization of protoplast
fusion-derived calli between tansy (Tanacetum vulgare L.) and pyrethrum (Tanacetum cinerarieafolium).
Theor. Appl. Genet. 98, 1335–1343.
Keskitalo, M., Kanerva, T., Pehu, E., 1995. Development of in vitro procedures for regeneration of petiole
and leaf explants and production of protoplast-derived callus in Tanacetum vulgare L. (Tansy). Plant
Cell Rep. 14, 261–266.
Keskitalo, M., Lindén, A., Valkonen, J.P.T., 1998. Genetic and morphological diversity of Finnish tansy
(Tanacetum vulgare L.). Theor. Appl. Genet. 96, 1141–1150.
Lawrence, B.M., 1992. Essential oils as source of natural aroma chemicals. Perfumer Flavorist 17, 15–28.
Levy, M., Romao, J., Marcetti, M.A., Hamer, J.E., 1991. DNA Fingerprinting with a dispersed repeated
sequence resolves pathotype diversity in rice blast fungus. Plant Cell 3, 95–102.
Lokki, J., Sorsa, M., Forsén, K., von Schantz, M., 1973. Genetics of monoterpenes in Chrysanthemum
vulgare. Hereditas 74, 225–232.
Millspaugh, C.F., 1974. American Medicinal Plants. An Illustrated and Descriptive Guide to Plants
Indigenous to and Naturalized in the United States which are used in Medicine. Dover Publications,
New York, 806p.
Mitich, L.W., 1992. Intriguing world of weeds } Tansy. Weed Technol. 6, 242–244.
Mordujovich-Buschiazzo, P., Balsa, E.M., Busciazzo, H.O., Mandrile, E., Rosella, M., Schinella,
G., Fioravanti, D., 1996. Anti-inflammatory activity of Tanacetum vulgare. Fitoterapia LXVII,
319–322.
Nano, G.M., Bicchi, C., Frattini, C., Gallino, M., 1979. Wild Piedmontese Plants II. A rare chemotype of
Tanacetum vulgare L., abundant in Piedmont (Italy). Planta Med. 35, 270–274.
National Landsurvey Institute, 1996. Geographic Data Center.
Németh, É.Z., Héthelyi, É., Bernàth, J., 1994. Comparison studies on Tanacetum vulgare L. chemotypes. J.
Herb Spices Med. Plants 2, 85–92.
Neszmélyi, A., Milne, G.W.A., Héthelyi, É., 1992. Composition of the essential oil of clone 409 of
Tanacetum vulgare and 2D NMR investigation of trans-chrysanthenyl acetate. J. Essent. Oil Res. 4,
243–250.

M. Keskitalo et al. / Biochemical Systematics and Ecology 29 (2001) 267–285

285

Nei, M., Li, W.H., 1979. Mathematical model for study genetic variation in terms of restriction
endonucleases. Proc. Natl. Acad. Sci. USA 74, 5267–5273.
Nottingham, S.F., Hardie, J., Dawson, G.W., Hick, A.J., Picett, J.A., Wadhams, L.J., Woodcock, C.M.,
1991. Behavioral and electrophysiological responses of aphids to host and nonhost plant volatiles. J.
Chem. Ecol. 17, 1231–1242.
Ognyanov, I., Todorova, M., 1983. Sesquiterpene lactones and flavonoids in flowers of Tanacetum vulgare.
Planta Med. 48, 181–183.
Ognyanov, I., Todorova, M., Dimitrov, V., Ladd, J., Irngartinger, H., Kurda, E., Rodewald, H., 1983.
Cis-Longipinane-2,7-dione, a sesquiterpene diketone in flowers of Tanacetum vulgare. Phytochemistry
22, 1775–1777.
Panasiuk, O., 1984. Response of Colorado potato beetles, Leptinotarsa decemlineata (Say), to volatile
components of tansy, Tanacetum vulgare. J. Chem. Ecol. 10, 1325–1333.
De Pooter, H.L., Vermeesch, J., Schamp, N.M., 1989. The essential oils of Tanacetum vulgare L. and
Tanacetum parthenium (L.) Schultz-Bip. J. Essent. Oil Res. 1, 9–13.
von Rudloff, E., Underhill, E.W., 1965. Gas-liquid chromatography of terpenes}XII. Seasonal variation
in the volatile oil from Tanacetum vulgare L. Phytochemistry 4, 11–17.
Rustaiyan, A., Zare, K., Habibi, Z., Hashemi, M., 1990. Germacranolides from Tanacetum polycephalum.
Phytochemistry 29, 3022–3023.
Saggar, P., Wamicha, W.N., Chhabra, S.C., Ndalut, P., 1997. Isolation, identification and bioassay of
repellent factors in the essential oil of pyrethrum for grain protection against Sitophilus zeamais
(Molts.). Pyrethrum Post 19 (4), 126–130.
Sanz, J.F., Marco, J.A., 1991. NMR studies of tatridin A and some related sesquiterpene lactones from
Tanacetum vulgare.