Acta agriculturae Slovenica, 87 - 2, september 2006 str. 415 - 425 
Agrovoc descriptors: cichorium intybus, organogenesis, callus, flowering, 
regeneration, in sacco experimentation, seedlings 
 
Agris category code: F60, F62 
 
COBISS Code 1.01 
 
Adenine sulphate induced high frequency shoot 
organogenesis in callus and in vitro flowering of Cichorium 
intybus L. cv. Focus - a potent medicinal plant 
 
S. NANDAGOPAL
1
, B.D. RANJITHA KUMARI
2
 
 
Received: May 28, 2006; accepted: August 18, 2006. 
Prispelo: 28. maja 2006; sprejeto: 18. avgusta 2006. 
 
ABSTRACT 
 
An efficient protocol has been developed for the in vitro plant regeneration and flowering 
achieved from young leaf explants of chicory (Cichorium intybus L. cv. Focus). The callus 
induction and shoot multiplication was obtained on Murashige and Skoog (MS) and 
 
Gamborg
 s (B
5
) media supplemented with different concentrations and combinations of 
Indole-3-acetic acid, 6-Benzylaminopurine, 6-Furfurylaminopurine and Adenine sulphate. The 
highest percentage of callus induction and multiple shoots proliferation was observed on 
MS+B
5
 medium containing 6-benzylaminopurine (6.66 µM) + indole-3-acetic acid (2.852 µM) 
+ adenine sulphate (1.360 µM). For root induction regenerated shoots were transferred to 
MS+B
5
 medium containing Indole-3-acetic acid, Indole-3-butyric acid and α-Naphthalene 
acetic acid that were developed roots. In vitro flowers were also noticed in the in vitro raised 
plantlets in the same medium under 16 h light and 8 h dark condition. The regenerated 
plantlets have been successfully established in vermiculite and subsequently shifted to the 
field. 
 
Key words: leaf callus; growth regulators; plant regeneration; in vitro flowering; hardening. 
 
IZVLEČEK 
 
USPEŠNO IN VITRO INDUCIRANJE ORGANOGENEZE POGANJKOV RADIČA 
Cichorium intybus L. cv. Focus Z ADENIN SULFATOM 
 
Razvita je bila metoda uspešne in vitro regeneracije radiča (Cichorium intybus L. cv. Focus) iz 
listnih delov; doseženo je bilo cvetenje regeneriranih rastlin. Induciranje kalusa in 
razmnoževanje poganjkov sta bili doseženi z Murashige in Skoog (MS) ter 
 
Gamborgovim 
(B
5
) gojiščem, dopolnjenima z različnimi koncentracijami indol-3-ocetne kisline, 6-
benzilaminopurina, 6-furfurilaminopurina in adenin sulfata. Največji odstotek induciranja 
kalusa ter največje število poganjkov sta bila dosežena na gojišču MS+B
5
, ki je vsebovalo 6-
benzilaminopurin (6.66 µM) + indol-3-ocetno kislino (2.852 µM) + adenin sulfat (1.360 µM). Za 
                                                 
1
  Ph. D., student, Department of Plant Science, Bharathidasan University, Tiruchirappalli - 
620024, Tamil Nadu, India. 
2
  Reader, Department of Plant Science, Bharathidasan University, Tiruchirappalli-620024, 
Tamil Nadu, India. Corresponding author: e-mail: ranjithakumari2004@yahoo.co.in phone: 
00-91-431-2407061, fax: 00-91-431-2407045. 

Acta agriculturae Slovenica, 87 - 2, september 2006 
 
416 
indukcijo korenin na poganjkih so ti bili prenešeni na gojišče MS+B
5
, ki je vsebovalo indol-3-
ocetno kislino, indol-3-masleno kislino in α-naftalen ocetno kislino, kar je omogočilo uspešno 
zasnovo korenin. Opazili smo tudi in vitro cvetove pri in vitro vzgojenih rastlinicah v istem 
gojišču, pri razmerah 16 h osvetlitve in 8 h teme. Rast regeneriranih rastlinic se je uspešno 
nadaljevala v vermikulitu in kasneje po presaditvi tudi na prostem. 
 
Ključne besede: listni kalus; rastni regulatorji; regeneracija rastlin; in vitro cvetenje; utrditev. 
 
 
 
1 INTRODUCTION 
 
Chicory (Cichorium intybus L.) is a medicinally important plant that belongs to the 
family Asteraceae. Root, leaf and seed contains a number of medicinally important 
compounds such as inulin, sesquiterpene lactones, coumarins, flavonoids and 
vitamins. It is used as antihepatotoxic, antiulcerogenic, antiinflammatory, appetizer, 
digestive, stomachic, depurative, diuretic (Varotto et al., 1997). Recent 
pharmacological investigation of the root extract of this plant revealed 
immunomodulator and anticancer properties (Angelina et al., 1999). 
 
Previous study reported by Varotto et al. (2000) on plant regeneration from leaf-
protoplasts derived callus has been obtained in Italian red chicory (C. intybus) on 
medium containing 0.1 mg l
-1 
NAA. Rehman et al. (2003) developed a protocol for 
the regeneration of plantlets from leaf explants of witloof chicory. The obtained 
regeneration via callus on modified MS semisolid medium containing 2 µM IAA in 
combination with 5 µM KIN and 1000 mg l
-1 
casein hydrolyzate. 
 
Flowering is considered as a complex process regulated by external and internal 
factors. In many plants, in vitro flowering were normally achieved by the application 
of exogenous hormones to the culture medium. The ability of explants to form flowers 
in vitro depends on numerous internal, chemical and physical factors and virtually all 
these interact in various complex and unpredicted ways (Tran than van, 1973; Scorza 
and Janick, 1980; Croes et al., 1985; Compton and Vielleux, 1992). Combination of 
genetic and environmental factors also plays a role in flowering response in vitro 
(Tisserat and Galletta, 1993). Flowering in vitro can occur on explants from flowering 
plants of some woody species (Scorza, 1982).  
 
Cichorium intybus is a biennial plant whose requirements for floral initiation are not 
fully understood. Hartman (1956) and Harrington et al. (1959) showed that chicory 
plants require both vernalization and long days to flower. In order to induce florally, 
chicory must undergo an exposure to low temperature (3 weeks at 4
o
C) that can be 
applied to the germinating seeds or to the entire plant (Rappaport and Wittwer, 1956). 
Studies of in vitro cultured explants from chicory roots initially led to the conclusion 
that the explants also must be vernalized before they will initiated inflorescences 
(Nitsch and Nitsch, 1964; Paulet and Nitsch, 1964). In contrast with these findings, it 
was found that the initial root explants do not always need to be vernalized (Margara, 
1965; Margara and Rancillac, 1966). Mikou and Badila (1992) produced 
inflorescences under short-day conditions of 9 hr if long days of 16 hr were applied 
initially. This indicates that the duration of light commits the meristem to the 
subsequent formation of flower primordia. Under natural conditions, chicory plants 

NANDAGOPAL, S., RANJITHA KUMARI, B.D.: Adenine sulphate induced high … 
 
417
are vernalized during the winter, and subsequently bolt and flowers in the second 
growing season from May until August (Demeulemeester and De Proft, 1999).  
 
The purpose of the study was to develop an efficient in vitro regeneration system from 
leaf derived callus of Cichorium intybus L. cv. Focus, an economically important 
medicinal plant and to induce flowering from the in vitro regenerated plants. In the 
present work, we have established a reproducible method for high frequency callus 
induction, regeneration and flowering from leaf segments, followed by successful 
establishment of regenerated plants in soil.  
 
 
2 MATERIALS AND METHODS 
 
2.1 Plant material  
 
The seeds of Chicory (Cichorium intybus L. cv. Focus) obtained from Wageningen 
Agricultural University, Netherlands were used as initial explants for plant source. 
 
2.2 Seed sterilization and germination 
 
Seeds were surface cleaned by washing in running tap water for 30 min. Then they were 
washed in an agitated solution of liquid detergent (1% v/v Teepol) for 5 min and distilled water 
for 2-3 times. After thorough washing, the seeds were taken into the Laminar Flow Chamber 
where they were disinfected with 70% (v/v) ethanol for 60-70 s followed by treatment with 
0.1% (w/v) aqueous mercuric chloride (HgCl
2
) solution for 3-5 min and then rinsed 3-5 times 
in sterile distilled water. Finally, the seeds were germinated under sterile conditions on moist 
cotton in test tubes (150x25 mm). The tubes were incubated in dark initially for 48 h and after 
transferred to light conditions of 3000 lux (Philips, India) light intensity provided by cool white 
fluorescent tubes. 
 
2.3 Basal medium and incubation condition 
 
The basal medium used in this investigation consisted of MS (Murashige and Skoog, 1962) 
and B
5 
medium
 
(Gamborg et al., 1968) supplemented with 30 g l
-1
and 20 g l
-1
 sucrose 
(Himedia, India), respectively. 0.7% and 0.68% agar (Himedia, India) was used as the gelling 
agent on respective media. The pH of all media were adjusted between 5.7 - 5.8 using 0.1N 
NaOH or 0.1N HCl before solidification. The media were autoclaved at 121°C for 15 minutes 
for sterilization. All cultures were incubated in a controlled temperature of 25 ± 2°C. IAA, NAA, 
IBA, BAP, KIN and ADS were the different hormone sources taken for the present study. 
 
2.4 In vitro culture conditions and shoots regeneration 
 
The young leaf from 7 days old seedlings were inoculated on MS and B
5 
media. Different 
concentration and combinations of hormones including 5.71-17.13 µM IAA, 0.492-14.76 µM 
IBA, 0.537-16.11 µM NAA, 0.444-13.32 µM BAP, 0.464-13.92 µM KIN and 0.272- 8.15 µM 
ADS were tested. The pH of all media was adjusted between 5.7-5.8 before it was autoclaved 
for 15 min at 121°C. The cultures were incubated at 25 ±2°C under 16/8 h (light/dark) 
photoperiod provided by cool white fluorescent tubes with 3000 lux intensity and relative 
humidity of 60–70%. The effects of different combinations of growth regulators on callus 
formation and regeneration were recorded at regular intervals (Table 1). Shoot cuttings 
excised from the regenerants were transferred to MS+B
5
 medium supplemented with 0.571-
14.27 µM IAA, 0.537-16.11 µM IBA and 0.492-14.76 µM NAA to induce rooting. Rooted 
plantlets were transferred to sterile vermiculite supplemented with half-strength MS salts and 
incubated under saturated humidity conditions. After 2 wk of hardening, plants were 
transferred to pots and grown at 25 ± 2°C initially and subsequently established in the field 
condition. 

Acta agriculturae Slovenica, 87 - 2, september 2006 
 
418 
Table 1: Effect of different concentrations of growth regulators on callusing, regeneration, 
number, height of regenerated shoots and flowers of Cichorium intybus L. cv. 
Focus. 
 
 
 
Sl 
No 
 
 
 
BAP(µM) 
 
 
 
IAA(µM) 
 
 
 
ADS(µM) 
 
 
Callus 
percentage 
(mean ± 
SE) 
 
 
Regeneration 
percentage 
(mean ± SE) 
 
 
 
Number of 
shoots 
 (mean ± 
SE) 
 
 
Height of 
shoots 
(mean ± 
SE) 
 
 
Number of 
flower/per 
shoot apex 
(mean ± 
SE) 
 
1 
2 
3 
4 
5 
6 
7 
8 
 
1 
2 
3 
4 
5 
6 
7 
8 
 
2.22 
4.44 
6.66 
8.88 
2.22 
4.44 
6.66 
8.88 
KINµM 
2.32 
4.64 
6.96 
9.28 
2.32 
4.64 
6.96 
9.28 
 
0.571 
1.142 
2.852 
5.710 
0.571 
1.142 
2.852 
5.710 
IAAµM 
0.571 
1.142 
2.852 
5.710 
0.571 
1.142 
2.852 
5.710 
 
1.360 
1.360 
1.360 
1.360 
- 
- 
- 
- 
 
1.360 
1.360 
1.360 
1.360 
- 
- 
- 
- 
 
57.3±1.65
d 
83.0±1.88
b 
94.3±0.98
a 
76.0±0.94
bc 
52.6±1.90
e 
40.3±1.90
f 
37.0±2.05
fg 
32.3±1.18
g 
 
56.0±1.24c 
82.0±1.24
ab 
90.6±0.54
a 
74.6±1.96
b 
52.0±2.05
cd 
39.0±1.24
e 
30.3±0.54
ef 
21.6±0.54
f 
 
54.0±2.49
d 
79.0±b1.69
b
 
93.0±bc1.36
a 
74.0±1.88b
c 
51.0±2.84
de 
38.0±1.63
f 
34.3±1.08
fg 
28.6±0.72
g 
 
37.3±2.17
e 
75.6±2.41
b 
88.0±1.24
a 
69.3±0.27
bc 
48.3±0.36
d 
37.3±2.17
e 
26.0±0.47
f 
20.0±1.24
fg 
 
 
16.3±1.08
d 
23.6±1.96
bc 
33.0±1.69
a 
25.6±0.54
b 
11.3±1.36
e 
09.0±0.47
ef 
07.6±0.54
f 
06.3±0.27
fg 
 
06.6±0.72
e 
18.6±1.51
bc 
29.0±0.47
a 
21.6±1.44
b 
09.0±1.47
d 
6.66±0.72
de 
6.03±0.27
ef 
0.50±0.47
g 
 
3.63±0.10
c 
5.90±0.24
d 
7.53±0.13
a 
6.73±0.11
bc 
9.56±0.05
d 
3.00±0.10
de 
2.83±1.13
e 
2.33±0.13
ef 
 
2.66±0.13
e 
5.23±0.11
b 
6.66±0.13
a 
5.00±0.23
bc 
3.50±0.40
d 
2.66±0.13
e 
2.50±0.23
ef 
2.16±0.36
f 
 
 
- 
1.69±0.11
b 
5.21±0.9
a 
0.75±0.2
c 
- 
- 
0.52±0.52
cd 
- 
 
- 
- 
- 
- 
- 
- 
- 
- 
 
Each value represents the mean ± standard error (S.E.) of ten replicates per treatment in 
three repeated experiments. Means within a row followed by the s a m e l e t t e r s a r e n o t 
significant at P=0.05 according to DMRT. 
 
 
2.5 Statistical analysis 
 
The percentage of response, callus induction frequency, regeneration frequency of callus, 
number of shoots, shoot length, number of roots, root length and number of flowers were 
monitored as growth parameters. Data of three independent experiments represented by 10 
replicates from each experiment were subjected to statistical analysis (Mean   SE), 
according to New Duncan’s Multiple Range Test (Gomez and Gomez, 1976).  
 
 
3 RESULTS 
 
3.1 Callus induction and shoot regeneration 
 
Callus formation from leaf explants could be observed in all hormone combinations 
after 10-15 days. Young leaf from the 7 days old seedlings exhibited higher response 

NANDAGOPAL, S., RANJITHA KUMARI, B.D.: Adenine sulphate induced high … 
 
419
for callus induction when cultured on MS+B5 medium supplemented with different 
combinations and concentrations of BAP, IAA, KIN and with or with out ADS. The 
callus obtained with MS+B5 medium supplemented with 2.22-8.80 µM BAP, 0.571-
5.710 µM IAA, 2.32-9.28 µM KIN and 1.360 µM ADS were greenish compact and 
pale yellow but were friable on MS+B5 medium supplemented with 2.32-9.28 µM 
KIN and 0.571-5.710 µM IAA. The percentage of callusing response was 57.3-94.3% 
on MS+B5 medium fortified with 2.22-8.88 µM BAP+0.571-5.710 µM IAA+1.360 
µM ADS; it was 32.3 - 52.6% on 2.22-8.88 µM BAP+0.571-5.710 µM IAA. Without 
ADS in the medium, the callusing percentage was low. Maximum callusing (94.3%) 
was obtained on MS+B5 medium with 6.66 µM BAP, 2.852 µM IAA and 1.360 µM 
ADS (Table 1; Fig 1A). 
 
Regeneration of shoot buds initiated in 30 days old callus. The buds transformed into 
green, healthy shoots in the presence of growth regulators in the medium on MS+B5 
with 2.22 - 8.88 µM BAP + 0.571-5.710 µM IAA+1.360 µM ADS. The regeneration 
percentage was 54.0-93.0. The maximum regeneration of 93.6% was observed on 
MS+B5 medium with 6.66 µM BAP+2.852 µM IAA+1.360 µM ADS (Table 1). The 
number of shoots per callus and height of regenerants was monitored in all the 
combinations after 45 days. The number of shoots ranged between 16.3 and 33.0 on 
MS+B
5
 media with 2.22-8.88 µM BAP+0.571-5.710 µM IAA+1.360 µM ADS and 
between 06.3-11.3 on MS+B5 media with 2.22-8.88 µM BAP+0.571-5.710 µM IAA. 
The maximum number of shoots 33.0 was observed on MS+B5 media fortified with 
6.66 µM BAP+2.852 µM IAA+1.360 µM ADS. The shoot height ranged between 5.5 
and 5.90 cms on media supplemented with ADS and between 2.66 and 3.50 cms on 
media without ADS. The maximum height of shoots was 9.5 cms on MS+B
5
 media 
with 6.66 µM BAP+2.852 µM IAA+1.360 µM ADS on MS+B
5 
with media 2.32-9.28 
µM KIN+0.571-5.710 µM IAA+1.360 µM ADS the number or shoots ranged between 
16.3 and 33.0 and the shoot height between 5.90 and 9.56 cms (Table 1; Fig 1 B, C) 
MS+B5 basal medium supplemented with 6.66 µM BAP+2.852 µM IAA+6.66 µM 
BAP+1.360 µM induced the best callus and shoot- forming response. These 
observations indicate that these media are at concentrations favorable for promoting 
callus and shoot proliferation in chicory cv. Focus. 
 
3.2 Root induction and in vitro flowering 
 
For rooting of 45 days old in vitro raised callus regenerated shoots, different 
concentrations of (02.85-14.27 µM) IAA, (2.460-1230 µM) IBA and (02.69-13.43 
µM) NAA were tested. All the hormones responded well in inducing the roots (Fig. 
1C, Table 2). Of the three hormones at different concentrations tested for root 
induction, the best response was obtained with 04.92 µM IBA, 05.71 µM IAA and 
05.37 µM NAA. Maximum length of root was obtained with IBA (85.6 mm) followed 
by IAA (77.0 mm) and NAA (61.0 mm) (Table 2). The maximum number of flowers 
(5) was observed on MS+B
5 
medium supplemented with 6.66 µM BAP, 2.852µM 
IAA and 1.360 µM ADS. These results indicate that without ADS in the culture 
medium, the number of flowers (1) produced was low. This result indicates that the 
media supplemented with ADS favours high frequency of in vitro flowering (5) in 
chicory plant (Table 1; Fig 1 E, F).  
 

Acta agriculturae Slovenica, 87 - 2, september 2006 
 
420 
Table 2: Effect of different concentrations of auxins on rooting of microshoots and 
root length in Cichorium intybus L. cv. Focus. 
 
 
S. No 
 
Growth Regulators (µM) 
 
 
 
Percentage of Response 
(Mean ± SE) 
 
Root Length (mm) 
(Mean ± SE) 
 
1 
 
 
 
 
 
2 
 
 
 
 
 
3 
 
IAA   01.14 
IAA   02.85 
IAA   05.71 
IAA   08.56 
IAA   11.42 
 
IBA   00.98 
IBA   02.46 
IBA   04.92 
IBA   07.38 
IBB   09.84 
 
NAA  01.07 
NAA  02.69 
NAA  05.37 
NAA 18.06 
NAA 10.74 
 
40.0 ± 0.94
cd 
52.0 ± 0.47
c 
92.3 ± 0.27
a 
79.9 ± 0.47
ab 
69.6 ± 0.27
a 
 
54.6 ± 1.18
e 
66.3 ± 0.54
d 
100  ± 0.00
a 
92.0 ± 0.47
ab 
79.3 ± 0.72
c 
 
20.6 ± 0.27
de 
52.1 ± 0.47
d 
78.6 ± 1.08
a 
37.3 ± 1.18
c 
21.3 ± 0.72
d 
 
40.3 ± 0.72
c 
41.6 ± 0.27
bc 
77.0 ± 0.47
a 
44.0 ± 0.47
b 
37.3 ± 0.27
cd 
 
54.0 ± 0.47
d 
75.3 ± 0.27
b 
85.6 ± 0.27
a 
69.6 ± 0.27
bc 
50.6 ± 0.27
de 
 
23.3 ± 0.72
d 
56.6 ± 0.27
ab 
61.0 ± 0.00
a 
42.0 ± 0.47
c 
20.5 ± 0.27
de 
Each value represents the mean ± standard error (S.E.) of ten replicates per treatment in three repeated 
experiments. Means within a row followed by the same letters are not significant at P=0.05 according 
to DMRT. 
 
3.4 Establishment of plantlets   
 
The plantlets with or without flower heads were successfully subjected to hardening 
on the foam cups for 15 days and then transferred to pots and grown at 25 ±2°C under 
60- 70% relative humidity. 70-80% of plantlets were survived in the hardening 
procedure. These plants were then transferred to the field and were well established 
(Fig 1 D, G and H). 

NANDAGOPAL, S., RANJITHA KUMARI, B.D.: Adenine sulphate induced high … 
 
421
 

Acta agriculturae Slovenica, 87 - 2, september 2006 
 
422 
4  DISCUSSION 
 
The early studies reported that (Vermeulen et al., 993) micro calluses produced from 
the leaf tissue of Cichorium intybus in liquid medium supplemented with 1.0 mg/l 
NAA 0.5 mg/l 2, 4-D and 1mg/l BAP. Wagner and Gailing (1996) induced callus 
from the leaf vein segments of chicory on MS medium supplemented with 2.0 mg/l 
IAA and 0.26 mg/l BAP. Earlier reports on Cichorium intybus plant regeneration from 
the leaf explants were reported to be better for callus formation and shoot 
development (Eung et al., 1999). The leaf protoplasts derived callus has been 
obtained in Italian red chicory by Varotto et al. (2000). Rehman et al. (2001) cultured 
leaf segments of Cichorium intybus on MS medium supplemented with various 
growth regulators in different concentrations and obtained callus in the combination 
of 1.0 mg/l IAA with 5.0 mg/l KIN. 
 
Similar results were reported in Vitex where they used 1.5 mg/l BAP in combination 
with 0.1 mg/l NAA (Thiruvengadam and Jayabalan, 2001). Various factors such as 
carbohydrates, growth regulators, light and pH of the culture medium are playing a 
major role in flowering (Heylon and Vendrig, 1998). Jumin and Ahmed (1999) 
recommended 0.01 mg/l BAP to induce flowers in Murraya. Stephen and Jayabalan 
(1998) induced maximum number of flowers from Coriandrum on culture with 0.15 
mg/ l NAA and 0.5 mg/l GA
3
. Patil et al. (1993) indicated that the exogenous 
cytokinin stimulates flowering by activation of endogenous cytokinin in ascending 
xylem sap. Earlier studies indicated that during budding and early flowering stages 
the basil oil was rich in monoterpenes (Lemberkovics et al., 1998). In vitro flowering 
in sunflower was also reported with low concentration of sucrose and 1 mg/l each of 
NAA and GA
3, 
and 0.5 mg/l activated charcoal (Patil et al., 1993). In our experiment 
high frequency in vitro flowering was achieved without GA
3 
in the culture medium. 
However, increase in sucrose concentration (30 g/l) in the culture medium did not 
result in flowering in groundnut (Narasimhalu et al., 1984). But, in our investigation 
in vitro flowering was observed with higher sucrose concentration (30 g/l). 
 
In chicory in vitro flowering was also influenced by the addition of 5-azacytidine, a 
DNA demethylation agent (Demeulemeester et al., 1999), putrescine and silver nitrate 
(Bais et al., 2000). In India, chicory cv. Focus flower initiation usually takes place 
during July-September and flowering takes almost after 521-551 days after planting. 
In the present investigation, flower production was observed from the cultured shoots 
after 45 days of culture. More number of flowers was observed in the hardened plants, 
which were growing in the field than that of normal plants. To our knowledge this is 
the first report on in vitro callus induction, plantlet regeneration and flowering of 
chicory cv. Focus.  
 
Chicory flower contains a number of medicinally important compounds such as 
sesquiterpene lactones, coumarins, flavonoids, vitamins and also used as dye. Since 
chicory is a absolute long day plant, during the first year the plant shows only the 
vegetative growth and produces flowers and completes its life cycle in the second 
year (Varotto et al., 2000). In our experiment, in vitro flowering was initiated after 45 
days of culture that reduces the breeding cycle and thus plays a key role in the field of 
agriculture, horticulture and pharmaceuticals. 

NANDAGOPAL, S., RANJITHA KUMARI, B.D.: Adenine sulphate induced high … 
 
423
Acknowledgments 
 
The authors are thankful to Maurice C. R. Franssen, Department of Organic 
Chemistry, Wageningen Agricultural University, Netherlands, Harro J. Bouwmeester, 
Institute of Organic Chemistry, Hamburg University, Germany for providing the seed 
samples of the Cichorium intybus L. cv. Focus. 
 
 
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