Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol83.Issue1.Jan2000:

Scientia Horticulturae 83 (2000) 1±10

Micropropagation of Cynara scolymus L. employing
cyclodextrins to promote rhizogenesis
C. Brutti*, N.M. ApoÂstolo, S.A. Ferrarotti,
B.E. Llorente, N. Krymkiewicz
Laboratorio de Cultivo de Tejidos Vegetales y Laboratorio de QuõÂmica BioloÂgica, Departamento de
Ciencias BaÂsicas, Universidad Nacional de LujaÂn. C.C. 221. LujaÂn 6700, Buenos Aires, Argentina
Accepted 9 April 1999

Abstract
This work describes a micropropagation protocol for plants of Cynara scolymus cv. Early French,
employing cyclodextrins during the rhizogenesis stage. Shoot apices were established in basic
culture medium consisting of Murashige Skoog mineral salts modified by 50% reduction in
NH4NO3 and KNO3, Heller's micronutrients, Gamborg's vitamins, 100 mg lÿ1 myo-inositol,
30 g lÿ1 saccharose and 8.0 g lÿ1 agar, supplemented with 1.0 mg lÿ1 6-(g,g-dimethylallylamino)purine and 0.1 mg lÿ1 naphthalene acetic acid. In the multiplication stage, to achieve a rate of 3 : 1,
basic medium was supplemented with 10 mg lÿ1 6-(g,gÿdimethylallylamino)-purine, 2.0 mg lÿ1
kinetin, 0.5 mg lÿ1 naphthalene acetic acid and 80 mg lÿ1 adenine sulphate. The incorporation of aor b-cyclodextrins to the rhizogenesis medium supplemented with 3 mg lÿ1 naphthalene acetic acid,
allowed tripling the rooting percentage and doubling the number of differentiated roots per explant
compared to medium without cyclodextrins. The exflasking survival rate of the plants was 70%.
# 2000 Elsevier Science B.V. All rights reserved.

Keywords: Artichoke; Bioactive carbohydrates; Plant tissue culture; Rooting

1. Introduction
The Cynara scolymus L. artichoke is a perennial plant with nutritional and
medicinal application (choleretic, diuretic, gastrointestinal stimulant), widely
* Corresponding author. Tel.: +54-2323-420380 int. 273; fax: +54-2323-425795.
E-mail address: apostolo@mail.unlu.edu.ar (C. Brutti)
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 6 7 - 9

2

C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10

cultivated in the Mediterranean region. Its cultivation extends to Northern Africa
and America, with USA (CA) and Argentina as the main American producers
(Ordas et al., 1990; Rossi and De Paoli, 1992).
The Early French cultivar was introduced in Argentina during the 1970s,
derived from European cultivars of unknown varietal identity. This cultivar
represents the greatest cultivation area of the artichoke in Argentina due to its

aptitude to produce edible inflorescence (capitulum) during winter and to its
hardiness (Avila, 1987; Zembo, 1996). The spread of this species through in vitro
culture of shoot apices leads to plants free from systemic pathogens, with a
greater multiplication rate than that obtained by traditional agamic multiplication.
Research on the in vitro propagation of the artichoke is mainly limited to
European cultivars (Ancora et al., 1981; Moncousin, 1981; Pecaut et al., 1983;
Bigot and Foury, 1984; Rossi and De Paoli, 1992; Morzadec and Hourmant,
1997) and to the Green Globe cultivar (Lauzer and Vieth, 1990), but there is no
literature on the micropropagation of the Early French cultivar. The abovementioned authors have indicated that rhizogenesis is the critical stage of
artichoke in vitro cultivation and even though in some cultivars this problem has
been partly solved, it is still essential to investigate the subject (Rossi and De
Paoli, 1992; Morzadec and Hourmant, 1997).
Taking into account this background and studies that show the rhizogenesispromoting effect of cyclodextrins (CD) in vivo as well as in vitro (Mura et al.,
1995; ApoÂstolo et al., unpublished work), their action on the artichoke was
studied. This work describes a micropropagation protocol for plants of Cynara
scolymus L. cv. Early French employing CD during the rhizogenesis stage.

2. Material and methods
2.1. Initiation
Buds of Cynara scolymus L. cv. Early French field-gathered during the spring

were employed as vegetative material and disinfected as follows: in a solution of
5 g lÿ1 mercury dichloride for 5 min, in a solution of 17% sodium hypochlorite
(1.0 g lÿ1 of active chlorine) for 10 min and three rinsings with sterile distilled
water. In order to avoid their oxidation, disinfected buds were maintained in a
sterile solution of 150 mg lÿ1 citric acid and 100 mg lÿ1 ascorbic acid. From
these buds were isolated shoot apices 0.8 to 1 mm in length and placed on basic
medium (BM) supplemented with 0.1 mg lÿ1 a-naphthaleneacetic acid (NAA)
and diverse cytokinins (Table 1). BM contained Murashige Skoog macronutrients
(Murashige and Skoog, 1962), modified by 50% reduction in NH4NO3 and
KNO3; Heller's micronutrients (Heller, 1953); Gamborg's vitamins (Gamborg
et al., 1968), 100 mg lÿ1myo-inositol, 30 g lÿ1 saccharose and 8 g lÿ1 agar

C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10

3

(Sigma). Explants were cultured in 55 ml flat-bottomed glass tubes (one explant
per tube) containing 10 ml of culture medium.
After two subcultures, the percentage of apices with normal growth was
evaluated (rosettes with three foliar primordia and without basal callus

proliferation). Forty apices were used per treatment and each assay was
performed three times.
2.2. Multiplication
Rosettes from the medium supplemented with 1 mg lÿ1 6-(g,g-dimethylallylamino)-purine (2iP) of the initiation stage were cultured in BM supplemented with
0.5 mg lÿ1 NAA and diverse cytokinins (Table 2). The effect of incorporating
80 mg lÿ1 adenine sulphate to the treatment previously selected (BM supplemented with 0.5 mg lÿ1 NAA, 2 mg lÿ1 kinetin (Kn) and 10 mg lÿ1 2iP) was
tested. Rosettes were cultured in 120-ml glass flasks (three rosettes per flask)
containing 30 ml of culture medium.
Monthly multiplication rate was evaluated (number of final rosettes over
number of initial rosettes), as well as the quality of the rosettes obtained.
Twenty rosettes were used per treatment and the experiment was conducted
three times.
2.3. Rhizogenesis
Prior to the rhizogenesis stage, rosettes from the medium supplemented with
10 mg lÿ1 2iP, 2 mg lÿ1 Kn and 80 mg lÿ1 adenine sulphate of the multiplication stage were subcultured in BM during four weeks. During the stage
of rhizogenesis BM was modified to rooting medium (RM) by reducing
the concentration of agar to 6 g lÿ1 and of saccharose to 20 g lÿ1. Each rosette
was cultured in 55-ml flat-bottomed glass tubes containing 10 ml of culture
medium.
2.3.1. Effect of auxins

Rosettes with a length equal to or >1 cm were cultured in RM supplemented
with diverse concentrations of NAA or of indole-3-butyric acid (IBA) during one
week. They were then cultured in RM free of growth regulators during three
weeks. After four weeks culture rhizogenesis and survival percentages were
evaluated. Fifteen rosettes were used per treatment and the experiment was
conducted three times.
2.3.2. Effect of cyclodextrins
Rosettes with a length equal to or >1 cm were cultured in RM supplemented
with 3 mg lÿ1 NAA and diverse concentrations of a-CD (cyclohexamylose) or

4

C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10

b-CD (cycloheptamylose) from Sigma, St. Louis Mo, USA (Table 3). Culture
conditions were identical to those previously described. On completion of culture,
rhizogenesis percentage, number of roots per rosette, basal proliferation callus
and quality of the produced plant were evaluated. Ten rosettes were used per
treatment and the experiment was performed twice.
2.4. Culture conditions

Cultures were incubated with a 16 h photoperiod at 24  28C. A light intensity
of 25±26 mmol mÿ2 sÿ1 was provided by Philips daylight fluorescent tubes. Prior
to the initiation stage, the culture was maintained in darkness during a week to
reduce oxidation.
2.5. Acclimatisation
In vitro plants were transferred to sterile potting mixture (peat : perlite 3 : 1).
Transplanted plants were exposed to gradually decreasing humidity for a period
of 30 days in the greenhouse. The humidity regime was managed with the help of
nylon bags, which initially covered the entire plant and were gradually removed
to increase exposure to the air.
2.6. Statistical analysis
Experiments were performed on the basis of a thoroughly randomised design.
For data analysis, ANOVA with Duncan's multiple range test and 2 as nonparametric test were used.

3. Results
3.1. Initiation
The methodology employed for disinfection allowed 75% of sterile explants to
be obtained without oxidation. After two subcultures, BM supplemented with
1 mg lÿ1 2iP and 0.1 mg lÿ1 NAA showed the greatest number of shoots with
normal growth. When other cytokinins such as kinetin (Kn) or 6-benzylaminopurine (BAP) were employed, the number of apices capable of continuing normal

growth was lower (Table 1). The percentage of shoots with abnormal growth
(with a single or expanded primordium of deformed and vitreous appearance and
basal callus proliferation) ranged from 44% to 96%, according to the cytokinin
and the concentration employed (Table 1).

5

C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10

Table 1
Effect of three cytokinins on shoot apex development of Cynara scolymus L. cv. Early French after
four weeks culture. The medium was supplemented with 0.1 mg lÿ1 naphthaleneacetic acid and
diverse concentrations of cytokinins
Cytokinin

Concentration (mg lÿ1)

Normal shoots (%)a

Kinetin

0.1
0.5
1.0
0.1
0.5
1.0
1.0

27bb
26b
4c
28b
26b
18b
56a

BAPc

2iPc

a

Normal shoots: rosettes obtained with three foliated primordia and without basal callus
proliferation.
b
Different letters indicate significant differences for p  0.05 according to the 2 test.
c
BAP, 6-benzylaminopurine; 2iP, 6-(g,g-dimethylallylamino)-purine.

3.2. Multiplication
With BAP a multiplication rate was obtained greater to that with Kn and 2iP
(Table 2). However, BAP alone (Table 2) or combined with Kn or 2iP (data not
shown) showed a lower percentage of normal rosettes. Using a combination of
2 mg lÿ1 Kn and 10 mg lÿ1 2iP, normal rosettes with a monthly multiplication
rate of 3.0 were obtained. On incorporating adenine sulphate to this treatment
rosette growth was doubled, maintaining the multiplication rate (three final
rosettes per each initial rosette) (Table 2).
3.3. Rhizogenesis
3.3.1. Effect of auxins
NAA proved the most effective auxin assayed, leading to 21% rhizogenesis and

100% survival when used at a concentration of 3 mg lÿ1. Treatments with IBA
failed to differentiate roots and showed higher mortality compared to NAA
(50%).
3.3.2. Effect of cyclodextrins
The presence of 2 and 4 g lÿ1 a- or b-CD in RM supplemented with 3 mg lÿ1
NAA, showed a significant increase versus controls (22%) with rhizogenesis
percentages ranging from 50% to 100% (Table 3). On employing 4 g lÿ1 a- or bCD, the greatest percentages of rhizogenesis were obtained, but basal callus
proliferation was observed (Fig. 1(A) and (C)), while in treatments including
2 g lÿ1 of 1a- or b-CD there was a significant increase in rhizogenesis percentage
without observing callus (Table 3; Fig. 1(E)). The use of CD concentrations

6

C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10

Table 2
Effect of diverse cytokinins on in vitro multiplication of Cynara scolymus L. cv. Early French after
four weeks culture. Medium was supplemented with 0.5 mg lÿ1 naphthaleneacetic acid
Kinetin
(mg lÿ1)

BAPf
(mg lÿ1)

2iPf
(mg lÿ1)

Adenine
sulphate
(mg lÿ1)

Shoot
length
(cm)

MultiplicatioÂn
ratea

Normal
rosettesb
%

80.0

1.25bc
0.92bc
0.72bc
0.46c
1.12b
1.10b
1.20b
0.47c
0.57bc
NDe
NDe
NDe
NDe
NDe
NDe
NDe
0.93bc
1.88a

1.0
1.0
1.0
1.0
1.0
1.0
1.5
3.0
5.0
1.0
1.0
1.2
1.5
1.0
1.0
1.8
3.0
3.0

100ad
100a
100a
100a
100a
100a
22b
12b
13b
100a
100a
100a
100a
100a
100a
100a
100a
100a

0.1
0.5
1.0
2.0
0.1
0.2
0.5
1.0
2.0

2.0
2.0
2.0
2.0
2.0

1.0
2.5
5.0
10.0
1.0
2.5
5.0
10.0
10.0

a

Multiplication rate: number of final rosettes over number of initial rosette per month.
Normal rosettes: rosettes with expanded leaves and without basal callus proliferation.
c
Different letters indicate significant differences among treatments for p  0.05 according to
Duncan's test.
d
Different letters indicate significant differences among treatments for p  0.05 for 2 test.
e
Not done.
f
BAP, 6-benzylaminopurine; 2iP, 6-(g,g-dimethylallylamino)-purine.
b

higher than 4 g lÿ1 led to a decrease in the number of rooted rosettes and
malformations in shoots and roots (Fig. 1(B) and (D). Rooting percentages
obtained for each one of the tested concentrations demonstrated that b-CD was
more effective than a-CD. Whereas with a concentration of 1 g lÿ1 b-CD
rhizogenesis percentage was tripled and the number of differentiated roots per
rosette doubled, 2 g lÿ1 of a-CD were required to obtain the same result
(Table 3). In the absence of auxin, CD failed to show a positive effect on root
formation in artichoke.
3.4. Acclimatisation
Micropropagated artichoke plants obtained with 2 mg lÿ1 b-CD showed a
survival percentage of 70%. In contrast, plants with basal callus proliferation or

7

C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10

Table 3
Effect of the incorporation of a- or b-cyclodextrin (CD) on in vitro artichoke rhizogenesis in
medium supplemented with 3 mg lÿ1 naphthaleneacetic acid after four weeks culture
a-CD (g lÿ1)

b-CD (g lÿ1)

Rhizogenesis (%)

Number of rootsa

Callusb

0
0.1
0.5
1.0
2.0
4.0
8.0
Ð
Ð
Ð
Ð
Ð
Ð

0
Ð
Ð
Ð
Ð
Ð
Ð
0.1
0.5
1.0
2.0
4.0
8.0

22cc
17c
20c
20c
50b
71b
66b
20c
33c
60b
60b
100a
50b

1.5bd
1.0b
1.5b
1.5b
3.4b
3.4a
2.9a
1.5b
3.0a
3.6a
3.6a
3.0a
3.3a

ÿ
ÿ
ÿ
ÿ
ÿ
‡
‡
ÿ
ÿ
ÿ
ÿ
‡
‡

a

Number of roots: average number of roots per rosette.
Callus: ÿ, absent; ‡, present.
c
Different letters indicate significant differences among treatments for p  0.05 according to
2 test.
d
Different letters indicate significant differences among treatments for p  0.05 according to
Duncan's test.
b

malformations in shoots and roots (4±8 mg lÿ1 a- or b-CD) were unable to
survive potting.

4. Discussion and conclusions
The methodology described allowed the micropropagation of Cynara scolymus
cv. Early French. Protocols designed for various cultivars of this species were not
effective for the cultivation under study (Ancora et al., 1981; Moncousin, 1981;
Harbaoui et al., 1982; Pecaut et al., 1983; Bigot and Foury, 1984; Rossi and De
Paoli, 1992).
While Ancora et al. (1981), Moncousin (1981) and Bigot and Foury (1984)
used a cytokinin alone for the proliferation of rosettes in other cultivars, the
results of the present work indicate that a combination of 2iP and Kn is necessary
to achieve a 3.0 multiplication rate. Debergh et al. (1981) also used a similar
combination of cytokinins for Violet d'HuyeÂres cultivar, but without the
80 mg lÿ1 adenine supplement.
Rhizogenesis, the critical stage in artichoke micropropagation, is clearly
stimulated by the addition of CD to the culture medium. This enhancing effect on

8

C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10

Fig. 1. (A±E) Effect of cyclodextrins (CD) on rooted Cynara scolymus cv. Early French rosettes
at final rooting stage. Morphology: A, 4 g lÿ1 a-CD; B, 8 g lÿ1 a-CD; C, 4 g lÿ1 b-CD; D, 8 g lÿ1
b-CD; E, 2 g lÿ1 b-CD. Bar scale: 1 cm.

the part of CD has been described in olive cuttings (Mura et al., 1995) and in
jojoba sprouts cultured in vitro (ApoÂstolo et al, unpublished). Mura et al. (1995)
attributed the promotion of rhizogenesis to the increase in solubility of the auxin
employed. However, the in vitro increase in rooted sprouts of the cv. Early French

C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10

9

artichoke cannot be explained in this way, since the auxin is soluble at the
concentration used, such as happens in jojoba (ApoÂstolo et al., unpublished). As
CD increases cell-membrane permeability (Uekama et al., 1998) it may be
speculated that altered interaction between auxins and their receptors, could
explain the enhancing effects of CD on rhizogenesis of Cynara scolymus cv.
Early French.
To conclude, the application of CD seems useful for the micropropagation of
diverse artichoke cultivars, as well as those of other species having horticultural
interest, which present rooting difficulties.

References
Ancora, G., Belli-Donini, M.L., Cuozzo, L., 1981. Globe artichoke obtained from shoot apices
through rapid in vitro micropropagation. Scientia Horticulturae 14, 207±213.
Avila, F.A., 1987. El alcaucil o alcachofa: planta hortõÂcola y medicinal. Ed. El Ateneo, Buenos
Aires.
Bigot, C., Foury, C., 1984. Multiplication in vitro d'artichaut (Cynara scolymus L.) a partir de
semences: comparaison au champ de quelques clones a la ligneÂe dont ils sont issus. Agronomie
4(8), 699±710.
Debergh, P., Harboui, Y., Lemeur, R., 1981. Mass propagation of Globe artichoke (Cynara
scolymus). Evaluation of different hypothesis to overcome vitrification with special reference to
water potential. Physiologia Plantarum 53, 181±187.
Gamborg, O.L., Miller, R., Ojima, K., 1968. Nutrients requirements of suspension cultures of
soybean root cells. Experimental Cell Research 50, 151±158.
Harbaoui, Y., Smaijn, G., Welvaert, W., Debergh, P., 1982. Assainissement viral de l' artichaut
(Cynara scolymus L.) par la culture in vitro d' apex meÂristeÂmatiques. Phytopathologie
Mediterrane 21, 15±19.
Heller, R., 1953. Recherches sur la nutrition minerales des tissues veÂegetaux cultives, in vitro. Ann.
Sci. Nat. Bot. Biol. Veg. 14, 1±223.
Lauzer, D., Vieth, J., 1990. Micropropagation of seed-derived plants of Cynara scolymus L. cv.
Green Globe. Plant Cell Tissue and Organ Culture 21, 237±244.
Moncousin, C., 1981. Multiplication veÂgeÂtative acceÂleÂreÂ. Revue Horticole Suisse 54, 105±116.
Morzadec, J.M., Hourmant, A., 1997. In vitro rooting improvement of globe artichoke (cv. Camus
de Bretagne) by GA3. Scientia Horticulturae 72(1), 59±62.
Mura, P., Ceccarelli, L., Faucci, M.T., Rinaldelli, E., Mancuso, S., 1995. Improvement of solubility
of indolbutyric acid by complexation with alpha-cyclodextrin and rhizogenic activity in Olea
europea L cv Leccio del Corno. . Advance Horticultural Science 9, 119±121.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco
tissue culture. Physiologia Plantarum. 15, 473±497.
Ordas, R., Tavazza, R., Ancora, J., 1990. In vitro morphogenesis in globe artichoke (Cynara
scolymus L.). Plant Science 71, 233±237.
Pecaut, P., Dumas de Vaulx, R., Lof, H., 1983. Virus-free clones of globe artichoke (Cynara
scolymus) obtained after in vitro propagation. Acta Horticulturae 131, 303±309.
Rossi, V., De Paoli, G., 1992. Micropropagation of artichoke (Cynara scolymus). In: Bajaj, Y.P.S.
(Ed.), Biotechnology in Agriculture and Forestry: High-Tech and Micropropagation II, Vol 19.
Springer-Verlag, BerlõÂn, pp. 118±134.

10

C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10

Uekama, K., Hirayama, F., Irie, T., 1998. Cyclodextrin drug carrier systems. Chemical Review 98,
2045±2076.
Zembo, J.C., 1996. El cultivo del alcaucil en el Gran La Plata. BoletõÂn HortõÂcola Facultad de
Ciencias AgronoÂmicas y ForestalesÐUNLP. Argentina 4(11), 10±14.