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 a-

or 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 andmedicinal application (choleretic, diuretic, gastrointestinal stimulant), widely

Scientia Horticulturae 83 (2000) 1±10

* 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

cultivated in the Mediterranean region. Its cultivation extends to Northern Africaand 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 cultivarrepresents the greatest cultivation area of the artichoke in Argentina due to itsaptitude to produce edible inflorescence (capitulum) during winter and to itshardiness (Avila, 1987; Zembo, 1996). The spread of this species through in vitroculture of shoot apices leads to plants free from systemic pathogens, with agreater multiplication rate than that obtained by traditional agamic multiplication.Research on the in vitro propagation of the artichoke is mainly limited toEuropean 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 noliterature on the micropropagation of the Early French cultivar. The above-mentioned authors have indicated that rhizogenesis is the critical stage ofartichoke in vitro cultivation and even though in some cultivars this problem hasbeen partly solved, it is still essential to investigate the subject (Rossi and DePaoli, 1992; Morzadec and Hourmant, 1997).

Taking into account this background and studies that show the rhizogenesis-promoting 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 wasstudied. This work describes a micropropagation protocol for plants of Cynarascolymus 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 springwere employed as vegetative material and disinfected as follows: in a solution of5 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 distilledwater. In order to avoid their oxidation, disinfected buds were maintained in asterile solution of 150 mg lÿ1 citric acid and 100 mg lÿ1 ascorbic acid. Fromthese buds were isolated shoot apices 0.8 to 1 mm in length and placed on basicmedium (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 andKNO3; Heller's micronutrients (Heller, 1953); Gamborg's vitamins (Gamborget al., 1968), 100 mg lÿ1myo-inositol, 30 g lÿ1 saccharose and 8 g lÿ1 agar

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

(Sigma). Explants were cultured in 55 ml flat-bottomed glass tubes (one explantper tube) containing 10 ml of culture medium.

After two subcultures, the percentage of apices with normal growth wasevaluated (rosettes with three foliar primordia and without basal callusproliferation). Forty apices were used per treatment and each assay wasperformed three times.

2.2. Multiplication

Rosettes from the medium supplemented with 1 mg lÿ1 6-(g,g-dimethylallyla-mino)-purine (2iP) of the initiation stage were cultured in BM supplemented with0.5 mg lÿ1 NAA and diverse cytokinins (Table 2). The effect of incorporating80 mg lÿ1 adenine sulphate to the treatment previously selected (BM supple-mented with 0.5 mg lÿ1 NAA, 2 mg lÿ1 kinetin (Kn) and 10 mg lÿ1 2iP) wastested. 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 overnumber of initial rosettes), as well as the quality of the rosettes obtained.Twenty rosettes were used per treatment and the experiment was conductedthree times.

2.3. Rhizogenesis

Prior to the rhizogenesis stage, rosettes from the medium supplemented with10 mg lÿ1 2iP, 2 mg lÿ1 Kn and 80 mg lÿ1 adenine sulphate of the multipli-cation stage were subcultured in BM during four weeks. During the stageof rhizogenesis BM was modified to rooting medium (RM) by reducingthe concentration of agar to 6 g lÿ1 and of saccharose to 20 g lÿ1. Each rosettewas cultured in 55-ml flat-bottomed glass tubes containing 10 ml of culturemedium.

2.3.1. Effect of auxinsRosettes 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 oneweek. They were then cultured in RM free of growth regulators during threeweeks. After four weeks culture rhizogenesis and survival percentages wereevaluated. Fifteen rosettes were used per treatment and the experiment wasconducted three times.

2.3.2. Effect of cyclodextrins

Rosettes with a length equal to or >1 cm were cultured in RM supplementedwith 3 mg lÿ1 NAA and diverse concentrations of a-CD (cyclohexamylose) or

C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10 3

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

2.4. Culture conditions

Cultures were incubated with a 16 h photoperiod at 24 � 28C. A light intensityof 25±26 mmol mÿ2 sÿ1 was provided by Philips daylight fluorescent tubes. Priorto the initiation stage, the culture was maintained in darkness during a week toreduce 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 periodof 30 days in the greenhouse. The humidity regime was managed with the help ofnylon bags, which initially covered the entire plant and were gradually removedto 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 non-parametric test were used.

3. Results

3.1. Initiation

The methodology employed for disinfection allowed 75% of sterile explants tobe obtained without oxidation. After two subcultures, BM supplemented with1 mg lÿ1 2iP and 0.1 mg lÿ1 NAA showed the greatest number of shoots withnormal growth. When other cytokinins such as kinetin (Kn) or 6-benzylamino-purine (BAP) were employed, the number of apices capable of continuing normalgrowth was lower (Table 1). The percentage of shoots with abnormal growth(with a single or expanded primordium of deformed and vitreous appearance andbasal callus proliferation) ranged from 44% to 96%, according to the cytokininand the concentration employed (Table 1).

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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 notshown) showed a lower percentage of normal rosettes. Using a combination of2 mg lÿ1 Kn and 10 mg lÿ1 2iP, normal rosettes with a monthly multiplicationrate of 3.0 were obtained. On incorporating adenine sulphate to this treatmentrosette growth was doubled, maintaining the multiplication rate (three finalrosettes 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 and100% survival when used at a concentration of 3 mg lÿ1. Treatments with IBAfailed to differentiate roots and showed higher mortality compared to NAA(50%).

3.3.2. Effect of cyclodextrinsThe 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 rhizogenesispercentages ranging from 50% to 100% (Table 3). On employing 4 g lÿ1 a- or b-CD, the greatest percentages of rhizogenesis were obtained, but basal callusproliferation was observed (Fig. 1(A) and (C)), while in treatments including2 g lÿ1 of 1a- or b-CD there was a significant increase in rhizogenesis percentagewithout observing callus (Table 3; Fig. 1(E)). The use of CD concentrations

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 27bb

0.5 26b

1.0 4c

BAPc 0.1 28b

0.5 26b

1.0 18b

2iPc 1.0 56a

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.

C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10 5

higher than 4 g lÿ1 led to a decrease in the number of rooted rosettes andmalformations in shoots and roots (Fig. 1(B) and (D). Rooting percentagesobtained for each one of the tested concentrations demonstrated that b-CD wasmore effective than a-CD. Whereas with a concentration of 1 g lÿ1 b-CDrhizogenesis percentage was tripled and the number of differentiated roots perrosette 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 rootformation in artichoke.

3.4. Acclimatisation

Micropropagated artichoke plants obtained with 2 mg lÿ1 b-CD showed asurvival percentage of 70%. In contrast, plants with basal callus proliferation or

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

rateaNormal

rosettesb

%

0.1 1.25bc 1.0 100ad

0.5 0.92bc 1.0 100a

1.0 0.72bc 1.0 100a

2.0 0.46c 1.0 100a

0.1 1.12b 1.0 100a

0.2 1.10b 1.0 100a

0.5 1.20b 1.5 22b

1.0 0.47c 3.0 12b

2.0 0.57bc 5.0 13b

1.0 NDe 1.0 100a

2.5 NDe 1.0 100a

5.0 NDe 1.2 100a

10.0 NDe 1.5 100a

2.0 1.0 NDe 1.0 100a

2.0 2.5 NDe 1.0 100a

2.0 5.0 NDe 1.8 100a

2.0 10.0 0.93bc 3.0 100a

2.0 10.0 80.0 1.88a 3.0 100a

a Multiplication rate: number of final rosettes over number of initial rosette per month.b 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.

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

4. Discussion and conclusions

The methodology described allowed the micropropagation of Cynara scolymuscv. Early French. Protocols designed for various cultivars of this species were noteffective 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 DePaoli, 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, theresults of the present work indicate that a combination of 2iP and Kn is necessaryto achieve a 3.0 multiplication rate. Debergh et al. (1981) also used a similarcombination of cytokinins for Violet d'HuyeÂres cultivar, but without the80 mg lÿ1 adenine supplement.

Rhizogenesis, the critical stage in artichoke micropropagation, is clearlystimulated by the addition of CD to the culture medium. This enhancing effect on

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 22cc 1.5bd ÿ0.1 Ð 17c 1.0b ÿ0.5 Ð 20c 1.5b ÿ1.0 Ð 20c 1.5b ÿ2.0 Ð 50b 3.4b ÿ4.0 Ð 71b 3.4a �8.0 Ð 66b 2.9a �Ð 0.1 20c 1.5b ÿÐ 0.5 33c 3.0a ÿÐ 1.0 60b 3.6a ÿÐ 2.0 60b 3.6a ÿÐ 4.0 100a 3.0a �Ð 8.0 50b 3.3a �

a Number of roots: average number of roots per rosette.b 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.

C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10 7

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

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.

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artichoke cannot be explained in this way, since the auxin is soluble at theconcentration used, such as happens in jojoba (ApoÂstolo et al., unpublished). AsCD increases cell-membrane permeability (Uekama et al., 1998) it may bespeculated that altered interaction between auxins and their receptors, couldexplain the enhancing effects of CD on rhizogenesis of Cynara scolymus cv.Early French.

To conclude, the application of CD seems useful for the micropropagation ofdiverse artichoke cultivars, as well as those of other species having horticulturalinterest, which present rooting difficulties.

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