Micropropagation of Cynara scolymus L. employing cyclodextrins to promote rhizogenesis

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1 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 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 NH 4 NO 3 and KNO 3, Heller's micronutrients, Gamborg's vitamins, 100 mg l 1 myo-inositol, 30 g l 1 saccharose and 8.0 g l 1 agar, supplemented with 1.0 mg l 1 6-(g,g-dimethylallylamino)- purine and 0.1 mg l 1 naphthalene acetic acid. In the multiplication stage, to achieve a rate of 3 : 1, basic medium was supplemented with 10 mg l 1 6-(g,g dimethylallylamino)-purine, 2.0 mg l 1 kinetin, 0.5 mg l 1 naphthalene acetic acid and 80 mg l 1 adenine sulphate. The incorporation of a- or b-cyclodextrins to the rhizogenesis medium supplemented with 3 mg l 1 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.: int. 273; fax: address: apostolo@mail.unlu.edu.ar (C. Brutti) /00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S (99)

2 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 5gl 1 mercury dichloride for 5 min, in a solution of 17% sodium hypochlorite (1.0 g l 1 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 l 1 citric acid and 100 mg l 1 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 l 1 a-naphthaleneacetic acid (NAA) and diverse cytokinins (Table 1). BM contained Murashige Skoog macronutrients (Murashige and Skoog, 1962), modified by 50% reduction in NH 4 NO 3 and KNO 3 ; Heller's micronutrients (Heller, 1953); Gamborg's vitamins (Gamborg et al., 1968), 100 mg l 1 myo-inositol, 30 g l 1 saccharose and 8 g l 1 agar

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 Multiplication Rosettes from the medium supplemented with 1 mg l 1 6-(g,g-dimethylallylamino)-purine (2iP) of the initiation stage were cultured in BM supplemented with 0.5 mg l 1 NAA and diverse cytokinins (Table 2). The effect of incorporating 80 mg l 1 adenine sulphate to the treatment previously selected (BM supplemented with 0.5 mg l 1 NAA, 2 mg l 1 kinetin (Kn) and 10 mg l 1 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 Rhizogenesis C. Brutti et al. / Scientia Horticulturae 83 (2000) 1±10 3 Prior to the rhizogenesis stage, rosettes from the medium supplemented with 10 mg l 1 2iP, 2 mg l 1 Kn and 80 mg l 1 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 l 1 and of saccharose to 20 g l 1. Each rosette was cultured in 55-ml flat-bottomed glass tubes containing 10 ml of culture medium 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 Effect of cyclodextrins Rosettes with a length equal to or >1 cm were cultured in RM supplemented with 3 mg l 1 NAA and diverse concentrations of a-cd (cyclohexamylose) or

4 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 Culture conditions Cultures were incubated with a 16 h photoperiod at 24 28C. A light intensity of 25±26 mmol m 2 s 1 was provided by Philips daylight fluorescent tubes. Prior to the initiation stage, the culture was maintained in darkness during a week to reduce oxidation 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 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 1mgl 1 2iP and 0.1 mg l 1 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 5 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 l 1 naphthaleneacetic acid and diverse concentrations of cytokinins Cytokinin Concentration (mg l 1 ) Normal shoots (%) a Kinetin b b b 1.0 4c BAP c b b b 2iP c a 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 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 2mgl 1 Kn and 10 mg l 1 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) Rhizogenesis 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 l 1. Treatments with IBA failed to differentiate roots and showed higher mortality compared to NAA (50%) Effect of cyclodextrins The presence of 2 and 4 g l 1 a-orb-cd in RM supplemented with 3 mg l 1 NAA, showed a significant increase versus controls (22%) with rhizogenesis percentages ranging from 50% to 100% (Table 3). On employing 4 g l 1 a-orb- CD, the greatest percentages of rhizogenesis were obtained, but basal callus proliferation was observed (Fig. 1(A) and (C)), while in treatments including 2gl 1 of 1 a-orb-cd there was a significant increase in rhizogenesis percentage without observing callus (Table 3; Fig. 1(E)). The use of CD concentrations

6 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 l 1 naphthaleneacetic acid Kinetin (mg l 1 ) BAP f (mg l 1 ) 2iP f (mg l 1 ) Adenine sulphate (mg l 1 ) Shoot length (cm) MultiplicatioÂn rate a Normal rosettes b % b c a d bc a bc a c a b a b a b b c b bc b 1.0 ND e a 2.5 ND e a 5.0 ND e a 10.0 ND e a ND e a ND e a ND e a bc a a a 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. higher than 4 g l 1 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 l 1 b-cd rhizogenesis percentage was tripled and the number of differentiated roots per rosette doubled, 2 g l 1 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 Acclimatisation Micropropagated artichoke plants obtained with 2 mg l 1 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 7 Table 3 Effect of the incorporation of a- or b-cyclodextrin (CD) on in vitro artichoke rhizogenesis in medium supplemented with 3 mg l 1 naphthaleneacetic acid after four weeks culture a-cd (g l 1 ) b-cd (g l 1 ) Rhizogenesis (%) Number of roots a Callus b c c 1.5b d 0.1 Ð 17c 1.0b 0.5 Ð 20c 1.5b 1.0 Ð 20c 1.5b 2.0 Ð 50b 3.4b 4.0 Ð 71b 3.4a 8.0 Ð 66b 2.9a Ð c 1.5b Ð c 3.0a Ð b 3.6a Ð b 3.6a Ð a 3.0a Ð b 3.3a a Number of roots: average number of roots per rosette. b Callus:, 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. malformations in shoots and roots (4±8 mg l 1 a-orb-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 l 1 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 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 l 1 a-cd; B, 8 g l 1 a-cd; C, 4 g l 1 b-cd; D, 8 g l 1 b-cd; E, 2 g l 1 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

9 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., Globe artichoke obtained from shoot apices through rapid in vitro micropropagation. Scientia Horticulturae 14, 207±213. Avila, F.A., El alcaucil o alcachofa: planta hortõâcola y medicinal. Ed. El Ateneo, Buenos Aires. Bigot, C., Foury, C., Multiplication in vitro d'artichaut (Cynara scolymus L.) aâ partir de semences: comparaison au champ de quelques clones aâ la ligneâe dont ils sont issus. Agronomie 4(8), 699±710. Debergh, P., Harboui, Y., Lemeur, R., 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., Nutrients requirements of suspension cultures of soybean root cells. Experimental Cell Research 50, 151±158. Harbaoui, Y., Smaijn, G., Welvaert, W., Debergh, P., Assainissement viral de l' artichaut (Cynara scolymus L.) par la culture in vitro d' apex meâristeâmatiques. Phytopathologie Mediterrane 21, 15±19. Heller, R., Recherches sur la nutrition minerales des tissues veâegetaux cultives, in vitro. Ann. Sci. Nat. Bot. Biol. Veg. 14, 1±223. Lauzer, D., Vieth, J., Micropropagation of seed-derived plants of Cynara scolymus L. cv. Green Globe. Plant Cell Tissue and Organ Culture 21, 237±244. Moncousin, C., Multiplication veâgeâtative acceâleâreâ. Revue Horticole Suisse 54, 105±116. Morzadec, J.M., Hourmant, A., In vitro rooting improvement of globe artichoke (cv. Camus de Bretagne) by GA 3. Scientia Horticulturae 72(1), 59±62. Mura, P., Ceccarelli, L., Faucci, M.T., Rinaldelli, E., Mancuso, S., 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., A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum. 15, 473±497. Ordas, R., Tavazza, R., Ancora, J., In vitro morphogenesis in globe artichoke (Cynara scolymus L.). Plant Science 71, 233±237. Pecaut, P., Dumas de Vaulx, R., Lof, H., Virus-free clones of globe artichoke (Cynara scolymus) obtained after in vitro propagation. Acta Horticulturae 131, 303±309. Rossi, V., De Paoli, G., 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.

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