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 ofCynara scolymuscv. 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 l
ÿ1
myo-inositol, 30 g lÿ1saccharose and 8.0 g lÿ1agar, supplemented with 1.0 mg lÿ16-(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 ofa -orb-cyclodextrins to the rhizogenesis medium supplemented with 3 mg lÿ1naphthalene 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)
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 above-mentioned 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 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 was studied. This work describes a micropropagation protocol for plants of Cynara scolymusL. cv. Early French employing CD during the rhizogenesis stage.
2. Material and methods
2.1. Initiation
Buds ofCynara 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
(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 -dimethylallyla-mino)-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 supple-mented 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 multipli-cation 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
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 2428C. A light intensity of 25±26mmol 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 non-parametric 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
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- orb-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- orb -CD, the greatest percentages of rhizogenesis were obtained, but basal callus proliferation was observed (Fig. 1(A) and (C)), while in treatments including 2 g lÿ1of1a- orb-CD there was a significant increase in rhizogenesis percentage without observing callus (Table 3; Fig. 1(E)). The use of CD concentrations Table 1
Effect of three cytokinins on shoot apex development ofCynara scolymusL. 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
Normal shoots: rosettes obtained with three foliated primordia and without basal callus proliferation.
b
Different letters indicate significant differences forp0.05 according to the2test.
cBAP, 6-benzylaminopurine; 2iP, 6-(
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 thatb-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 Table 2
Effect of diverse cytokinins on in vitro multiplication ofCynara scolymusL. cv. Early French after four weeks culture. Medium was supplemented with 0.5 mg lÿ1naphthaleneacetic acid
Kinetin
2.0 10.0 0.93bc 3.0 100a
2.0 10.0 80.0 1.88a 3.0 100a
aMultiplication 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 p0.05 according to Duncan's test.
d
Different letters indicate significant differences among treatments forp0.05 for2 test.
eNot done. f
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 ofCynara 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 Table 3
Effect of the incorporation of a- or b-cyclodextrin (CD) on in vitro artichoke rhizogenesis in medium supplemented with 3 mg lÿ1naphthaleneacetic acid after four weeks culture
a-CD (g lÿ1
) b-CD (g lÿ1
) Rhizogenesis (%) Number of rootsa Callusb
0 0 22cc 1.5bd ÿ
Number of roots: average number of roots per rosette.
b
Callus:ÿ, absent;, present.
c
Different letters indicate significant differences among treatments for p0.05 according to
2test.
d
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 Fig. 1. (A±E) Effect of cyclodextrins (CD) on rootedCynara scolymuscv. 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
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.
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