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Establishment of Aster sedifolius and Aster caucasicus callus cultures as a potential source of antioxidants
Maria Minutolo , Immacolata Caruso , Gianluca Caruso , Pasquale Chiaiese & Angela Errico
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Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples Federico II, Via Universit 100, 80055, Portici, NA, Italy Available online: 01 Dec 2011

To cite this article: Maria Minutolo, Immacolata Caruso, Gianluca Caruso, Pasquale Chiaiese & Angela Errico (2012): Establishment of Aster sedifolius and Aster caucasicus callus cultures as a potential source of antioxidants, Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana, 146:1, 41-46 To link to this article:

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Plant Biosystems, Vol. 146, No. 1, March 2012, pp. 4146

Establishment of Aster sedifolius and Aster caucasicus callus cultures as a potential source of antioxidants

Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples Federico II, Via Universita ` 100, 80055 Portici (NA), Italy

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Abstract Callus cultures were established for Aster sedifolius and Aster caucasicus, two Aster species used in natural medicine for their anticancer, antibacterial and antiviral activities attributed to the high content of antioxidant compounds such as polyphenols and ascorbate. The effects of growth medium and light condition on the induction and growth rate of callus from leaf, petiole and root explants are reported. Callus induction and proliferation depended on the genotype and the experimental conditions. In particular, a profuse callus culture was obtained from leaf explants grown in the light on medium supplemented with 2,4-D (0.1 mg l71) for A. caucasicus and on medium supplemented with 2,4-D (0.44 mg l71) plus 6-benzil-ammino-purine (BAP) (0.22 mg l71) for A. sedifolius. The content of total polyphenol and ascorbic acid was estimated in leaf and petiole explants of in vivo plants and in the relative derived calli. In calli, polyphenol content was lower than in the corresponding in vivo organs. Furthermore, the total ascorbic acid content decreased in calli while the reduced ascorbic acid pool increased. These ndings demonstrate that Aster callus cultures produce antioxidant compounds and as such might be a model system to investigate the regulation and production of these important metabolites.

Keywords: Ascorbic acid, in vitro culture, polyphenols

Introduction The genus Aster (Asteraceae) is widely distributed in nature, including about 600 species adapted to various niches. A large number of species and interspecic hybrids are grown as ornamentals plants or for cut owers; however, theyre predominantly used for their therapeutic properties known since ancient times. In traditional Chinese medicine, Aster species have been used for the treatment of cough, fever, and tonsillitis. In recent years, diuretic, antitumor, antibacterial, antiviral and anti-ulcer activities have been attributed to these plants (Ng et al. 2003). The Aster therapeutic features have been widely ascribed to the high content of antioxidant compounds such as polyphenols and ascorbic acid which exhibit antibacterial, antiviral, anti-inammatory, anti-allergic, antithrombotic and vasodilator actions and are useful in the treatment of arteriosclerosis, cancer, diabetes, neurodegenerative diseases, and

arthritis, as well as for the prevention of other diseases (Tiwari 2001; Pourmorad et al. 2006). Antioxidant content is variable in plants because their synthesis and storage is a direct consequence of plantenvironment interaction. Although much research has been carried out on the isolation and determination of antioxidants from plants, little is known about their regulation and production in plants growing in vitro or under tissue culture conditions. Callus and cell suspension cultures are an attractive model system and represent an interesting tool to increase the knowledge of antioxidant production and accumulation (Bauer et al. 2004; Keskin & Kunter 2008). Cell cultures have been established for many plant genera but have been reported only for a few species of the Aster genus (Cammareri et al. 2001, 2002; Uno et al. 2009). Therefore, the aims of the present study were to develop an efcient protocol for callus culture from A. sedifolius and A. caucasicus by nding the most suitable explant source and the best callus

Correspondence: Dr. Maria Minutolo, Dipartimento di Scienze del Suolo, della Pianta, dellAmbiente e delle Produzioni Animali, Universita ` di Napoli Federico II, Via Universita ` 100, 80055 Portici (NA), Italia. Tel: 39 0812539430. Fax: 39 0812539186. Email: The rst two authors contributed equally to this work. ISSN 1126-3504 print/ISSN 1724-5575 online 2012 Societa ` Botanica Italiana


M. Minutolo et al. sterilized with a solution of ethanol 70% (v/v) for 5 min, and then left for 15 min in a solution of sodium hypochlorite 2% (v/v) plus 0.1% (v/v) of Tween-20. Finally, they were rinsed three times in sterile distilled water. For each tissue (P, L, LSF and so on) and tested experimental condition, 30 explants were collected from in vivo and 30 from in vitro growing plants. They were cut into rectangular small pieces (ca. 1 cm2) and cultivated on Petri dishes containing MS0 medium supplemented with 2,4-dichlorophenoxyaceti acid (2,4-D) and 6-benzil-ammino-purine (BAP) at various concentrations: 0.1 mg l71 of 2,4-D (medium A); 1 mg l71 of 2,4-D (medium B); 10 mg l71 of 2,4-D (medium C); 0.94 mg l71 of 2,4-D and 0.18 mg l71 of BAP (medium D); 0.44 mg l71 of 2,4-D, and 0.22 mg l71 of BAP (medium E). All cultures were incubated at 258C+2 under a 14/10 (light/dark) photoperiod at 125 mEm72 s71 irradiance provided by cool white uorescent tube (Philips) or in complete darkness. Subcultures were performed monthly. Callus growth (CG), was recorded after 15, 30, and 45 days of in vitro growth, and estimated as explants area showing callus. A score was assigned at each explant according to the following rank: 0, no visible callus; 1, appearance of callus only at cut edges; 2, callus on 20% of explant area; 3, callus on 50% of explant area and 4, callus on 70% and more of explant area (Figure 2).

growth conditions, and to evaluate the production of antioxidant metabolites.

Materials and methods Plant material A. sedifolius, accession number no. 151, and A. caucasicus, accession no. 81, were kindly provided by the Botanical Garden of Stuttgart (Germany) and the Botanical Garden of Bayreuth (Germany), respectively. A set of three plants per species were grown in vivo, in a non-conditioned greenhouse, and in vitro on hormone free medium containing Murashige and Skoog (1962) mineral salt and vitamins formulation supplemented with sucrose 3% (w/v), agar 0.9% (w/v), and adjusted to pH 5.8 (MS0). Plant tissues culture In A. sedifolius, leaf explants of three different developmental stages, namely rosette (L), initial stem elongation phases (LIS) and nal stem elongation phases (LFS), were used while for A. Caucasicus, leaves were taken only at rosette stage (L) (Figure 1). For both species, petiole (P) and leaf explants were collected from in vitro and in vivo grown plants, while root (R) explants were collected just from in vitro plants. Explants from in vivo plants were surface-

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Figure 1. Tissues sampled from A. sedifolius and A. caucasicus plants.

Figure 2. Callus development of A. sedifolius petiole explants. Score 0 (a), score 1 (b), score 2 (c), score 3 (d), and score 4 (e).

Aster sedifolius and Aster caucasicus callus cultures Antioxidant extraction and quantication Antioxidants extraction was performed only from 45 days old calli developed in medium E for A. sedifolius and in medium A for A. caucasicus, both under light condition. Three extraction experiments were performed for each sample; for each extraction, antioxidants quantication was performed in triplicate. Total polyphenols were extracted and assayed according to FolinCiocalteus procedure (Singleton and Rossi 1965). Total ascorbic acid (total-AsA), as sum of reduced and oxidized forms, and reduced ascorbic acid (AsA) were extracted and quantied using the procedure described by Kampfenkel et al. (1995). Statistical analysis Statistical analysis was performed by using the Statistical Package for Social Sciences Package version 14 (SPSS Inc Chicago, Illinois). The effects of experimental factors on callus growth were estimated by the ANOVA procedure and the Duncan test was used for mean separation referring to p  0.01 and p  0.05 probability levels. Results and discussion Callus growth rate was highly variable for both species and for all explants assayed (Figure 3a and b). In the rst 15 days of culture, the highest CG rate was detected in A. sedifolius from leaf explants at initial (LIS) and at nal stem elongation stage (LFS) followed by leaves at rosette stage (L), petioles (P) and nally roots (R). Root explants turned brown and died within 3 weeks. At day 45, a CG rate of 2.6 was detected for leaf explants (L, LIS and LFS) while the lowest rate (1.5) was calculated for petiole explants (Figure 3a). On the contrary, in A. caucasicus, root explants showed a high CG rate after 15 and 30 days. Furthermore, at day 45 of culture, a CG rate of 3.84.0 was detected for all explant types (Figure 3b) suggesting that callus production in Aster is species and genotype dependent (Cammareri et al. 2001). In plant species such as Aster tripolium L. (Uno et al. 2009), Saussurea obvallata (Dhar & Joshi 2005) and Actinidia deliciosa (Akbas et al. 2009) profuse callus production and proliferation were obtained using leaf explants. On the contrary, for Changium smyrnioides, petioles were the best explants for obtaining callus culture (Jiang et al. 2009). On medium B (2,4-D 1 mg/l), no callus development was observed in both species (Figure 3c and d). This is in contrast with the observations of Uno et al. (2009) reporting that friable white callus was obtained after cultivation of A. tripolium leaf explants on medium containing 1 mg l71 of 2,4-D.


The herbicide 2,4-D has been commonly used for obtaining friable callus culture; however, in our experiments, an increase of its concentration in the growth medium negatively affected callus induction and development. All explants in medium C (2,4-D 10 mg l71) died after 3 weeks of culture. On medium A, in which 2,4-D concentration was tenfold less, explants have a higher CG, comparable to that obtained by Cammareri et al. (2001). In contrast, in other Asteraceae such as Inula racemosa, rapid browning of callus was reported for explants cultured on medium containing 0.8 mg l71 of 2,4-D and further increase in 2,4-D concentration led to death within 20 days (Jabeen et al. 2007). In our condition, after 45 days of in vitro culture the best CG rate was observed using medium D (0.94 mg l71 of 2,4-D and 0.18 mg l71 of BAP) and medium E (0.44 mg l71 of 2,4-D and 0.22 mg l71 of BAP) (Figure 3c). Also, an increase in callus induction rate and callus proliferation was observed in A. tripolium when using 2,4-D plus citochinins rather than auxin alone (Uno et al. 2009). A similar trend was observed in A. caucasicus cultures, in which no induction was observed on explants cultured on medium B (1 mg/l 2,4-D), whereas callus grown on medium C (2,4-D 10 mg/l) turned brown and died. The best CG rate was recorded on medium A (Figure 3d). These data suggest that Aster cells are susceptible to high 2,4-D concentrations. The effect of light on callus induction and growth depends on the species. In A. caucasicus, light positively affected callus cultures obtained from all media assayed (Figure 3e). On the other hand, in A. sedifoilius a positive effect was recorded only during the rst 15 days of in vitro culture (Figure 3f). In barley, no differences were observed in callus induction of leaf explants under light or dark conditions (Zapata et al. 2004) while Lemna gibba callus induction and growth required light (Moon and Stomp 1997). The physiological stage of the mother plant affects callus development and growth. In A. sedifolius, at 15 days, the CG of all explants collected from in vivo grown plants was higher than that from in vitro grown plants (Figure 3g) although after 45 days of culture no differences were detected. On the contrary, for A. caucasicus a higher and faster CG was recorded on explants collected from in vitro tissues rather than on explants collected from in vivo plants (Figure 3h). A. sedifolius calli were dark green brownish and generally compact; they became partially friable after 5 months of culture (Figure 4a, b, and c) on medium E. Calli developed on A. caucasicus explants, on the other hand, were generally white yellowish and friable (Figure 4d, e, and f). Evaluation of total polyphenols (Phe), reduced ascorbic acid (AsA), and total ascorbic acid

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M. Minutolo et al.

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Figure 3. Effect of tissue type (panels a and b), media (panels c and d), light conditions (panels e and f) and explant source (panels g and h) on callus growth rate in A. sedifolius and A. caucasicus. For each culture day, different letters represent statistical differences (p 50.05) among treatments. Callus growth (CG) at 15, 30, and 45 days of in vitro culture is reported. L, leaf at rosette stage; LIS, leaf at initial stem elongation stage; LFS, leaf at nal stem elongation stage; P, petiole; R, root.

(total-AsA) contents was performed on in vivo LIS and LFS of A. sedifolius, and leaf and petiole of A. caucasicus and the relative in vitro callus cultures. In A. sedifolius, Phe accumulation in callus cultures obtained from LIS and LFS was lower than that found in their original tissues. The lower content of Phe in callus culture is in accordance with that observed in other species and might be due to the negative effect of these compounds on cell division. In cell culture, an increase of Phe content is generally observed at the end of the exponential or steady growth phase of the growth curve (Phillips & Henshaw 1977). Gurney et al. (1999) reported that caffeine and theobromine production in Theobroma cacao callus obtained from leaf explants were 10% lower than those found in vivo. In Vitis cell cultures,

anthocyanins were produced and accumulated just after the end of cell division, when an increase of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) activities were also recorded (Kakegawa et al. 1995). For Aster, total-AsA and the percentage of reduced AsA on total-AsA (AsA%) in LIS were not statistically different from those recorded in LFS (Table I). These results are in contrast with studies in other species; in Arabidopsis thaliana, Medicago sativa, and Impatiens walleriana, ascorbic acid content was higher in mature leaves than in younger leaves (Franceschi & Tarlyn 2002). The reduced AsA% found in callus culture from LIS and LFS was doubled compared to those of differentiated tissues. This is not surprising because an increase of the

Aster sedifolius and Aster caucasicus callus cultures


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Figure 4. Leaf derived calli of A. sedifolius in vivo grown plants cultured (under light conditions) on media A (a), D (b) and E (c), and of A. caucasicus in vitro grown plants cultured on medium A (light conditions) (d) and medium E (light or dark conditions, respectively) (e, f).

Table I. Content of total polyphenol, total-ASA and percentage of reduced ASA on total-ASA (AsA%), in in vivo grown tissues and derived calli of A. sedifolius and A. caucasicus. Polyphenols (mg/g) Aster sedifolius Callus from Callus from LIS* LFS** Callus from Callus from Leaf Petiole LIS* LFS** 0.22 (0.08) c 0.15 (0.01) c 6.13 (0.05) b 7.80 (0.15) a 0.17 (0.02) B 0.28 (0.06) B 6.4 (0.20) A 6.2 (0.23) A Total AsA (umol/g) 0.33 0.17 0.83 0.84 0.18 0.17 0.39 0.61 (0.05) (0.05) (0.15) (0.10) (0.02) (0.02) (0.01) (0.03) b c a a B B A A AsA% 24.1 22.3 9.6 10.7 22.7 21.3 7.7 10.1 (4.07) (6.76) (1.47) (1.86) (2.59) (2.85) (1.38) (1.01) a b c c A A B B

Aster caucasicus

leaf petiole

Mean and standard error values are reported. Different letters represent statistical differences (p 5 0.05) among tissues. *LIS, leaf at initial stem elongation stage; **LFS, leaf at nal stem elongation stage.

endogenous reduced ascorbic acid level and of the relative redox status was generally recorded during cell division, suggesting that an intracellular decrease in the amount of dehydroascorbate could be a signal for the cell to proceed from G1 into S phase (Kato and Esaka 1999). On the other hand, root quiescent cells (mitotically inactive cells) have generally low or undetectable levels of reduced ascorbic acid (Kerk and Feldman 1995). These ndings are in agreement with our observations on Phe accumulation conrming that A. sedifolius calli were still dividing and growing under the optimized in vitro culture conditions. For A. caucasicus, no differences in metabolite amounts were observed between leaf and petiole (collected from in vivo plants) (Table I). These ndings are in contrast with data reported in other species where polyphenol and ascorbic acid levels are

generally higher in leaves than in petioles (Baba et al. 1956; Zainol et al. 2003; van der Rest et al. 2006; Zadeh et al. 2007). These discrepancies could be due to A. caucasicus leaf morphology; the separation between leaf and petiole is not well delineated and explants could have been arbitrarily collected (Figure 1). On the other hand, polyphenol content was similar in different tissues of Pisum sativum (Singh et al. 2002). In any case, although polyphenols are generally a constitutive component of plant tissues, their presence is also inuenced by environmental conditions and plant phenological stages. Similarly to what observed for A. sedifolius, a decrease of Phe and total-AsA and an increase of reduced AsA% in callus tissues compared with the original tissues were also found for A. caucasicus (Table I). In conclusion, we established a valuable callus culture procedure for two Aster species: for


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A. Sedifolius, the best result was obtained with leaf explants collected from in vivo grown plants and cultivated on medium containing 0.44 mg l71 of 2,4D and 0.22 mg l71 of BAP under light conditions; for A. Caucasicus, the best callus was obtained from in vitro root explants growing on medium containing 0.1 mg l71 of 2,4-D. Furthermore, our experiments demonstrated that Aster callus cultures produce antioxidants and might be used as a model system to investigate the regulation and production of these metabolites. Acknowledgments The authors thank Dr. Di Matteo Antonio for assistance in metabolites analysis and Mrs. Lotti Elvira for her technical assistance. This work was supported by a grant from the Ministry of Agricultural, Food and Forestry Policies (MiPAAF), Italy, as part of the BIOPEST project and contribution from DISSPAPA n. 218 is also acknowledged. References
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