In this work, a protocol for the establishment of callus cultures from Opuntia robusta, a wild and medicinal cactus, was developed. The effects of plant growth regulators and culture media composition on callus development were evaluated.The best response was observed on Murashige and Skoog medium added with 2,4-dichlorophenoxyacetic acid, benzyladenine, biotin, casein hydrolysate and proline. The exposure of O. robusta callus to jasmonic acid increased 1.3-fold and 3-fold total phenolic acids and flavonoids concentration, respectively. The in vitro culture from O. robusta could be a new approach for the obtainment of metabolites with pharmaceutical and/or nutraceutical value.Keywords: Callus, flavonoids, jasmonic acid, wild Opuntia, phenolsAfrican Journal of Biotechnology Vol. 12(21), pp. 3204-320

Astello-García, Marizel Georgina

Barba-de la Rosa, Ana Paulina

Robles-Martínez, Marissa

Santos-Díaz, María del Socorro

English

AJOL - African Journals Online

    Vol. 12(21), pp. 3204-3207, 22 May, 2013  DOI: 10.5897/AJB2013.12000 ISSN 1684-5315 ©2013 Academic Journals  http://www.academicjournals.org/AJB African Journal of Biotechnology        Full Length Research Paper  Establishment of callus from Opuntia robusta Wendl., a wild and medicinal cactus, for phenolic compounds production  Marizel Georgina Astello-García1, Marissa Robles-Martínez1, Ana Paulina Barba-de la Rosa2 and María del Socorro Santos-Díaz1  1Faculty of Chemistry, Autonomous University of San Luis Potosí, Manuel Nava 6, CP 78210 San Luis Potosí, México. 2IPICyT, Institute of Scientific and Technological Research, Camino a la Presa San José, No. 2055, Lomas 4ª sección, 78216 San Luis Potosí, México.  Accepted 22 April, 2013  In this work, a protocol for the establishment of callus cultures from Opuntia robusta, a wild and medicinal cactus, was developed. The effects of plant growth regulators and culture media composition on callus development were evaluated.The best response was observed on Murashige and Skoog medium added with 2,4-dichlorophenoxyacetic acid, benzyladenine, biotin, casein hydrolysate and proline. The exposure of O. robusta callus to jasmonic acid increased 1.3-fold and 3-fold total phenolic acids and flavonoids concentration, respectively. The in vitro culture from O. robusta could be a new approach for the obtainment of metabolites with pharmaceutical and/or nutraceutical value.   Key words: Callus, flavonoids, jasmonic acid, wild Opuntia, phenols.   INTRODUCTION  Cactus prickly pear belongs to Opuntia genus and has been used as a source of food, building material, natural pigment production and as a medicinal plant. Opuntia plants produce tender cladodes, consumed as vegetable and fruits (prickly pear) both highly appreciated due its high nutritional value and nutraceutical properties. Their therapeutic effects have been associated to its antioxi-dant constituents like vitamin C, vitamin E, carotenoids, glutathione and phenolic compounds such as flavonoids and phenolic acids (Santos-Zea, 2011). It  is  known  that  metabolite  and   chemical   profile  in Opuntia depends on harvesting season, culture condi-tions, growth stage and species. In addition, production of young cladodes and prickly pear fruits is affected by plagues and diseases that attack both commercial crops and wild populations of Opuntia (Méndez-Gallegos et al., 2008). Plant cell culture (PCC) represents an alternative with biotechnological potential for metabolite production.  In addition, using PCC is possible to increase yield using strategies like optimization of culture conditions, selection of high-producing cell lines, bioreactor design and elicitation.  *Corresponding author. *Corresponding autor. E-mail: ssantos@uaslp.mx. Tel: +52 (444) 826-2440. Fax +52 (444) 826-2371/2372.  Abbreviations: BA, Benzyladenine; CH, casein hydrolysate; 2,4-D, 2,4-dichlorophenoxyacetic acid; DICAMBA, 3,6-dichloro-2-methoxybenzoic acid; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; KIN, Kinetin; NAA, 1-naphthaleneacetic acid; PGR, plant growth regulators; PICLORAM, 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid; PPM, plant preservative mixture; MS, Murashige and Skoog medium.     During elicitation, in vitro cultures are exposed to com-pounds that induce metabolite synthesis as a defense mechanism like jasmonic acid (JA) and its methylated form (MeJA). These elicitors increase the expression of phenylpropanoid biosynthetic genes and enzymes and have been used in plant cell cultures to enhance the synthesis of health promoting metabolites (Karuppusamy, 2009). For the establishment of in vitro cultures, the participation of PGR is essential. It has been described that auxins affect development, cell growth, embryo formation and callus induction in different species. Kinetin (KIN) and benzyladenine (BA) are synthetic cytokinin commonly used in plant cell culture to generate somatic embryogenesis, organogenesis and callus formation in cacti (Shedbalkar et al., 2010). Addition of amino acids (proline and glutamine) and organic supplements (CH and peptone) also have a promoting effect on in vitro culture development stimulating cell proliferation. Vita-mins, like biotin improves the physiological response of in vitro cultured recalcitrant species (Al-Khayri, 2001).  Previous reports of in vitro culture and micropropaga-tion protocols of Opuntia ficus-indica and its cultivars have been described (Llamoca-Zárate et al., 1999; García-Saucedo et al., 2005;) but this is the first report of a protocol for in vitro culture establishment of Opuntia robusta, a wild species, as a potential metabolite produc-tion source. O. robusta has long and succulent cladodes, highly appreciated for human consumption, in both fresh and pickled forms and effects on glucose and lipid metabolism have been reported (Wolfram et al., 2002). In this study, a protocol for the establishment of callus culture from O. robusta for the phenolic compounds production, was developed. An optimized sterilization protocol for O. robusta cladodes was designed and the effect of plant growth regulators (PGR) and different organic nitrogen sources (casein hydrolysate, glutamine, and proline) was evaluated. Since we are particularly interested in the production of flavonoid and phenolic acids (due to their antioxidant properties), the induction of these metabolites by elicitation with JA is also described.    MATERIALS AND METHODS  Collection of plant material  O. robusta donor plants were provided by the National Institute of Agricultural, Livestock and Forest Research at San Luis Potosí. After collection, plants were put in pots and maintained under greenhouse conditions for young cladodes production.   Surface sterilization protocol  Prior to collection of young cladodes, O. robusta donor plants were treated with a biocide mixture (containing 3 mg l-1 Captan, 3 mg l-1 Benlate, 1 ml l-1 Previcur, 0.5 g l-1 amoxicillin, and 0.4 g l-1 keto-conazol). Collected young cladodes were washed with commercial antibacterial detergent and running tap water for 45 min, followed by immersion in the biocide mixture (described above) for 36 h, rinsed in sterile water, and  treated  with  10% calcium  hypochlorite  Astello-García et al.         3205    Ca(ClO)2 during 10 min, followed by 8% Ca(ClO)2 during 5 min. Finally, cladodes were rinsed in sterile water three times.   Culture conditions and callus induction  Sterile explants (1.5 cm2) were cultured on Murashige and Skoog (MS) basal media (Murashige and Skoog, 1962) supplemented with116 μM myo-inositol, 1.2 μM thiamine-HCl and 30 g l-1sucrose. To evaluate the auxin type effect on callus induction, explants were cultured on MS basal media with 2 mg l-1 (IAA), (IBA), (NAA) or (2,4-D). To evaluate the effect of PGR combinations on callus deve-lopment, explants were cultured on MS basal media with 3 mg l-1 IAA or 2,4-D in combination with 1 mg l-1 Picloram, Dicamba, BA or KIN. The effect of media supplements (0.1 mg l-1 biotin/0.4 g l-1 CH, 0.1 mgl-1 biotin/500 M glutamine or proline) on callus development was also tested. In order to prevent or decrease oxidation of explant, all culture media contained 1% (w/v) activated charcoal (AC), 0.3% polyvinyl-pyrrolidone (PVP), 0.025 mg l-1ascorbic acid and 0.025 mg l-1citric acid. All media were adjusted to pH 6.7 with 0.1 N KOH solution, solidified with 4.5 g L-1 phytagel (SIGMA) and autoclaved at 121°C for 20 min (1.37 x 105 Pa). The explants were cultured under dark conditions and callus formation was evaluated three weeks later.   Induction of phenolic compounds production  To promote phenolic compounds production, O. robusta calli were cultured on MS basal media with 50 M MeJA. Elicited and control calli cultures were maintained under same conditions during 15 days and then were lyophilized and stored at -20°C until analysis.  Total phenolic compounds content determination was performed according to Luximon-Ramma et al. (2002). Gallic acid (GA) was used as standard and results were expressed as μmol of GA equi-valents (GAE) per g of sample (dry weight). Data correspond to mean value ± standard deviation (SD). Total flavonoids content was performed according to the method reported by Oltica et al. (2007) with brief modifications. Quercetin (Q) was used as standard and the results were expressed as μmol of quercetin equivalents (QE) per g of sample (dry weight). Data were reported as a mean value ± standard deviation (SD). All determinations were made in triplicate.  A completely random experimental design was selected using 20 to 30 explants per treatment. Statistical analysis were performed using the Instat III program the Tukey test (P=0.05) to compare differences between means.    RESULTS AND DISCUSSION   The sterilization of O. robusta cladodes surface was a challenging process due to the huge variety of micro-organisms including insects, fungus, bacteria and viruses hosted in spines, areoles, hairs and waxes, which density is higher than in O. ficus-indica cladodes (Méndez-Gallegos et al., 2008). In order to obtain sterile explants, washing with detergent, immersion in a biocide solution, followed by the application of Ca(ClO)2 solution were required. The sterilization efficiency was 90%. Auxin type and concentration are determinant in callus development. Therefore, the effect of natural (IAA, IBA) and synthetic auxins (NAA, and 2,4-D) on callus induction from O. robusta was primarily evaluated. Callus formation was obtained in 65 to 75% of explants cultured in presence of IAA, IBA or 2,4-D (no significant  differences,3206        Afr. J. Biotechnol.    Table 1. Effect of auxins on callus induction from cladodes of Opuntia robusta  Auxin (2 mg l-1) Callus induction (%) Callus morphology IAA 75a Moderate friable callus IBA 75a Green compact callus NAA 33.3b Green compact callus 2,4-D 66.6a Green compact callus  Data with different letters are statistically different (p<0.05).    Table 2. Effect of PGR combinations and supplements on callus formation from Opuntia robusta.  PGR (mg l-1) Callus induction (%) Size of callus (mm)  IAA3 100a 5-10  IAA3-DICAMBA1 22c < 5  IAA3-PICLORAM1 100a < 5  IAA3- PICLORAM1-KIN1 100a 5-10  IAA3- PICLORAM1-KIN1-Bio-gln 100a 5-10  IAA3- PICLORAM1-KIN1-Bio-CH 100a 10-13 2,4-D 3 66b 5-10 2,4-D 3-PICLORAM1-BAP1-Bio-CH 20c 6-14  2,4-D 3- BAP1-Bio-CH-pro 50b 6-14  Bio, biotin (0.1 mg l-1); CH, casein hidrolysate (0.4 g l-1); gln, glutamine (500 M); pro, proline (500 M). Values with different letters are statistically different (p<0.05).    p<0.05). The lowest callus formation (33%) was observed in explants cultured in the presence of NAA (Table 1). However, IAA induced the generation of friable callus, which is a desirable feature in order to obtain suspension cultures. Therefore, sterile explants were cultured on media with 0.5, 1, 2 and 3 mg l-1 of IAA to evaluate callus formation. Callus formation occurred in100% of explants cultured with 3 mg l-1IAA, nevertheless, development rate was slow. It is well known that recalcitrant plants require combina-tions of PGR, higher concentration of macro-nutrients, additional organic nitrogen sources or vitamins to induce growth or development. To improve growth rate of callus, PGR combinations and addition of media supplements (biotin and CH, glutamine) were tested (Table 2). Callus induction was observed in 100% of explants maintained on media containing IAA alone or in combination with PICLORAM, KIN, biotin/glutamine and biotin/CH but callus formation decreased on medium with IAA/DICAMBA combination.The highest callus amount was obtained in media supplemented with CH/biotin (10 to 12 mm), however, after subcultures, the development  of callus was arrested. It has been shown that 2,4-D promote callus formation and increases growth rates in several cacti species.Thus, 3 mg l-1 2,4-D in combination with PICLORAM and BA, and supplemented with biotin/CH or proline were tested (BA used instead KIN because higher oxidation rates were observed in presence of KIN). Data (Table 2) sug-gest that 2,4-D, PICLORAM and BA combination induced callus formation, but was lower than callus formation in explants cultured with 2,4-D alone (statistically different, p<0.05), however, biomass was similar.On the other hand, callus formation occurred in 50% of explants cultured on media containing 2,4-D and BA, biotin/CH and proline. This calli were friable and its development continued after subcultures. Then, this media was consi-dered as the optimal for the establishment of callus culture of O. robusta. The beneficial effect of CH and proline have been attributed to a higher availability of organic nitrogen source, due to the ammonium, derived of transamination reactions of amino acids. CH is also a source of calcium, phosphate, micronutrients, vitamins, and several growth factors that improve shooting and callus development (Khaleda and Al-Forkan, 2006). In addition, proline is an energy source (the oxidation of one molecule of proline yields 30 ATP equivalents), has a reactive oxygen species (ROS) scavenging activity, an osmoprotective role, is considered as a molecular chape-rone able to protect protein integrity, and enhance the activities of different enzymes (Szabados and Savoure, 2009). Biotin is a heterocyclic compound that binds cova-lently to specific carboxylases to facilitate the transfer of CO2 during carboxylation and decarboxylation reactions, improves the physiological response of in vitro cultured recalcitrant species, and embryo development (Al-Khayri, 2001). The content of phenolic compounds was determined inAstello-García et al.         3207    Table 3. Production of phenolic acids and flavonoids by callus from Opuntia robusta1.  Metabolite Control callus (μmol g-1) Elicited callus ( μmol g-1) Increment Phenolic acids2 7.50 ± 0.33b 9.81± 0.38a 1.30 Flavonoids3 1.91± 0.08b 5.84 ± 0.34a 3.06  1Values between columns with different letters are statistically different (p<0.05).  2Phenolic acids content determined as gallic acid equivalents.  3Flavonoids content determined as quercetin equivalents.    O. robusta callus. The results show that callus produced 2.3 times more phenolics acids (GAE) than flavonoids (QE). Ellicitation with JA increased 1.3-folds the phenolic acids content and 3-folds the flavonoids concentration in comparison to control callus (Table 3). JA has been widely used in promoting the biosynthesis of both inducible and constitutive secondary metabolites, including several anti-oxidants as -tocopherol, cyanidineglucosides, resveratrol and flavonoids (Matkowski, 2008).  Comparing our results with other data from literature, the content of phenolic acids in O. robusta cultures was 11 times higher than in callus from Lepidum meyenii (Wang et al. 2007), while the flavonoids content was two times higher than in cellular suspensions of Pueraria tuberose (Goyal and Ramawat, 2008). Further studies must be done to identify and characterize the metabolites from in vitro cultures of O. robusta species and know if they correspond or not to those present in O. robusta plant.In addition, the levels of metabolites could be improved using biotic and abiotic stress.   ACKNOWLEDGMENTS   This work was supported by the FOMIX-GTO Grant No. 2006-CO1-31809, CONACYT- Fondos Bilaterales México Francia (No. 142873) and FAI –UASLP.   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Establishment of callus from Opuntia robusta Wendl., a wild and medicinal cactus, for phenolic compounds production

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Establishment of callus from Opuntia robusta Wendl., a wild and medicinal cactus, for phenolic compounds production

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