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Direct and indirect regeneration from tuberosa (Spreng.) Hicken calli and its β-ecdysone production

FLORES, R.1*; MALDANER, J.2; BRONDANI, D.3; CEZAROTTO, V.4; GIACOMELLI, S.R.4; GARLET, T.M.B.5; NICOLOSO, F.T.6 1Instituto Federal Farroupilha, Campus São Vicente do Sul. RS, Brasil. 2Fundação Estadual de Pesquisa Agropecuária. Santa Maria, RS, Brasil. 3Universidade Federal de Santa Catarina, Campus Blumenau. SC, Brasil. 4Universidade Regional Integrada, Campus Frederico Westphalen. RS, Brasil 5Universidade Federal de Santa Maria (UFSM), Campus Palmeira das Missões. RS, Brasil. 6UFSM, Campus sede. Santa Maria, RS, Brasil. *E-mail: [email protected]

ABSTRACT: This study aimed to establish calli from nodal segments of P. tuberosa and to evaluate the effect of growth regulators (PGR) on plant regeneration and production of β-ecdysone from in vitro-grown tissues. Calli were induced from nodal segments on Murashige and Skoog (MS) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D)

combined with N6-benzylaminopurine (BAP). The β-ecdysone concentrations [on a dry weight (DW) basis] in in vitro grown and field-grown tissues were measured by high performance liquid chromatography. Direct regenerants (ranging from 4.0 to 75.0%) arose from sprouts present in the explants only in the medium containing 2,4-D. Maximum frequency of indirect shoot regeneration from calli (20.0%) was observed in the medium containing 2,4-D and BAP (1.0 µM at each). The accumulation of β-ecdysone in the calli was associated with the presence of shoots and influenced by the concentrations of 2,4-D. Higher amounts of β-ecdysone were observed in the calli grown with 10.0 µM 2,4-D [0.516% (w/w; DW)], whose content was almost twice that found in the shoots [0.333% (w/w; DW)] from field-grown . Calli grown under 1.0 to 5.0 µM 2,4-D presented lower β-ecdysone concentrations than those found in the shoot, but higher than those detected in the roots from field-grown plants [0.022% (w/w; DW)]. Our study is the first report on the presence of ecdysteroids in the organogenic calli of P. tuberosa, and may be useful for several biotechnological applications and/or in vitro propagation approaches.

Keywords: In vitro culture, ecdysteroid, Brazilian ginseng, .

RESUMO: Regeneração direta e indireta em Pfaffia tuberosa (Spreng.) Hicken e sua produção de β-ecdisona. Este estudo teve como objetivo estabelecer calos em P. tuberosa e avaliar o efeito de reguladores vegetais sobre a regeneração e a produção de β-ecdisona. Os calos foram induzidos a partir de segmentos nodais em meio de Murashige e Skoog, acrescido de 2,4-D e BAP. O teor de β-ecdisona (com base na biomassa seca), nos tecidos in vitro e em plantas a campo, foi avaliado por cromatografia liquida de alta eficiência. Observou-se regeneração direta de brotos (4 a 75%) somente nos explantes cultivados na presença do 2,4-D. A maior frequência de regeneração indireta a partir dos calos (20%) foi registrada em meio com 2,4-D e BAP (1 µM de cada). O teor de β-ecdisona nos calos foi associado com a presença de brotos e influenciado pelas concentrações de 2,4-D. Maiores quantidades de β-ecdisona foram observadas nos calos cultivados com 10 µM de 2,4-D (0,516%), cujo teor foi quase duas vezes maior que o encontrado nos brotos (0,333%) das plantas a campo. Calos cultivados com concentrações de 1 a 5 µM de 2,4-D apresentaram menores teores de β-ecdisona quando comparado com aqueles encontrados nos brotos, mas maiores que o detectado nas raízes (0,022%) das plantas a campo. Este é o primeiro estudo que mostra a presença de ecdisteróides em calos organogênicos de P. tuberosa.

Palavras-chave: cultivo in vitro, ecdisteróides, ginseng brasileiro, Amaranthaceae.

Recebido para publicação em 13/06/2016 Aceito para publicação em 20/03/2017 10.1590/1983-084X/0052

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INTRODUCTION acetic acid (NAA) or 2,4-Dichlorophenoxyacetic In , the Amaranthaceae family is made acid (2,4-D) (Flores et al., 2006). Furthermore, up of 20 native genera and about 100 species, chemical analysis of plants grown in Southern Brazil including the medicinal species belonging to the has shown that both roots and aerial parts contain Pfaffia genus, popularly known as Brazilian ginseng. a significant amount of b-ecdysone [range 0.09 - It is a neotropical genus and Brazil is considered to be 0.63% (w/w) of the dry mass] (Flores et al., 2009; its main center of diversity (Marchioretto et al., 2010). Flores et al., 2010). However, to our knowledge, In general, the Pfaffia genus is used in situations information regarding the production of β-ecdysone of stress or fatigue because of its tonic, fortifying, from in vitro callus and organs is very limited in the aphrodisiac and anti-stress properties, among literature, despite the importance of obtaining new other adaptogenic properties (Mendes, 2011). This physiological and phytochemical information about term was coined by N. Lazarev in the Soviet Union the Pfaffia genus. Thus, the aims of this study were to classify plants and/or compounds that augment to establish calli from nodal segments of P. tuberosa non-specific resistance of the body (Brekhman & and to evaluate the effect of 2,4-D and BAP on plant Dardymov, 1969). The adaptogen effects of the regeneration and the production of β-ecdysone Brazilian ginsengs are attributed to a number of from in vitro-grown tissues. The level of production metabolites, such as the phytoecdysteroids (PEs) of β-ecdysone in in vitro-tissues was compared to (Lafont & Dinan, 2003). field-grown plants. Pfaffia tuberosa (Spreng.) Hicken is an herb or subshrub plant called corango-de-batata due to its tuberous roots. It is native to the Pampa and MATERIALS AND METHODS Cerrado biomes, which are recognized as priority Plant material ecosystems for conservation in Brazil due mainly to Pfaffia tuberosa (Spreng.) Hicken the destruction of their habitats and the presence (Amaranthaceae) plants (n = 20) used in this study of a great number of endemic species (Boldrini, were collected during the flowering stage (October 2009). In several regions, the tuberous roots of P. to March) of growth at the Botanical Garden of the tuberosa are used in folk medicine, mainly as an Universidade Federal de Santa Maria (UFSM, Rio infertility treatment. The main chemical constituents Grande do Sul, Santa Maria, Brazil) and identified isolated from roots of P. tuberosa are saponins (i.e., by Fernando Teixeira Nicoloso. A sample (voucher oleanolic acid) and PEs (i.e., β-ecdysone) (Nishimoto specimen SMDB 9840) was placed at the Herbarium et al., 1986), indicating the pharmaceutical potential of the Department of Biology, at UFSM, Santa Maria, of this plant. RS, Brazil. However, contrary to P. glomerata, considered to be one of the most economically In vitro establishment and shoot important Brazilian ginsengs (Daniel et al., 2005; multiplication Neto et al., 2005; Serra et al., 2012), there are very Young shoots (50 mm-long) of P. tuberosa few chemical and pharmacological studies, as well were surface-disinfected according to the method as propagating strategies in P. tuberosa. Propagation described by Flores et al. (2010). Disinfected nodal by seed is not recommended because of its high segments (10 mm long micro-cuttings consisting of genetic and morphological variability (Taschetto & two buds) were transferred to glass culture tubes (25 Pagliarini, 2003) and propagation by cuttings is not mm x 150 mm), each containing 10 ml of solidified viable due to the fragility of its stem and the presence Murashige and Skoog medium (MS; Murashige & of very short internodes. Skoog, 1962), containing 100 mg L−1 myo-inositol, Moreover, the production of secondary 30 g L−1 sucrose and 6 g L−1 agar (Sigma-Aldrich, metabolites via field cultivation of plants has St. Louis, MO, USA). The pH of the medium was various disadvantages (low yields and fluctuations adjusted to 5.8 with 1N HCl or NaOH, and autoclaved in concentrations due to geographical, seasonal at 121°C and 1.15 kg cm−2 for 20 min. Explants were and environmental variations) (Murthy et al., 2014). incubated at 25 ± 2°C under a 16 h photoperiod at Therefore, a tissue culture technique has emerged a light intensity of 35 μmol m−2s−1 provided by 40 W as an attractive alternative for the propagation of cool-white fluorescent tubes. homogeneous plants of this species of Brazilian After 30 days in culture, the regenerated ginseng as well as for the in vitro production of shoots were excised and cut into single nodal secondary metabolites. segments and grown in the same medium described Studies have previously reported the in for in vitro establishment, as described by Flores vitro clonal propagation of plants using thidiazuron et al. (2010). These plants served as the source of (TDZ) (Flores et al., 2010) and callus culture using explants for callus induction. 6-benzylaminopurine (BAP) plus α-naphthalene Callus induction and organogenesis

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Nodal segments (n = 600; each 10 mm-long equipped with a UV-VIS detector and a Perkin-Elmer and two buds) were excised and grown horizontally in Nova-Pak RP-18 column (5 μm particle size; 220 MS medium supplemented with various combinations mm × 4.6 mm) protected by a pre-column Nova-Pak of 2,4-Dichlorophenoxyacetic acid (2,4-D; 1.0, 5.0, RP-18. The mobile phase consisted of HPLC-grade 10.0, or 20.0 µM) and 6-benzylaminopurine (BAP; 100% (v/v) methanol, degassed in an ultrasonic bath, 0, 1.0, or 10.0 µM), according to Flores et al. (2006). at a flow rate of 1 ml min-1. A 20 μl sample of each The pH of all media was adjusted to 5.8 using 1.0 N methanol extract was injected and the absorbance NaOH or HCl, followed by autoclaving at 121°C at of the eluate was monitored at 245 nm. 1.15 kg cm-2 for 20 min. For quantification, solutions containing Cultures were maintained at 25 ± 2°C 5, 10, 15, 20 or 25 µg ml-1 authentic β-ecdysone and for 10 d in continuous darkness. They were (ChromaDex Inc., Irvine, CA, USA) were prepared in then placed and kept in a growth room with a 16 h 100% (v/v) methanol (HPLC-grade), and a standard photoperiod at 35 µmol photons m-2 s-1. curve was generated by linear regression. The At 35 d after culture initiation, the numbers experimental results represent the average of three of explants forming calli were scored. Callus injections of each concentration of β-ecdysone. induction percentages were calculated according β-Ecdysone was identified in tissue samples to the formula: (number of explants with calli/total by comparing its UV spectra and retention times with number of explants) × 100. Different callus types those of the authentic standards, by chromatography were categorized according to consistency (i.e., of the samples with authentic β-ecdysone, and by compact or soft), color (i.e., white translucent or thin layer chromatography (TLC; Flores et al., 2010). white opaque) and organogenic response (i.e., calli with shoots and/or roots or non-organogenic). The Statistical analysis organogenic responses were evaluated at 35 and The experiment was arranged in a 55 d after culture initiation. Callus type percentages completely randomised design. Each treatment was were calculated according to the following formula: repeated five times and each replicate consisted of (number of calli in each callus category/total number ten explants. β-Ecdysone concentrations [in % of dry of calli) × 100. mass (w/w)] were expressed in 100 g-1 of triturated raw plant material. Statistical analyses were carried Measurement of β -ecdysone out using ANOVA, followed by the Duncan’s multiple concentrations range test. A significance level of P ≤ 0.01 was Organogenic and non-organogenic calli used for all statistical analysis. Data expressed induced in the different callus induction media as percentages were arcsine transformed before were used for quantification of β-ecdysone by performing ANOVA. high performance liquid chromatography (HPLC). The β-ecdysone concentration in these calli was compared with field-grown plants of P. tuberosa (n RESULTS = 20). Callus induction and organogenesis The extraction procedures and the HPLC In all callus induction media, the proliferation conditions used for β-ecdysone detection were of callus was observed after the first week of culture, optimized after preliminary assays using extracts especially in apical and basal ends of the nodal of P. tuberosa (Flores et al., 2009). All samples, segments. A higher callus proliferation was observed including calli (whole calli) and field-grown plants in medium containing only 2,4-D when compared (aerial parts plus roots analysed separately), were with the calli induced in medium with 2,4-D plus BAP dried in a circulating air oven at 40°C. The dried (data not shown). samples (200 mg of each) were then ground with The concentration and, especially, the a pestle and mortar and extracted twice with 5 ml type of PGR affected the pattern of regeneration of 100% (v/v) MeOH in an ultrasonic bath (LS21D; and both direct organogenesis (i.e., shoot and root LimpSonic, São Paulo, SP, Brazil) at 55°C for 20 min. regeneration from buds present in the explant) and Each supernatant was collected after centrifugation indirect organogenesis (i.e., adventitious shoot and at 1,000 x g for 10 min at 25°C. The sequential root, regenerated from callus tissues) were observed methanol extracts were combined (giving 10 ml (Table 1). per sample), filtered through a 0.45 μm membrane The development of axillary shoots from (Millipore, Bedford, MA USA), and injected into the buds present in the explants was observed after HPLC apparatus. the first week of in vitro culture only in media Quantitative analysis of β-ecdysone was supplemented with 2,4-D (Table 1). However, there carried out at room temperature in a Perkin-Elmer was a reduction in the regeneration percentage HPLC (Model S 200; P-E, Boston, MA, USA) and in the number and length of axillary shoots with

Rev. Bras. Pl. Med., São Paulo, v.19, n.2, p.266-273, 2017. 269 increasing concentrations of 2,4-D (Table 1; Figure calli grown in media with 2,4-D (1.0 at 10.0 µM). 1A and 1B). Additionally, axillary shoot regeneration High levels of 2,4-D (20.0 µM) and the use of BAP with abnormal leaf morphology occurred with a (all concentrations) inhibited the accumulation of frequency of 4% in medium supplemented with 5.0 β-ecdysone in the calli (Table 1). In the medium µM of 2,4-D (data not shown). On the other hand, containing 2,4-D alone, 100% of the calli showed BAP completely inhibited the formation of axillary soft consistency and white translucent color. On shoots from explants (Table 1). the other hand, the addition of BAP in the medium In calli without axillary shoots, a low favored (100%) the formation of calli with both soft frequency of indirect shoot regeneration was and compact white opaque areas. observed. The culture medium with 2,4-D plus BAP The aerial part of the field-plants showed (both at a concentration of 1.0 µM) provided the fifteen times higher content ofβ -ecdysone (0.333%) greatest indirect shoot regeneration (average 20.0% when compared with the roots (0.022%) (Table 1). of organogenic calli) (Table 1, Figure 1C). However, A higher amount of β-ecdysone was observed in in calli cultivated in media supplemented with both the calli grown with 10.0 µM 2,4-D (0.516%) and its BAP and 2,4-D, a longer culture time was required content was almost twice that found in the shoots for shoot regeneration (55 days) when compared (0.333%) from field-grown plants. The calli grown with treatments containing only 2,4-D (35 days). in medium with 1.0 to 5.0 µM 2,4-D showed lower In general, each callus formed an average of 1.5 β-ecdysone concentrations than those found in the adventitious shoots (3.0 mm-long) (Figure 1C). shoot (0.333%), but higher than detected in the roots Non-organogenic calli were observed in medium from field-grown plants (0.022%) (Table 1). containing 10.0 µM BAP (Table 1). In general, BAP also inhibited the regeneration of roots from calli, with the best results obtained in medium containing DISCUSSION low concentrations of 2,4-D (Table 1). In the present study, friable calli with different organogenic responses were produced Characteristics of callus and β-ecdysone from nodal segments of P. tuberosa by supplying concentration different combinations of 2,4-D and BAP. Increased β-Ecdysone was detected only in the proliferation of calli was observed in medium with

TABLE 1. Effect of BAP and 2,4-D concentrations on the direct and indirect organogenesis and β-ecdysone concentration [% (w/w)] from calli and field-grown plants of Pfaffia tuberosa. GR (µM)* Direct organogenesis Indirect organogenesis β-ecdysone Number of axil- Axillary Axillary Shoots Roots # BAP 2,4-D ** lary shoots per shoots [% (w/w) DW] shoots (%) ** ** explant** length (cm) ** (%) (%) 1.0 75.0a 1.2a 3.8a 8.0b 65.5a 0.075 ± 0.001d 5.0 58.4b 0.9b 1.9b 12.0ab 70.3a 0.164 ± 0.001c 0 10.0 18.8c 0.3c 0.7c 12.0ab 34.5b 0.516 ± 0.001a 20.0 4.0d 0.1c 0.1d 12.0ab 10.0c n.d. 1.0 0.0d 0.0c 0.0d 20.0a 3.0d n.d. 5.0 0.0d 0.0c 0.0d 12.0ab 0.0d n.d. 1.0 10.0 0.0d 0.0c 0.0d 8.0b 0.0d n.d. 20.0 0.0d 0.0c 0.0d 4.0bc 0.0d n.d. 1.0 0.0d 0.0c 0.0d 0.0c 0.0d n.d. 5.0 0.0d 0.0c 0.0d 0.0c 0.0d n.d. 10.0 10.0 0.0d 0.0c 0.0d 0.0c 0.0d n.d. 20.0 0.0d 0.0c 0.0d 0.0c 0.0d n.d. 0.333 ± Aerial parts 0.002b Field-grown plants 0.022 ± Roots 0.001e *GR: growth regulators (BAP, 6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic). **Values are the means of five replicates of 10 explants for each treatment (n = 50). #Data are reported as means ± standard error of three replicates HPLC measurement (n = 15). Mean values followed by the same lower-case letters in each column are not significantly different by Duncan’s multiple range test atP ≤ 0.01. n.d.: not detected.

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2,4-D alone, whereas there was a reduction in the growth of those grown in the presence of BAP. Both 2,4-D and BAP are growth regulators often used to initiate calli from explants, but contrary to findings in P. tuberosa, BAP has been shown to be effective in stimulating callus proliferation in a wide range of species (Beyl, 2011). Several studies have shown cytokinins to be an important regulatory agent of plant meristem activity and organogenesis, with opposing roles in shoots and roots (Chang et al., 2013). For example, in most species, cytokinins have a positive role in the proliferation of shoots (Beyl, 2011), whereas it tends to inhibit root formation and elongation (Werner et al., 2010; Beyl, 2011). However, in this study with P. tuberosa, the addition of BAP to the medium reduced the regeneration of adventitious shoots and completely inhibited the growth of axillary shoots. These effects of BAP were observed in a preliminary assay using P. tuberosa (Flores et al., 2006; Flores & Nicoloso, 2007). In other Amaranthaceae species, such as P. glomerata (Flores et al., 2015), Alternanthera philoxeroides (Gao et al., 2011) and Gomphrena officinalis (Mercier et al., 1992), BAP is one of the cytokinins most commonly used in in vitro propagation. These conflicting results confirm that the organogenic ability is influenced by genotype sensitivity to hormones and suggest that P. tuberosa buds are not responsive to adenine derivatives, such as BAP. However, this species was propagated efficiently with thidiazuron (TDZ: N-phenyl-N0 (1,2,3-thidiazol-5-yl)urea) (Flores et al., 2010). Several studies have shown that hormones with different structures exert different physiological effects, due to variations in affinity to their receptors. According to Beyl (2011), this may explain why, in some cases, cytokinin-binding receptors have better affinity for the phenylurea-type (as TDZ) than for adenine-type (as BAP). Depending on the hormonal combinations used, there were two regeneration pathways observed (i.e., direct and indirect organogenesis). Depending on the purpose of a particular application, both regeneration pathways are important. For example, the regeneration by direct organogenesis FIGURE 1. Callus induction and shoot regeneration is an alternative for large-scale cloning of selected from Pfaffia tuberosa (Spreng.) Hicken. (A) Friable genotypes (Flores et al., 2010), whereas indirect callus and shoot axillary originated from nodal organogenesis is advantageous for expanding segments cultivated on MS medium supplemented genetic variability (Bairu et al., 2011). with 10.0 µM 2,4-D, after 35 days of in vitro culture. In relation to direct organogenesis, an (B) Friable callus and shoot axillary cultivated on efficient formation of axillary shoots (75%) was medium with 1.0 µM 2,4-D, after 35 days of culture. obtained in medium with a low level of 2,4-D (1.0 (C) Shoot adventitious regenerated from friable µM). However, increasing concentrations of 2,4-D callus on medium with 1.0 µM 2,4-D plus 1.0 µM BAP, after 55 days of in vitro culture. Bars: 5 mm (A) led to decreased shoot regeneration percentage, (B); 2 mm (C). BAP: 6-benzylaminopurine; 2,4-D: number and length of regenerated shoots and 2,4-dichlorophenoxyacetic. root regeneration. These responses are possibly

Rev. Bras. Pl. Med., São Paulo, v.19, n.2, p.266-273, 2017. 271 associated with the genic expression changes in (Oluk et al., 2010). cells caused by the presence of high concentrations Organ-specific accumulation of of a strong auxin like 2,4-D (Beyl, 2011); thereby, the β-ecdysone found in previous studies in P. regeneration of axillary shoots in the presence of 2,4- glomerata (Flores et al., 2010; Serra et al., 2012) D does not rule out the possibility of genetic and/or and P. tuberosa (Flores et al., 2010) corroborated epigenetic changes. In this study with P. tuberosa, the results obtained in this study where greater shoots with malformed leaves were observed in the accumulation of this metabolite was observed presence of 5.0 µM 2,4-D. Although there was a in the aerial part of the plant than in the roots. small occurrence of these morphological changes, Moreover, the accumulation of β-ecdysone in this result shows that axillary shoots formed in the calli was associated with the presence of medium with 2,4-D should not be used for clonal axillary shoots and tended to increase with higher propagation in this species. concentrations of 2,4-D in the medium. The highest Adventitious shoots occurred both in medium β-ecdysone content observed in calli was higher with 2,4-D alone (all concentrations) or with 2,4-D (all than that found in the shoots from field-grown concentrations) in combination with low levels of BAP plants. According to Flores et al. (2015), high (1.0 µM). On the other hand, the medium with 10.0 levels of ecdysteroids in the shoots may mean a µM BAP inhibited adventitious shoot regeneration. protective role against insect predation. Interestingly, the need for further growth time for In the present study, despite the the regeneration of adventitious shoots in the regeneration of adventitious shoots from calli combination of 2,4-D plus BAP (1.0 µM of each) grown in medium with 20.0 µM 2,4-D or 2,4-D (all was offset by a higher regeneration frequency (20% concentrations) plus 1.0 µM BAP, β-ecdysone of regenerative calli) when compared to the results was not detected, which may be due to the small obtained with 2,4-D alone (11% regenerative calli). number and size of the shoots (average of 1.5 The different PGRs used in tissue cultures have shoots per callus with 3.0 mm length) (Figure 1C). unique effects, and yet they are capable of working Thus, the quantity of adventitious shoots may not together synergistically or antagonistically to exert have been sufficient forβ -ecdysone accumulation different responses in plants. This may explain within the detection limit used in this study. why only low concentrations of both hormones Shoots from field-plants showed the favored indirect regeneration. In other plants highest β-ecdysone content of 0.333% (w/w), but of the Amaranthaceae, such as Alternanthera this content can be in vitro optimized using 2,4-D. phyloxeroides (Gao et al., 2011), BAP was the Concentrations of β-ecdysone from in vitro-calli best cytokinin for indirect shoot organogenesis. increased from 0.075% to 0.516% in the presence In P. tuberosa 2,4-D was found to be of 1.0 µM and 10.0 µM of 2,4-D, respectively. suitable for the proliferation of soft calli as well Therefore, this study demonstrated that this as for the rapid induction of shoots and/or roots. synthetic auxin appears be an effective stimulator On the other hand, the use of BAP together of β-ecdysone production in P. tuberosa callus with 2,4-D is indicated only for regeneration of cultures. Calli generally require an exogenous shoots via indirect organogenesis, due to the supply of growth regulators for growth and low cell proliferation observed in the presence of metabolite accumulation (Murthy et al., 2014). In BAP. Similar results were earlier obtained by our P. glomerata, the effect of 2,4-D on β-ecdysone research group for calli of P. tuberosa (Flores et content was associated with a high cellular al., 2006). proliferation rate (Flores et al., 2015). Adventitious shoots formed after culturing In this study, BAP was not effective with 2,4-D or 2,4-D plus BAP were isolated from for the production of organogenic calli able to the calli and grown in MS-free PGR medium. accumulate β-ecdysone. Contrary to these results, These shoots developed into complete plants (i.e., this cytokinin has been beneficial for both shoot with aerial parts and roots), demonstrating that proliferation and accumulation of secondary this methodology may be useful for the production metabolites in P. glomerata (Flores et al., 2015). of novel variants in P. tuberosa. Among factors In studies investigating the relationship between that induced in vitro variation, the callus culture plant growth regulators (including BAP) and organ and, especially the use of 2,4-D, are probably the differentiation with β-ecdysone concentrations most important determinants of genetic variation (Flores et al., 2015), increased BAP concentrations (Bairu et al., 2011; Beyl, 2011). Our results clearly improved β-ecdysone production in calli of P. indicate the possibility of somaclonal variation, glomerata, probably due to increased shoot which would provide the opportunity to select proliferation. plants with desirable features, i.e., increased In P. tuberosa, β-ecdysone concentrations secondary metabolite content in medicinal plants were also associated with the presence of aerial

Rev. Bras. Pl. Med., São Paulo, v.19, n.2, p.266-273, 2017. 272 parts, mainly axillary shoots; however, BAP should DANIEL, J.F.S. et al. Free radical scavenging not be used to produce β-ecdysone because activity of Pfaffia glomerata (Spreng.) Pedersen it inhibits the proliferation of axillary shoots in (Amaranthaceae). Indian Journal of Pharmacology, this species. According to Murthy et al. (2014), v.37, n.3, p.174-178, 2005. DINAN, L. et al. Phytoecdysteroids: diversity, PGRs are often a crucial factor in secondary biosynthesis and distribution. In: SMAGGBE, G. (Ed.). product accumulation, and auxins and cytokinins Ecdysone: Structures and Functions. London: have different effects (i.e., stimulate or inhibit) Springer, 2009. p.3-10. depending on the type of metabolite and genotype FLORES, R.; NICOLOSO, F.T. Effect of NAA and BAP concerned. on calogenesis and organogenesis of Pfaffia tuberosa In P. tuberosa, the β-ecdysone (Spreng.) Hicken. 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