266 Direct and indirect regeneration from Pfaffia 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 plant 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 plants. 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, Amaranthaceae. 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 Rev. Bras. Pl. Med., São Paulo, v.19, n.2, p.266-273, 2017. 267 INTRODUCTION acetic acid (NAA) or 2,4-Dichlorophenoxyacetic In Brazil, 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 Rev. Bras. Pl. Med., São Paulo, v.19, n.2, p.266-273, 2017. 268 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.