In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)

Abstract

RESUMO -(Morfogênese in vitro e estabelecimento de culturas de suspensão celular em Piper solmsianum C. DC. (Piperaceae)). Piper solmsianum é uma espécie herbácea do sudeste brasileiro onde vários compostos biologicamente ativos já foram identificados. O objetivo deste trabalho foi estabelecer suspensões celulares nesta espécie. Para tanto, foram utilizados explantes de pecíolos e folhas, retirados de plântulas cultivadas in vitro, os quais foram submetidos a diferentes combinações de reguladores de crescimento (AIA, ANA, 2,4-D e BAP). Foi obtida a neo-formação de raízes e brotos, estes últimos através do processo de organogênese indireta evidenciada por estudos histológicos. Para a indução e crescimento dos calos, foram avaliados, além das diferentes combinações de reguladores de crescimento, a suplementação ao meio de cultura de carvão ativado (1,5 mg.l -1 ) e o regime de luz. Culturas mantidas na luz, em meio de cultura suplementado com 0,2 mg.l -1 2,4-D e 2 mg.l -1 BAP e sem carvão ativado, resultaram em maior crescimento (massa fresca) dos calos. A partir destes calos, foram obtidas suspensões celulares, cuja dinâmica de crescimento e acúmulo de metabólitos foi estudado. Os resultados obtidos deverão ser utilizados para a caracterização de rotas biosintéticas em culturas in vitro em P. solmsianum. Palavras-chave: calogênese, morfogênese, pariparoba, reguladores de crescimento, suspensões celulares ABSTRACT -(In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae)).Piper solmsianum is a shrub from Southeast Brazil in which many biologically active compounds were identified. The aim of this work was to establish a cell suspension culture system for this species. With this in mind, petiole and leaf explants obtained from in vitro plantlets were cultured in the presence of different plant growth regulator combinations (IAA, NAA, 2,4-D and BA). Root and indirect shoot adventitious formation, detected by histological analysis, was observed. Besides the different combinations of plant growth regulators, light regime and the supplement of activated charcoal (1.5 mg.l -1 ) were tested for callus induction and growth. Cultures maintained in light, on a 0.2 mg.l -1 2,4-D and 2 mg.l -1 BA supplemented medium, and in the absence of activated charcoal, showed the highest calli fresh matter increment. From a callus culture, cell suspension cultures were established and their growth and metabolite accumulation studied. The achieved results may be useful for further characterization of the activated secondary metabolites pathways in in vitro systems of P. solmsianum.

Induction and growth pattern of callus from Piper permucronatum leaves

“…Leaf explants were individually transferred with the adaxial surface up, to test tubes (25 mm x 150 mm) containing 10 mL of an MS (Murashige & Skoog, 1962) basal culture medium supplemented with 3% (w/v) sucrose, 0.6% (w/v) agar and factorial combinations of 2,4-Dichlorophenoxyacetic acid (2,4-D) (0, 4.52, 9.05, or 18.10 µM) and 6-Benzylaminopurine (BA) (0, 4.44, 8.88, or 17.76 (Table 1) each one with three replicates of three explants. The growth regulators, their concentrations and combinations tested were based on successful studies on callus induction in Piper species; P. cernuum, P. crassinervum (Danelutte et al, 2005), P. solmsianum (Balbuena et al, 2009), P. aduncum (Delgado-Paredes et al, 2013), and P. hispidinervum (Santiago, 2013). The pH of the medium was adjusted to 5.8 before the addition of agar followed by autoclaving at 121°C for 20 minutes.…”

“…Valle (2003), studying the callus growth of the same species observed the deceleration phase starting on the 40 th day. Balbuena et al (2009) used callus cells of P. solmsianum on the 24 th day of culture to initiate cell suspension cultures. The appropriate time to subculture the calluses of P. permucronatum in order to establish cell suspension cultures is in the beginning of the deceleration phase, on the 56 th day of culture, when the cells have to be transferred to a new culture medium due to the reduction of nutrients, agar dryness and accumulation of toxic substances.…”

“…Several species of the Piper genus are native to the Amazon Rainforest and are notable producers of chemical compounds which have several biological effects in insects, fungi, bacteria, Trypanossoma cruzi (Navickiene et al, 2003;Dyer et al, 2004;Danelutte et al, 2005;Balbuena et al, 2009) and can also affect human health, as analgesic, anti-depressant, cytoprotective, anti-ulcer, anticonvulsant, anti-inflammatory, and antioxidant agents (Ahmad et al, 2010).…”

Antinociceptive properties of conocarpan and orientin obtained from Piper solmsianum C. DC. var. solmsianum (Piperaceae)

“…Only a few chemical or biological aspects of this plant have been investigated. Phytochemical studies of this species indicate the presence of aliphatic hydrocarbons, monoterpenes, sesquiterpenes, arylpropanoids and neolignans [9][10][11][12]. Moreira and coworkers [9,11] demonstrated that the essential oil from this plant has promising depressant effects.…”

Cultivo de callos <i>in vitro</i> a partir de las hojas de <i>Jodina rhombifolia</i> Hook. & Arn. (Reissek) para obtención de C-glicosilflavonas

“…Las ventajas de emplear biotécnicas en la producción de plantas de uso medicinal, respecto a la producción por agricultura tradicional son (Karuppusamy, S., 2009;Al-Sane, K. O. et al, 2005;Balbuena, T. S. et al, 2009):…”