ABSTRACT
Tubérculos del género Dioscorea comercializados con fines medicinales, fueron recolectados con el propósito de lograr su establecimiento a condiciones in vitro. Previamente se lograron identificar taxonómicamente las especies y por medio de análisis fitoquímicos demostrar su potencial farmacéutico. El material recolectado fue identificado como Dioscorea coriacea, D. lehmannii, D. meridensis, D. polygonoides y una especie comestible D. trifida. Tubérculos recolectados de centros de acopio y traídos de campo fueron lavados, desinfectados, asperjados con Ácido Giberélico (AG3) y sembrados en sustrato BM-2®, en invernadero a 18°C día y 10°C noche. Los tubérculos completos o por secciones fueron almacenados en bolsas herméticas a temperatura ambiente. Posteriormente se desinfectó material vegetal de las especies D. coriacea, D. lehmannii, D. meridensis y D polygonoides, seleccionando explantes de brotes sanos (D. coriacea / laboratorio) para su establecimiento. Se evaluaron tres medios de cultivo para establecimiento, el que presentó los mejores resultados fue Medio Murashige & Skoog (1962) suplementado con BAP 1 mL/L, AG3 1 mL/L y Putrescina 2 mL/L. Para la extracción y análisis de metabolitos secundarios se utilizaron tubérculos de D. coriacea, D. lehmannii y D. polygonoides, empleando como solvente de extracción metanol. Se encontró mayor concentración de extracto vegetal en D. coriacea (54%), y mediante cromatografía en capa delgada (CCD), se confirmó la presencia de saponinas, que resultó mayor en comparación con D. polygonoides especie reconocida por su alto contenido de saponinas. Estos resultados permitirán realizar análisis más avanzados de los compuestos presentes y plantear su propagación masiva en condiciones in vitro.
Wild tubers of the genus Dioscorea sold for medicinal use were collected for the purpose of achieving its establishment under in vitro conditions. First we taxonomically identified the species and through phytochemical analysis demonstrated pharmaceutical potential. The material collected was identified as Dioscorea coriacea, D. lehmannii, D. meridensis, D. polygonoides and the edible species D. trifida. Tubers collected from wholesale distributors and from the field were washed, disinfected, sprayed with Gibberellic Acid (GA3) and planted in substrate BM-2®, in a greenhouse at 18 ° C during the day and 10 ° C overnight. Whole tubers or sections thereof were stored in sealed bags at room temperature. Subsequently plant material of the species D. coriacea, D. lehmannii, D. meridensis and D. polygonoides was disinfected and healthy buds (D. coriacea / laboratory) were selected for in vitro establishment. Three different culture media were evaluated for establishment; that which presented the best results was the Murashige & Skoog (1962) medium, supplemented with BAP 1 mL / L, GA3 1 mL / L and Putrescin 2 mL / L. For the collection and analysis of secondary metabolites, tubers of D. coriacea, D. lehmannii and D. polygonoides were used, using methanol as the extraction solvent. The highest concentration of plant extract, 54%, was found in D. coriacea, a higher value than that of D. polygonoides, which had been reported previously; the presence of saponins was confirmed by thin layer chromatography (TLC). These results will enable more advanced analysis of the present compounds and enhance their mass propagation under in vitro conditions.
ABSTRACT
Dioscorea multiflora uma planta nativa do Sul do Brasil produz a diosgenina como metabólito secundário majoritário, uma substância potencialmente usada pela indústria farmacêutica para a produção de cortisona e substâncias com ação contraceptiva. Objetivou-se, neste trabalho otimizar o protocolo de micropropagação de D. multiflora, visando a produção de mudas em escala comercial. Segmentos nodais subcultivados em meio MS sólido foram transferidos para multiplicação em meio MS suplementado com BAP (0,01; 0,1; 0,5; 1,0 e 3,0 mg L-1)e meio MS suplementado com 0,1 mg L-1 ou 0,5 mg L-1 de BAP acrescido de diferentes concentrações de sacarose (2, 4, 6, 8 e 10 por cento). Para o enraizamento, as brotações foram cultivadas em meio MS suplementado com AIB (0,1; 0,5; 1,0 e 3,0 mg L-1) e meio MS suplementado com ANA (0,1; 0,5; 1,0 e 3,0 mg L-1). Os experimentos in vitro foram instalados em delineamento experimental inteiramente casualizado e cada tratamento constituiu-se de 3 repetições e 10 cubetas/parcela. Plântulas com e sem raízes foram aclimatizadas em casa de vegetação. Melhores resultados de multiplicação e enraizamento foram obtidos em meio MS + 0,1 mg L-1 de BAP (80 por cento) e em meio MS + 1,0 mg L-1 de AIB (42,6 por cento), respectivamente. Não houve diferença quanto à porcentagem de sobrevivência das plântulas in vitro e ex vitro durante a aclimatização (75 por cento). O protocolo de micropropagação para D. Multiflora é efetivo e pode ser usado para a produção em escala comercial.
Dioscorea multiflora is a plant native to southern Brazil that produces diosgenin as a major secondary metabolite, a substance which is used by the pharmaceutical industry for the production of cortisone and substances with contraceptive action. The objective of this work was to optimize the micropropagation protocol of D. multiflora, for the production of seedlings on a commercial scale. Nodal segments subcultured in solid MS medium were transferred for multiplication to MS medium supplemented with BAP (0.01, 0.1, 0.5, 1.0 and 3.0 mg L-1) and MS medium supplemented with 0.1 mg L-1 or 0.5 mg L-1 BAP plus different concentrations of sucrose (2, 4, 6, 8 and 10 percent). For rooting, the shoots were cultured on MS medium supplemented with IBA (0.1, 0.5, 1.0 and 3.0 mg L-1) and MS medium supplemented with NAA (0.1, 0.5, 1.0 and 3.0 mg L-1). A completely randomized design was used with treatment consisting of 3 replicates with 10 buckets per plot. Seedlings with and without roots were acclimatized in a greenhouse. The best results of multiplication and rooting were obtained in MS medium + 0.1 mg L-1 BAP (80 percent) and in MS medium + 1.0 mg L-1 IBA (42.6 percent), respectively. There was no difference in the survival percentage of seedlings in vitro and during ex vitro acclimatization (75 percent). The micropropagation protocol for production of D. multiflora is effective and can be used for commercial production.