Generación de cultivos de raíces transformadas de la planta medicinal Bidens odorata Cav (Compositae) y análisis fitoquímico preliminar

Abstract: An in vitro propagation protocol for Bidens odorata Cav. was developed by culturing nodal stem segments on Murashige and Skoog medium added with cytokinins. The best results were 5.5 ± 0.4 shoots per explant with benzyladenine, and 4.9 ± 0.3 with N6-(2-isopentenyl)-adenine. Their susceptibility to genetic transformation with three strains of Agrobacterium rhizogenes was also evaluated. The best was the A4/pESC4 strain, at a bacterial density of 107 CFU mL-1, which generated stable and active growth transformed roots cultures with a biomass accumulation of 4.8 times the initial inoculum at the third week of culture. The verification of the transformation was made histochemically by the GUS test, being positive from 7 to 82% of the evaluated roots; As well as molecularly, by amplifying the fragments corresponding to the rolB, gus and nptII genes. Phytochemical screening performed by thin layer chromatography revealed the ability of transformed roots to synthesize compounds of various types, suggesting the presence of alkaloids, anthracene derivatives, essential oils, flavonoids, saponins, steroids and triterpenes. These are present in the aerial part or roots of B. odorata, so that these results showed that the transformed roots may be a viable option for obtaining the compounds of pharmacological interest of this species.Resumen: Se desarrolló un protocolo de propagación in vitro para Bidens odorata Cav. mediante el cultivo de segmentos nodales en medio de Murashige y Skoog adicionado con citocininas. Los mejores resultados fueron 5.5 ± 0.4 brotes por explante con benciladenina y 4.9 ± 0.3 con N6-(2-isopentenil)-adenina. También se evaluó su susceptibilidad a la transformación genética con tres cepas de Agrobacterium rhizogenes. La mejor cepa fue la A4/pESC4, a una densidad bacteriana de 107 UFC mL-1, que generó cultivos de raíces transformadas estables y de crecimiento activo, con una acumulación de biomasa de 4.8 veces el inóculo inicial a la tercera semana de cultivo. La verificación de la transformación se hizo histoquímicamente mediante la prueba de GUS, siendo positiva desde un 7 hasta un 82% de las raíces evaluadas; así como molecularmente, mediante la amplificación de los fragmentos correspondientes a los genes rolB, gus y nptII. El escrutinio fitoquímico realizado mediante cromatografía en capa fina reveló la capacidad que tienen las raíces transformadas para sintetizar compuestos de diversos tipos, sugiriendo la presencia de alcaloides, derivados de antraceno, aceites esenciales, flavonoides, saponinas, esteroides y triterpenos. Estos metabolitos secundarios están presentes en la parte aérea o en raíces de plantas de B. odorata, por lo que los resultados demostraron que las raíces transformadas pueden ser una opción viable para la obtención de los compuestos de interés farmacológico propios de esta especie

Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments Transformação genética em Citrus sinensis e Citrus limonia mediada por Agrobacterium tumefaciens a partir de segmentos de epicótilo

Genetic transformation allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. The objective of this research was to establish a protocol for genetic transformation of Valencia and Natal sweet oranges (Citrus sinensis L. Osbeck) and Rangpur lime (Citrus limonia L. Osbeck). Epicotyl segments of germinated in vitro plantlets (three weeks in darkness and two weeks in a 16-h photoperiod) were used as explants. These were co-cultivated with Agrobacterium tumefaciens strain EHA-105 and different experiments were done to evaluate the transformation efficiency: explants were co-cultivated with Agrobacterium for one, three or five days; explants were incubated with Agrobacterium suspension for 5, 10, 20 or 40 minutes; co-cultivation medium was supplemented with acetosyringone at 0, 100 or 200 &micro;mol L-1; Explants ends had a longitudinal terminal incision (2-3 mm); co-cultivation temperatures of 19, 23 or 27&deg;C were imposed. The experimental design was completely randomized in all experiments with five replications, each consisted of a Petri dish (100 x 15 mm) with 30 explants and resulted in a total of 150 explants per treatment. Longitudinal terminal incision in the explant ends did not improve shoot regeneration. However, transgenic plants of all three cultivars were confirmed from explants that had been subjected to inoculation time of 20 minutes, co-culture of three days at 23-27&deg;C, in the absence of acetosyringone.<br>A transformação genética permite produzir cultivares com características específicas e pode, dessa forma ser associada a programas de melhoramento de citros. O objetivo deste trabalho foi estabelecer protocolos de transformação genética para as laranjas doce 'Valência' e 'Natal' (Citrus sinensis L. Osbeck), bem como para o limão 'Cravo'(Citrus limonia L. Osbeck). Segmentos de epicótilo de plântulas germinadas in vitro (três semanas no escuro e duas semanas sob fotoperíodo de 16h) foram utilizados como explantes. Estes foram co-cultivados com Agrobacterium tumefaciens (EHA-105), realizando-se vários experimentos para avaliar a eficiência do processo de transformação genética: explantes co-cultivados por um, três e cinco dias; tempo de inoculação com a bactéria de 5, 10, 20 e 40 minutos; co-cultivo em meio de cultura contendo 0, 100 e 200 mimol L-1 de acetoseringona; Incisão longitudinal (2-3 mm) nas extremidades do explante; temperatura de co-cultivo 19, 23 e 27&deg;C. Todos os experimentos consistiram de cinco repetições por tratamento, sendo cada repetição representada por uma placa de Petri contendo 30 explantes, perfazendo um total de 150 explantes por tratamento. Plântulas transgênicas dos três cultivares foram obtidas utilizando-se tempo de inoculação de 20 minutos, co-cultivo com Agrobacterium tumefaciens (EHA-105) por três dias, na ausência de acetoseringona no meio de cultura de co-cultivo e temperatura de co-cultivo de 23-27&deg;C. A incisão longitudinal na extremidade do explante favoreceu à organogênese in vitro, mas quando co-cultivado com Agrobacterium não houve regeneração de brotações