ABSTRACT
Using the White as basic medium, the effects of the exogenous IBA and endophytic fungal elicitor on the growth of in vitro roots cultures of Dysosma versipellis and production of podophyllotoxin were investigated in this study. The results showed that the IBA and the endophytic fungus Zasmidium syzygii elicitor could increase the content of podophyllotoxin of in vitro roots of D. versipellis after 3 weeks. The White medium added with 3 mg·L~(-1) IBA induced the highest increase of podophyllotoxin(1 830.86 μg·g~(-1)), which was 2.07 folds greater than the control, and followed by 1.5 mg·L~(-1) IBA, fungal elicitor, 1 mg·L~(-1) IBA, 0.5 mg·L~(-1) IBA and 4.5 mg·L~(-1) IBA, which was 1.82, 1.71, 1.63, 1.43 and 1.1 folds greater than the control, respectively. The results also showed that the growth of roots was certain positively correlated with the change of IBA concentration. Therefore, 3 mg·L~(-1) IBA was the most suitable for the production of podophyllotoxin in the in vitro roots of D. versipellis, and the stimulating effect of Z. syzygii fungal elicitor was between 1.5 mg·L~(-1) and 1 mg·L~(-1) IBA, which was a potential natural elicitor to induce the accumulation of podophyllotoxin in future production.
Subject(s)
Ascomycota , Berberidaceae , Chemistry , Endophytes , Plant Roots , Podophyllotoxin , Tissue Culture TechniquesABSTRACT
Methodologies were developed for the establishment and cultivation of Artemisia annua L (CPQBA 2/39 x PL5 hybrid) roots submitted to light conditions and genetic transformation performed with Agrobacterium rhizogenes (15834 and 8196 strains). The transgenic and non-transgenic (normal) roots were cultured in Murashige and Skoog (1962) medium, kept under different photoperiodic conditions and analyzed for evaluation of the antiulcerogenic dihydro-epideoxyarteannuin B (compound A) contents. The Dot Blot technique was used to confirm the transgenic nature of the roots. The plants¢s crude extracts were analyzed by Gas Chromatography coupled to Mass Spectrum (CG/MS). The chromatograms of the extracts taken from normal roots revealed the presence of dihydro-epideoxyarteannuin B and other compound (compound B). Photoperiods during cultivation influenced the production of these two compounds: under continuous darkness dihydro-epideoxyarteannuin B was intensely produced and the compound B present in small amounts, while on 16 h photoperiod, the inverse occurred. The quantification of dihydro-epideoxyarteannuin B by Gas Chromatography coupled to Flame Detector Ionization (CG/FID) revealed an approximately fivefold increase in the production of this compound by normal roots kept under continuous darkness compared to roots kept under 16 h light period. The terpene dihydro-epideoxiarteannuin B was not present in transgenic hairy roots.
Foram desenvolvidas metodologias para o estabelecimento e cultivo de raízes de Artemisia annua L. (híbrido CPQBA 2/39 x PL5). Estas raízes foram submetidas a diferentes condições de luz e a transformação genética com Agrobacterium rhizogenes (cepas 8196 e 15834). As raízes transgênicas e não-transgênicas (normais) foram cultivadas em meios de Murashige e Skoog (1962), mantidas sobre diferentes condições de fotoperíodo e analisadas para avaliação do conteúdo do composto antiulcerogênico dehidro-epideoxiarteanuína B (composto A). A confirmação do caráter transgênico das raízes foi obtida por Dot Blot. Os extratos dos materiais vegetais foram analisados por Cromatografia Gasosa acoplada a um Espectômetro de Massas (CG/EM). Os cromatogramas dos extratos das raízes normais revelaram a presença de dehidro-epideoxiarteanuína B e de um outro composto (composto B). As condições fotoperiódicas de cultivo influenciaram na produção destes dois compostos, sendo que sobre condição de escuro contínuo, dehidro-epideoxiarteanuína B foi intensamente produzido e o composto B foi detectado em pequenas proporções, enquanto que sob fotoperíodo de 16 horas, o inverso ocorreu. A quantificação de dehidro-epideoxiarteanuína B por Cromatografia Gasosa acoplada a um Detector de Ionização de Chamas (CG/FID) revelou um aumento de aproximadamente cinco vezes na produção deste composto pelas raízes normais cultivadas sobre escuro contínuo em relação às raízes cultivadas na presença de 16 horas de luz. O terpeno dehidro-epideoxiarteanuína B não estava presente nas raízes transgênicas.