RESUMEN
Roots of Rauwolfia serpentina, also known as "Sarpagandha" possess high pharmaceutical value due to the presence of reserpine and other medicinally important terpene indole alkaloids. Ever increasing commercial demand of R. serpentina roots is the major reason behind the unsystematic harvesting and fast decline of the species from its natural environment. Considering Agrobacterium rhizogenes-mediated hairy root cultures as an alternative source for the production of plant-based secondary metabolites, the present optimized protocol offers a commercially feasible method for the production of reserpine, the most potent alkaloid from R. serpentina roots. This end-to-end protocol presents the establishment of hairy root culture from the leaf explants of R. serpentina through the infection of A. rhizogenes strain A4 in liquid B5 culture medium and its up-scaling in a 5 L bench top, mechanically agitated bioreactor. The transformed nature of roots was confirmed through PCR-based rol A gene amplification in genomic DNA of putative hairy roots. The extraction and quantification of reserpine in bioreactor grown roots has been done using monolithic reverse phase high-performance liquid chromatography (HPLC).
Asunto(s)
Agrobacterium/fisiología , Reactores Biológicos , Técnicas de Cocultivo/métodos , Raíces de Plantas/microbiología , Rauwolfia/microbiología , Reserpina/metabolismo , Agrobacterium/genética , Cromatografía Líquida de Alta Presión/métodos , Medios de Cultivo/metabolismo , ADN de Plantas/genética , Amplificación de Genes , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/fisiología , Rauwolfia/genética , Rauwolfia/crecimiento & desarrollo , Rauwolfia/fisiología , Reserpina/análisis , Transformación GenéticaRESUMEN
The in vitro grown axillary micro shoots of Rauvolfia vomitoria were encapsulated in alginate beads. Following 6 months of normal storage at 25 +/- 2 degrees C the regrowth of encapsulated micro shoots, reached 95.2% within 40 days of incubation on MS medium containing 1.0 mg/L BAP and 0.1 mg/L NAA. Among the responding encapsulated explants 69.6% showed emergence of multiple shoots. The developing shoots showed rhizogenesis in two weeks following their transfer to rooting medium. Healthy plants were established in a glass house with 95% survival. Of the 50 RAPD primers tested, 10 produced 23 clear and reproducible amplicons, with an average of 2.3 bands per primer. Eleven ISSR primers produced a total of 42 bands, with a size range of 0.1-1.9 kb. The number of scorable bands for each primer varied from 2 to 6, with an average of 3.81. The similarity matrix, calculated individually from the results obtained from ISSR and RAPD analysis, showed similarity coefficients ranging from 1.0 for RAPD and 0.85 to 1.0 for ISSR.
Asunto(s)
Brotes de la Planta/genética , Plantas Medicinales/genética , Rauwolfia/química , Rauwolfia/genética , Especies en Peligro de Extinción , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/ultraestructura , Plantas Medicinales/crecimiento & desarrollo , Plantas Medicinales/ultraestructura , Reacción en Cadena de la Polimerasa , SemillasRESUMEN
A simple, repeatable and efficient protocol for direct multiple shoot regeneration from internodal explants has been defined in peppermint (Mentha x piperita var. Indus). In vitro regenerated shoots of peppermint were excised into 4 to 8 mm long internodes and cultured on Murashige and Skoog's medium supplemented with different cytokinins. In the hormonal assay, 3.0 mg L(-1) zeatin or 6-isopentenyl adenine independently supplemented to half strength MS medium exhibited multiple shoot regeneration, while thiaduzorn (0.1-3.0 mg L(-1)) showed no morphogenetic effect. A maximum of 85% in vitro cultured explants showed multiple shoot formation with an average of 7 shoots per explant on MS medium supplemented with zeatin. Multiple shoots were initiated within three weeks of cultivation. Internodes with regenerated multiple shoots were transferred to half- strength MS medium without supplementing with any plant growth hormone for shoot elongation and rhizogenesis. Rooted plants acclimatized and grew to maturity under glasshouse conditions. The plantlets developed were phenotypically identical to the parent plant and exhibited 96% survival.