ABSTRACT
Stevia rebaudiana Bert. is a plant that contains noncaloric sweeteners highly appreciated in the food industry. However, there is a high demand for propagules to establish commercial plantations, and the conventional reproduction types for this species are inefficient. Micropropagation is a technique that allows obtaining a large number of plants and can be used to meet the demand in the field. However, it requires in vitro propagation techniques such as temporary immersion systems (SIT) to increase yield and reduce production costs. This chapter describes an effective protocol for the large-scale micropropagation of S. rebaudiana using a TIS.
Subject(s)
Stevia , ReproductionABSTRACT
Guarianthe skinneri (Bateman) Dressler & W. E. Higgins is an orchid valued for its ornamental characteristics. However, it is an orchid classified as threatened with extinction due to the illegal extraction from its natural habitat. In addition, its propagation through seed germination is very low, as is the case with most members of the family Orchidaceae. Its asexual propagation through pseudobulb separation is slow and produces a few propagules. For this reason, in vitro propagation techniques are an alternative to increase the number of plants obtained and thus be able to recover this valuable plant genetic resource. Temporary immersion systems (TIS) offer the advantage of mass-propagating plants for different purposes. This chapter describes a large-scale micropropagation protocol for Guarianthe skinneri using temporary immersion bioreactors (TIB).
Subject(s)
Immersion , Orchidaceae , Bioreactors , Reproduction , Reproduction, AsexualABSTRACT
The effects of methyl jasmonate, salicylic acid and ethylene on alkaloid accumulation in in vitro cell suspension, hairy roots and rootless shoot cultures of Catharanthus roseus were analyzed. Ajmalicine, but not catharanthine, accumulation was promoted by jasmonate and ethylene treatments in cell suspensions. In hairy roots, jasmonate induced the accumulation of both alkaloids, whereas ethylene only induced catharanthine accumulation. In shoot cultures, positive effects of jasmonate and ethylene were recorded only in vindoline accumulation. Ethylene diminished catharanthine accumulation in these cultures. No effect of salicylic acid was observed in any of the studied in vitro culture systems.
Subject(s)
Acetates/metabolism , Catharanthus/drug effects , Catharanthus/metabolism , Cyclopentanes/metabolism , Ethylenes/metabolism , Oxylipins/metabolism , Salicylic Acid/metabolism , Secologanin Tryptamine Alkaloids/metabolism , Vinca Alkaloids/metabolism , Cells, Cultured , Plant Roots/chemistry , Plant Shoots/chemistryABSTRACT
BACKGROUND AND AIMS: The Madagascar periwinkle (Catharanthus roseus) produces the monoterpenoid alkaloid vindoline, which requires a specialized cell organization present only in the aerial tissues. Vindoline content can be affected by photoperiod and this effect seems to be associated with the morphogenetic capacity of branches; this association formed the basis of the study reported here. METHODS: Vindoline-producing in vitro shoot cultures were exposed either to continuous light or a 16-h photoperiod regime. New plantlet formation and alkaloid biosynthesis were analysed throughout a culture cycle. KEY RESULTS: In cultures under the photoperiod, the formation of new plantlets occurred in a more synchronized fashion as compared to those under continuous light. The accumulation of vindoline in cultures under the photoperiod occurred in co-ordination with plantlet formation, in contrast to cultures under continuous light, and coincided with a peak of activity of deacetylvindoline acetyl CoA acetyltransferase (DAT), the enzyme that catalyses the last step in vindoline biosynthesis. When new plantlet formation was blocked in cultures under the photoperiod by treatment with phytoregulators, vindoline synthesis was also reduced via an effect on DAT activity. No association between plantlet formation and other biosynthetic enzymes, such as tryptophan decarboxylase (TDC) and deacetoxyvindoline 4-hydroxylase (D4H), was found. Effects of light treatment on vindoline synthesis were not mediated by ORCA-3 proteins (which are involved in the induction of alkaloid synthesis in response to elicitation), suggesting that the presence of a different set of regulatory proteins. CONCLUSIONS: The data suggest that vindoline biosynthesis is associated with morphogenesis in shoot cultures of C. roseus.
Subject(s)
Catharanthus/growth & development , Catharanthus/metabolism , Plant Shoots/growth & development , Plant Shoots/metabolism , Vinblastine/analogs & derivatives , Acetyltransferases/metabolism , Aromatic-L-Amino-Acid Decarboxylases/metabolism , Catharanthus/enzymology , Light , Mixed Function Oxygenases/metabolism , Morphogenesis , Photoperiod , Plant Proteins/metabolism , Plant Shoots/enzymology , Transcription Factors/metabolism , Vinblastine/biosynthesisABSTRACT
A protocol for the establishment of in vitro shoot cultures of Catharanthus roseus is described. Shoots can be maintained for more than 1 yr without evidence of tissue vitrification, disaggregation, or callus formation. Vindoline was the main alkaloid accumulated, reaching values similar to those found in leaves from field-grown plants, after a long period of culture. An induction methodology to reduce such waiting time is also presented.
Subject(s)
Catharanthus/metabolism , Plant Shoots/metabolism , Secologanin Tryptamine Alkaloids/metabolism , Tissue Culture Techniques/methods , Catharanthus/growth & development , Culture Media , Cyclopentanes/metabolism , Oxylipins , Plant Shoots/growth & development , Seedlings/physiologyABSTRACT
Vindoline, the major alkaloid in cultures of Catharanthus roseus shoots, reached 2 mg g(-1) dry wt after 27 d in culture. Maximal vindoline accumulation coincided with maximum activities of deacetoxyvindoline 4-hydroxylase, deacetylvindoline acetyl-CoA acetyl transferase and tryptophan decarboxylase. Shoot exposure to jasmonate shortened the time required for the maximal vindoline accumulation to 14 d.