Narendra credit seminar 2
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Current approaches for Enhanced Expression of Secondary metabolites
productionSPEAKER: Narendra Singh BhandariId no. 10711ICAR-SRFDivision of Ornamental Crops
Credit Seminar IC
AR
-Ind
ian
Inst
itute
of H
ortic
ultu
ral R
esea
rch,
Ben
galu
ru
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Content• Introduction• Classification• Approaches• Case Study• Conclusion• Future Thrust
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Introduction• For million years, humankind is completely
dependent on plants as source of food and shelter.
• In addition, plants are a valuable source of a wide range of metabolites.
• Used as pharmaceuticals, agrochemicals, flavours, fragrances, colours, bio-pesticides and food additives.
• Nearly 70% - 80% of world population depends upon herbal drugs.(WHO) IC
AR
-Ind
ian
Inst
itute
of H
ortic
ultu
ral R
esea
rch,
Ben
galu
ru
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Metabolites
• Primary metabolitesPrimary metabolites are compounds that are directly involved in the growth and development of a plant whereas secondary metabolites are compounds produced in other metabolic pathways that, although important, are not essential to the functioning of the plant.
• Secondary metabolites1. Flavonoids and allied phenolic and polyphenolic compounds,2. Terpenoids and3. Nitrogen-containing alkaloids and sulphur-containing
compounds.
ICA
R-I
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Significance of Secondary metabolites
• Play important role in reinforcement of tissue and tree body (e.g. cellulose, lignin, suberin)
• Important for abiotic stresses and eenvironnemental adaptations
• Attract pollinators or seed dispersal agents• In human life, these compounds are used as
flavourings, medicines and relaxing drugs.
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Flow diagram of secondary metabolites production
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Methods....Current approaches...• Plant Tissue Cultures/ cell
and Organ Cultures• Elicitation of In vitro
products• Endophytes in in vitro
Production• Abiotic stress signals on
secondary metabolites• Genetic engineering in
Hairy Root Culture
Natural/ Wild
Rarely found in nature and
slow synthesis
The quantity obtained is in
trace amounts (0.01% of dry
weight).
Production affected by
environmental condition
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Cell Cultures for Secondary MetaboliteProduction
• Metabolite production is frequently higher in cell cultures
• Berberine production from Coptis japonica is about 5% of dry weight after 5 years of root growth, which equals 0.17 mg/g per week.
• However, in selected cell lines it can be 13.2% of the dry weight in cell culture after 3 weeks, which is about 44 mg/g/week or about 250 times higher.
ICA
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Organ Cultures for Secondary MetaboliteProduction
•Fritillaria unibracteata can be rapidly propagated, directly from
small cuttings of the bulb by the technique of organ culture
under in-vitro condition.
•The growth rate was about 30–50 times higher than that under
natural wild growth conditions.
•The content of alkaloid and beneficial micro-elements in the
cultured bulbs was higher than found in the wild bulb.
ICA
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luru
Namdeo, 2007
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Elicitation
• Elicitors are the compounds of biological origin which stimulate the production of secondary metabolites, and the phenomenon is called Elicitation.
• Physical agent: heat, cold, UV, osmotic pressure • Chemical agent: antibiotics, fungicide, etc.
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Endophytes in in vitro Production
The symbiotic association and effects of plants and endophytes on each other during the production of other important pharmacological bioactive natural products.
Claviceps sp: producing ergot alkaliods was a potential source of useful secondary metabolite (Clay, 1988).
Gibberella fujikouri an important endophytic fungus in rice (Oryza Sativa) was the source of the phytohormone Gibberellin (Steierle et al.,1993)
ICA
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Abiotic stress signals on secondary metabolites in plants.
• Accumulation of metabolites in plants subjected to stresses including various elicitors or signal molecules.
• Temperature, humidity, light intensity, the supply of water, minerals, and CO2 influence the growth of a plant and secondary metabolite production.
• Drought, high salinity, and freezing temperatures are environmental conditions that cause adverse effects on the growth of plants and the productivity of crops.
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Genetic manipulation in hairy root culture for secondary metabolite production
• Inoculation of plant with Agrobacterium rhizogenes produces infection in roots, exuding out the secondary metabolites with higher yields.
• A gene of interest with regard to secondary metabolism that was introduced into hairy roots is the 6-hydroxylase gene of Hyoscyamus muticus which was introduced to hyocyamin-rich Atropa belladonna by a binary vector system using A. Rhizogenes.
• Engineered roots showed an increased amount of enzyme activity and a five-fold higher concentration of scopolamine.
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Advantages of hairy root cultures
Hairy roots
high growth rateand genetic stability
produce high levels of secondary metabolites
produce stable levels of secondary
metabolites over long time
Enhance secondary metabolites production
scaling up in plant bioreactor
Elicitation
Metabolic Trapping
Metabolic engineering
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Bioreactor
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Case study 1
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ObjectiveTo characterize the effect of LED lighting• In horticultural ornamentals grown in
greenhouse facilities• On the photosynthetic characteristics and the
physiological mechanisms of selected plantspecies
• On secondary metabolites which play a crucial role in the biochemical defence of the plant
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Plant material• Roses (Rosa hybrida ‘Scarlet’)• Chrysanthemums (Chrysanthemum morifolium ‘Coral
Charm’)• Campanulas (Campanula portenschlagiana ‘BluOne’)• The plants were grown to flowering (except
chrysanthemums) and plant growth was recorded at the end of the experiment
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Greenhouse Setup• LED array yielding approximately 200 µmol /m2/for 16
hours per day• The temperature in the greenhouse compartments was set
to 24C and 18C during the day and night, respectively
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Chemical analysis• Leaf samples are taken randomly for
later analysis by HPLC/LCMS• Samples were ground with liquid
nitrogen and 80% MeOH was used forextraction
• Separations with a Zorbax EclipseXDB-C18 column (5μm, 150 X 4.6 mm;Agilent)
• Phenolic acids and flavonoids will bequantified in extracts by HPLC andLCMS at 320 nm and 360 nm,respectively
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Conclusions• The combination of RED and BLUE LED
lighting has a positive effect on growth and development of roses, chrysanthemum and campanulas
• BLUE/RED lighting treatment seem to have no significant effect on photosynthesis
• The amount of secondary metabolites increases with additional of BLUE light
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Objective:•To develop an efficient protocol for micro-propagation to improve secondary metabolite productions in C. officinalis in �vitro cultures•To investigate the influence of cytokinin, N6-benzyladenine on particular secondary metabolites as phenolic compounds, flavonoids and anthocyanins.
Case study 2
Gadzovska et al., 2007
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Material and methods• Seeds of Calendula officinalis L. were washed over
night, air dried and surface sterilized with 1 % NaOCl for 2 �minutes
• Apical segments, cotyledons and hypocotlswere isolated from 2 weeks old in vitro grown plants.
• The explants were cultured on MS/B 5 medium and supplemented with different concentrations of cytokyinin N6 benzyladenine. �
• Tissue cultures were maintained in a growth chamber at 26±1°C under a photo-period of 16 h light and 8 h dark, � �irradiance of 5 µmol/m2 /S and 60 % relative humidity.� �
• The in vitro cultures were harvested by vacuum filtration on Days 7, 14, 21, 2 and 35 of culture, weighted for growth �analysis
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Results
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Inferences
• Thus, secondary metaolite production under � in vitro condition can be partially changed � �
by phyto-hormone supplementation.��• Well controlled C. officinalis in vitro cultures
could be used as a source for rapid and �increased production of secondary metabolites �and associated pharmacological compounds.
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WHY in-vitro? The major advantages over the conventional cultivation :1. Compounds can be produced under controlled conditions as
per market demands. 2. Independent of environmental factors. The cells of any
plants, tropical or alpine, could easily be multiplied to yield their specific metabolites.
3. Quality will be consistent as the products are formed by a specific cell line.
4. Cultured cells would be free of microbes and insects.5. Easy recovery strategies can be applied6. Novel products can be produced via mutant cell lines. 7. Biotransformation can be done.
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Path ahead…..... Enhance basic knowledge of the biosynthetic routes, and mechanisms
responsible for the production of plant metabolites.
Need for screening of ornamental plants with regards to valuable
secondary metabolites.
Identification and characterization of genes for secondary metabolites.
Develop the tools and techniques that are the most convenient, efficient
and economical for obtaining the desired results.
Strain improvement, methods for the selection of high-producing cell
lines, and medium optimizations can lead to an enhancement in
secondary metabolite production.
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