Current approaches for Enhanced Expression of Secondary metabolites

productionSPEAKER: Narendra Singh BhandariId no. 10711ICAR-SRFDivision of Ornamental Crops

Credit Seminar IC

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Content• Introduction• Classification• Approaches• Case Study• Conclusion• Future Thrust

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

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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.

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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.

Flow diagram of secondary metabolites production

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

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.

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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.

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Namdeo, 2007

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.

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)

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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.

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.

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

Bioreactor

Case study 1

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

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

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

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

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

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

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

Results

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.

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.

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.