Current approaches for Enhanced Expression of Secondary metabolites production

1. Current approaches forEnhanced
Expression of Secondary metabolites
production
SPEAKER:
Narendra Singh Bhandari
Id no. 10711
ICAR-SRF
Division of Ornamental
Crops
Credit Seminar
ICAR-IndianInstituteofHorticulturalResearch,Bengaluru

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

3. 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)
ICAR-IndianInstituteofHorticulturalResearch,Bengaluru

4. Metabolites
• Primary metabolites
Primary 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 metabolites
1. Flavonoids and allied phenolic and polyphenolic
compounds,
2. Terpenoids and
3. Nitrogen-containing alkaloids and sulphur-containing
compounds.
ICAR-IndianInstituteofHorticulturalResearch,Bengaluru

5. 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.

6. Flow diagram of secondary metabolites production

7. 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

8. Cell Cultures for Secondary Metabolite
Production
• 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.
ICAR-IndianInstituteofHorticulturalResearch,Bengaluru

9. Organ Cultures for Secondary Metabolite
Production
•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.
ICAR-IndianInstituteofHorticulturalResearch,Bengaluru
Namdeo, 2007

10. 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.

11. 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)
ICAR-IndianInstituteofHorticulturalResearch,Bengaluru

12. 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.

13. 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.

15. Advantages of hairy root cultures
Hairy roots
high growth rate
and 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

16. Bioreactor

17. Case study 1

18. Objective
To characterize the effect of LED lighting
• In horticultural ornamentals grown in
greenhouse facilities
• On the photosynthetic characteristics and the
physiological mechanisms of selected plant
species
• On secondary metabolites which play a crucial
role in the biochemical defence of the plant

19. 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

20. 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

21. Chemical analysis
• Leaf samples are taken randomly for
later analysis by HPLC/LCMS
• Samples were ground with liquid
nitrogen and 80% MeOH was used
for
extraction
• Separations with a Zorbax Eclipse
XDB-C18 column (5μm, 150 X 4.6
mm;
Agilent)
• Phenolic acids and flavonoids will be
quantified in extracts by HPLC and
LCMS at 320 nm and 360 nm,
respectively

23. 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

24. 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

25. 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 hypocotls
were 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

26. Results

29. 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.

30. 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 applied
6. Novel products can be produced via mutant cell lines.
7. Biotransformation can be done.

31. 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.