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 A PRESENTATION ON THE
PRODUCION OF SECONDARY
METABOLITES THROUGH TISSUE
CULTURE

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Presenters
1. Tuba Yasmin (21-010025)
2. Md. Zahidur Rahman (21-010039)
3. Md. Tasnimul Hasan (21-010037)
4. Md. Imran Hosen (21-010101)
5. Mir Md. Shah Alam (21-010103)
6. Md. Daraj Uddin Prodhan (21-010033)

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 Contents
1. Introduction 8. Factors Affecting Secondary
2. Secondary Metabolites Metabolites Production
3. In-vitro Culture 9. Application
4. Secondary Metabolites & 10.Pros & Cons
In-vitro Culture
11.Future Prospects
5. Reason of Producing
Secondary Metabolites 12.Recent Works
6. Classification 13.Recommendation
7. Production Strategy

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Introduction
 Plants have a major role in human life. For millions
of years, humankind is completely dependent on
plants mainly as a source of food and shelter.

 Plants have the innate ability to manipulate organic

synthesis from simple molecules such as carbon
dioxide, water, and inorganic ions, giving a complex
array of natural products. This metabolic activity
leads to two classes of metabolites: primary
metabolites and secondary metabolites (Balandrin
and Klocke, 1985; Shimomura et al., 1997). 6
Introduction
 These plant metabolites determine the nutritional
quality of food, color, taste, smell, antioxidative,
anticarcinogenic, antihypertension, anti-inflammatory,
antimicrobial, immune-stimulating, and cholesterol-
lowering properties.

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 Primary Metabolites VS Secondary
Metabolites
 Primary metabolites are compounds that are directly
involved in the growth and development of plants.
Primary metabolites such as carbohydrates, vitamins,
proteins, lipids, nucleic acids, and hormones.

 Secondary metabolites are compounds that are not

directly involved in the growth and development of
plants but involved in plant defense mechanisms
against herbivores, pests, and pathogens.
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 Secondary Metabolites
 Secondary metabolites are organic chemical compounds
that are produced by plants known as phytochemicals.

 They are also widely used for pharmaceutical, medical, or

agricultural purposes (Calvo et. al., 2002) Moreover, they
are used as flavoring agents, food additives, cosmetics,
dyes, and antioxidant properties.

 They became the focus of plant science for commercial

and academic research purposes. At present, secondary
metabolites are a constant in our lives. As an example, we
have caffeine, a plant secondary metabolite found in 9

coffee, tea, or chocolate.

 In-Vitro Culture
 In vitro culture is a method applied for the growth and development of
plant cells, tissues and organs that use a nutritive culture medium under
controlled sterilized conditions.

 In-vitro culture is carried out in a test tube, Petri dish, or flask and it is
also referred to as the plant tissue culture technique. For in-vitro culture
explants from healthy and young part of the plant is used to regenerate
the whole plant.

 Plant tissue culture is an efficient method for the production of

secondary metabolites within a short duration for commercial
application and research purposes.
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Secondary metabolites & In vitro
culture
 Plants are a tremendous source for the discovery of new
products of medicinal value for drug development.

 Many of the drugs sold today are simple synthetic

modifications or copies of the naturally obtained
substances

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Secondary metabolites & In vitro
culture
 Evolving commercial importance of secondary
metabolites has in recent years resulted in the alteration
of the production of bioactive plant metabolites by
means of tissue culture technology.

 Plant cell culture technologies were introduced at the

end of the 1960’s as a possible tool for both studying and
producing plant secondary metabolites.

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Secondary metabolites & In vitro
culture
 In addition extraction from the in vitro tissues is
much simpler than extraction from organized,
complex tissues of a plant.

 Now In vitro culture becomes the most reliable

source of producing secondary metabolites in
stead of growing the whole plant.

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 Why Plant Produces
Secondary Metabolites?

 Secondary metabolites are essential part

of plant defense armament.

 These are some chemicals viz. terpenes,

phenolics etc. which are produced in
plants due to different stresses.

 It helps to withstand different kinds of

stress.
Fig. Role
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of plant secondary
metabolites
(Mendoza & Silva, 2018)
Why Plant Produces Secondary
Metabolites?
 Stress includes abiotic and biotic factor influenced
stress.
 Abiotic stress denotes to the stress initiated by
environmental factors like heat, humidity, acidity,
salinity, etc.
 Biotic stress is the stress that arises due to bio
organism. This stress can be a result of micro
organism attack, herbivorous attack or from the
competition with several other plants.
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Why Plant Produces Secondary
Metabolites?
 Secondary metabolites produces as the response
to these stresses and protect plants.

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Types of Secondary Metabolites

 There are three major classes of plant

metabolites based on the biosynthetic pathway.
 Phenolic groups (composed of simple sugars
and benzene rings).
 Terpenes and Steroids (composed mainly of
carbon and hydrogen).
 Nitrogen-containing compounds
 Sulphur-containing compounds

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Types of Secondary Metabolites
 Phenolic compounds derive from the phenylpropanoid
pathway in plants and they have diverse structures and
supply flowers, fruits and vegetables with color.

 A variety of phenolic metabolites seems to have

several properties, such as anti-oxidant, anti-
carcinogenic and anti-inflammatory effects.

 Alkaloids and flavonoids are large groups of secondary

metabolites with highly diverse biological functions.

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Table. Different classes of 2º metabolites

SECONDARY METABOLITE
PRODUCTION TECHNIQUE IN TISSUE
CULTURE
 These Technique includes the following:
 
 Callus Culture
 Cell Suspension Culture
 Organ Culture
 Hairy Root Culture
 Adventitious Root Culture

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Fig: Generalized scheme of Explant selection to Secondary metabolites21
production
SECONDARY METABOLITE
PRODUCTION TECHNIQUE
Cell Suspension Culture
 Suspension culture is a type of culture in which single
cells or small aggregates of cells (friable callus)
multiply while suspended in agitated liquid medium.

 First suspension culture in stirred tank reactor was used

to produce Shikonin From Lithospermume rythrorhizon

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Fig: Cell Suspension Culture

Advantages of Cell Suspension
Culture

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Organ Culture (Hairy Root Culture)

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Fig: Hairy Root Culture
Organ Culture (Adventitious Root
Culture)

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Fig: Adventitious Root Culture

Factors Enhance Secondary Metabolite
Production in Tissue Culture
 Biotransformation:
 Cell/Enzyme
A B
(Substrate) (Product)
 Biocatalyst can be micro organism, plant or animal cell either
growing or quiescent state or purified enzymes.
 Digitalis lantana produce much Methyl Digoxin in the presence of
Methyl Digitoxin in cell culture.
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Factors Enhance Secondary Metabolite
Production in Tissue Culture
 Immobilization:
Cells are physically immobilized by entrapment, besides
individual cells, it is also possible to immobilize the
callus.
Three type of Immobilization:
1. Entrapment Cells in Gels: Gel arrest the free
movement. E.g. Agar, Polyacrylamide gel.
2. Entrapment Cells in Net/Foams: Polyurethane foams
or net with various pore size are used. 29
Factors Enhance Secondary Metabolite
Production in Tissue Culture

3. Entrapment Cells in Hollow fiber membrane:

Cellulose acetate, Silicon polycarbonate composed
tubular hollow fiber is used to immobilize the cell

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Factors Enhance Secondary Metabolite
Production in Tissue Culture
 Elicitors: Elicitors are
the compound of
biological origin which
stimulate the
production of secondary
metabolites and the
phenomenon is called
Elicitation.

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FACTORS AFFECTING THE PRODUCTION
OF SECONDARY METABOLITES IN TISSUE
CULTURE
 Explant
 Media formulation
 Carbon sources
 Nitrogen
 Phosphate
 Agitation
 Plant growth hormones
 Precursor supplement 32
FACTORS AFFECTING THE PRODUCTION
OF SECONDARY METABOLITES IN TISSUE
CULTURE
 Media Formulation: Composition and types of media used
for the cell culture of plants impact the production of
secondary metabolites. For example, the use of MS
(Murashige and Skoog) media in the suspension culture of
Catharanthus roseus increases the production of
serpentine metabolite. The effect of media on metabolite
yield varies from one plant species to another.
 Carbon Source: Sucrose is one of the essential
components of plant tissue culture media. It has been
observed that altering the sugar concentration in the
media or using different carbon sources impacts the
production of secondary metabolites in some plants.
FACTORS AFFECTING THE PRODUCTION OF
SECONDARY METABOLITES IN TISSUE
CULTURE
 Nitrogen: Nitrogen is one of the essential components of
some tissue culture media, including MS, LS, or B5 media.
It has been observed that media containing amino acids
and proteins have better secondary metabolite yields.
 Phosphate: Several studies report that the level of
phosphate in tissue culture media affects the production
of secondary metabolites. For example, in many plants, it
has been observed that increasing the phosphate level in
the media increases cell growth, however, reduces the
secondary metabolite yields.
FACTORS AFFECTING THE PRODUCTION OF
SECONDARY METABOLITES IN TISSUE
CULTURE
 Agitation: During cell suspension culture, it has been
found that the speed of agitation impacts the cell
viability, resulting in impacting the production of
secondary metabolites. In Podophyllum hexandrum, a
speed of 200 rpm was found to be damaging for plant
cells, whereas more viable cells were obtained when
speed was in the range of 125- 150 rpm.
 Plant growth hormones: Several studies report that the
use of types of hormones and their concentration in the
media affect the plant’s growth in tissue culture, in
addition to the production of secondary metabolites.
FACTORS AFFECTING THE PRODUCTION OF
SECONDARY METABOLITES IN TISSUE
CULTURE
 Precursor supplementation: Some studies have found that
supplementing the cell cultures with compounds that are
also produced during secondary metabolite synthesis
increases their yield. This process of the addition of such
compounds to cell cultures is known as precursor feeding.
FUNCTION & APPLICATION of
Secondary Metabolites

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Fig. Function and applications of

Secondary Metabolites
Major application of secondary
metabolites
 Chemically they may be alkaloid,terpenoids,glycosides
etc.
 Pharmaceuticals,
flavors,perfumes,agrochemicals,insecticides and raw
materials for industries.
 Shikonine- A dye from Lithospermum erythrorhizon.
 Production of secondary metabolites is a multibillion
industry.
 One kg of vincristine and vinblastine cost
Uses of secondary metabolites
Uses of secondary metabolites
Uses of secondary metabolites
Uses of secondary metabolites
Pharmaceutical Uses

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Advantages of production of secondary
metabolites through plant tissue culture

1. Enables sustainable production of secondary

metabolites under a controlled condition.
2. No effect of climatic factors on the productions of
secondary metabolites.
3. Independent of geological and geographical
conditions conditions which enables incessant
production of secondary metabolites all around the
world.

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Advantages of production of secondary
metabolites through plant tissue culture
4. It’s a nondestructive mean of production of
secondary metabolites thus biodiversity loss and
genetic erosion can be avoided compared to
traditional means.
5. It’s an eco friendly alternative method for
production of secondary metabolites where natural
supply is limited thus conserving endangered plant
species when at present genetic erosion is aggressive
due to climate change.

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Advantages of production of secondary
metabolites through plant tissue culture
6. Takes almost no time compared to traditional
production which requires years to initiate
production.
7. It is a highly efficient, sustainable, reliable and
predictable method of production of secondary
metabolites compared to extraction from wild
population.

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 Advantages of production of secondary
metabolites through plant tissue culture

8. Economically more feasible because supply can be

controlled as per market demand thus no waste of excess
produces.
9. Sometimes there are products which cannot be extracted
from natural population but can be extracted from tissue
culture. Similarly some secondary metabolites are
difficult to extract from natural population but easy in
plant tissue culture method. e.g. some alkaloids.

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Advantages of production of secondary
metabolites through plant tissue culture

10. As aseptic condition is maintained there is no

incident of disease and pest infestations. So
herbicides and pesticides can be avoided reducing
environmental pollution protecting life on land and
water.
11. Cell growth can be controlled to facilitate improved
product formation.
12. Quality of product will be consistent as it is produced
by specific cell lines.
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Advantages of production of secondary
metabolites through plant tissue culture

13. Recovery from plant extract is easirer.

14. Mutant lines can be developed for the production of
novel compounds of commercial importance which
are not normally found in natural population.
15. Requires less land and labor for maintenance of
plant material. Thus cost efficient for large scale
production.

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Major Disadvantages

1. Cultured cells are genetically unstable so it is

susceptible to mutation.
2. Ageing of culture adversely subsequently reduces the
production level.
3. Some production procedure may damage cells. e.g.
stirring.

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Major Disadvantages

4. Sometimes various toxin is produced in plant tissue

culture which affects quality, purity and production
rate of secondary metabolites.
5. Production is low in for certain species compared to
natural population and permanent tissue.
6. Sometimes viability and productivity is not good due
to poor photosynthesis in the culture.

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Major Disadvantages
7. Maintaining a sterile condition is difficult and
expensive.
8. Expensive lab instruments are required which may or
may not be available wherever or whenever needed.
9. Water wastage is more in this method compared to
traditional production.

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Major Disadvantages

10. Economically not feasible if demand isn’t consistent

and continuous .
11. Requires highly trained and skilled persons to
maintain the production facility.
12. May have a negative impact on the economy of third
world countries as they would lose market to
efficient production of secondary metabolites in
industrial countries. This may increase the global
poverty and hunger.
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Future prospects
 Genome editing: Modern biotechnology tools like
CRISPR Cas, recombinant DNA technologies,
transcription activators like effector nucleases can be
used to produce lines which can enable more
efficient secondary metabolite production from more
species of natural resources.

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Future prospects
 Biotransformation: Biotransformation is the chemical
reaction catalyzed by enzymes cells and organs etc.
This has ability to form products more efficiently.
This is being used to an extent and research on it is
still going on extensively. There are some useful
secondary metabolites that is produced in plant cell
but can’t be accumulated by plants. Extensive
research is going on this subject which can open a
new leaf in the future.

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Future prospects
 Nanotechnology: Particles of 1-100 nm is called nano
particles. Nanotechnology can be applied in plant
tissue culture to boost plant growth and increase
production of biosynthetic materials. The effect of
certain metal oxides as nanoparticles like titanium
oxide, zinc oxide etc. has positive correlation with
the production of secondary metabolites. Extensive
research is ongoing to use more efficient nanotech in
secondary metabolite production.

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Future prospects
 Metabolic engineering: Metabolic engineering is the
amendment of metabolic routes using modern tools
like metabolomics, proteomics and genomics. It
allows manipulation of endogenous biochemical
pathways. For example phenylpropanoid pathway is
activated by MYB family protein which regulates
anthocyanin production. More research are going on
this matter and expected to formulate more
economically viable secondary metabolites
production strategy in future.

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Future prospects
 Artificial intelligence: Artificial intelligence is one of
the hot topics at present having massive potential in
possibly every developmental strategy. Attempts are
being made by scientists to apply this technology in
biological world. The potential is also being under
research in secondary metabolites production. Maybe
in near future sustainable eco friendly production of
secondary metabolites may be possible which will
require minimum human involvement and
economically viable production will be possible.

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 Way to overcome the limitations
1. Regular genetic testing and mutation screening
tests should be done. Mutant cultures shouldn’t
be kept in production of secondary metabolites
but should be checked if the mutation is
beneficial for secondary metabolites production.
2. Subculturing must be done on a regular basis to
optimize production.

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 Way to overcome the limitations
3. More research should be done on metabolic
pathways of secondary metabolites production
to avoid contamination and toxin production in
tissue culture process. Possible manipulation
should be made on the metabolic pathways
using modern biotechnology tools to eliminate
or reduce contamination or toxin production.
4. Water and other natural resources must be
recycled to reduce wastage.

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 Way to overcome the limitations
5. Protocol should be maintained strictly to optimize
photosynthesis in the cultures to increase viability
and productivity. However more research should
be made to boost photosynthesis and secondary
metabolite production.
6. Versatile and coordinated industrial use of
culture equipment and sterile culture
condition should be ensured to keep the cost
below economic threshold.

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Way to overcome the limitations
7. More people should be trained and skill development
program should be arranged to reduce the lack of
skilled workers.
8. Global cooperation should be set in motion to aid the
third world countries so that their people could
thrive. Policies should be made by global leaders to
formulate environmentally safe production
technology that would promote economic equity
among countries in global partnership. Global
partnership among developed, underdeveloped and
developing countries should be strengthen.
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 Current Work
 Production of bioactive plant secondary metabolites through in
vitro technologies—status and outlook ,
 Applied Microbiology and
Biotechnology volume 105, pages6649–6668 (2021)

 In Vitro Cultures for the Production of Secondary Metabolites

 Grazia Maria Scarpa, Vanda Prota, Nicola Schianchi and
Federica Manunta.

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 Current Work
 In vitro strategies for the enhancement of secondary metabolite
production in plants: a review; Mohammad Afaan Fazili, Irfan
Bashir, Mudasar Ahmad, Ubaid Yaqoob & Syed Naseem Geelani,
Bulletin of the National Research Centre volume 46,
Article number: 35 (2022) 

 Plant Cell and Tissue Culture as a Source of Secondary Metabolites

Author(s): Rodríguez-Sahagún A., Del Toro-Sánchez C.L.,
Gutierrez-Lomelí M. and Castellanos-Hernández O.A.

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Current Work

 Sustainable production of biomass and industrially

important secondary metabolites in cell cultures of
selfheal (Prunella vulgaris L.) elicited by silver and
gold nanoparticles
 Hina Fazal, Bilal Haider Abbasi, Nisar Ahmad,
Mohammad Ali, Syed Shujait Ali, Abbas Khan & Dong-
Qing Wei

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Current Work
 Plant tissue culture as a perpetual source for production of
industrially important bioactive compounds(June, 2020)
 Author: Chandran H, Meena M, Barupal T, and Sharma K.
 Findings: Plant tissue culture can serve as
sustainable, cost effective environmental friendly
method of secondary metabolite production and it’s
demand is rising as more medicinal plant species are
at risk of extinction due to climate change. More
research are going on to improve secondary
metabolites production by tissue culture.

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Current Work
 Title: Production of Plant Secondary Metabolites by
Using Biotechnological Tools( nov 2017)
Author: Sandra G. and Anabella R.
 Findings: Plant tissue culture is an attractive
mean to produce secondary metabolites. And
there is a great prospect to use biotechnology in
secondary metabolite production. But only very
few species are under this method for commercial
production. So more research are required to
improve the production of secondary metabolites
from tissue culture.
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Current Work
 Title: In vitro strategies for the enhancement of
secondary metabolite production in plants( feb, 2022)
Author: Fazili M A, Bashir I, Ahmad M and Gellani S N.
 Title: Factors affecting secondary metabolites
production in tissue culture. ( Feb, 2022)
Author: Anjali Singh.
 Title: Plant in vitro Culture Technologies; A Promise
Into Factories of Secondary Metabolites Against
COVID-19( March, 2021)
Author: Khan T, Khan M A, Karam K, Ullah N,
Mashwani Z R and Nadhman A. 68
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https://doi.org/10.1111/j.1469-8137.1994.tb02968.x
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with active principles for pharmacological importance. Phytochemicals - Source of Antioxidants and
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• S. Ramírez-Gómez, X. et al. (2020) “Plant metabolites in plant defense against pathogens,” Plant
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https://doi.org/10.1002/9783527619771.ch12
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Secondary Metabolites: Their Function and Evolution, 255–275.
https://doi.org/10.1002/9780470514344.ch15
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Any
Questions?

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Thank You

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