A seminar about Production of Plant Secondary Metabolites.

1. PRODUCTION OF
SECONDARY
METABOLITES
PHARMACEUTICAL BIOTECHNOLOGY
FACULTY OF PHARMACY, CLINICAL PROGRAM
MANSOURA UNIVERSITY

2. Done By
 Ahmed Megahed Al Jamal
 Ahmed Mohamed Al Sukkary
 Ahmed Osama Fathy
 Ahmed Raafat Alaa Eldin
 Mahmoud Saad Al Abbasy
 Moataz Hesham Al Sheikh
 Mohamed Ahmed El Sharkawy
 Mohamad Salah Mansour
 Mostafa Amgad Zeyada
 Tariq Hegazy Salama

3. PTC
Plant Tissue Culture

4. Definition
It is the growth of plant cells in vitro (in
glass, outside an intact plant) on an artificial
media under complete aseptic conditions or
axenic conditions (complete sterilized
conditions).

5. Advantages and Disadvantages
Advantages Disadvantages
Small part of plant is required Maintaining a sterile environment
Produce many plantlets in small areas Expensive lab facilities
Disease-free plants Poor photosynthesis
Pest-resistant Water loss
Need highly trained persons

6. Applications of Plant Tissue Culture
 Morphological Differentiation (micro propagation), which can be done by:
a- axillary buds
b- adventitious shoots:
i. Organogenesis
ii. Somatic embryogenesis
c- culture organogenesis
 Protoplast culture
 Production of plant secondary metabolites

7. Secondary Metabolite

8. Intro
 Secondary metabolites are organic compounds that are
not directly involved in the normal growth, development,
or reproduction of an organism.
 Unlike primary metabolites, absence of secondary
metabolites does not result in immediate death, but
rather in long-term impairment of the organism's
survivability, fecundity, or aesthetics, or perhaps in no
significant change at all.

9. Intro
 Secondary metabolites are often restricted to a narrow
set of species within a phylogenetic group.
 Secondary metabolites often play an important role in
plant defense against herbivores and other interspecies
defenses.
 Humans use secondary metabolites as medicines,
flavorings, and recreational drugs.

10. Types
 There is no fixed, commonly agreed upon system for
classifying secondary metabolites. Based on their
biosynthetic origins, plant secondary metabolites can be
divided into three major groups:
1. Flavonoids and allied phenolic and polyphenolic compounds,
2. Terpenoids
3. Nitrogen-containing alkaloids and sulphur-containing compounds.

11. Why culture lose its potential to
synthesize valuable chemicals?
 Competition between primary and secondary pathways for
key intermediates & enzymes [the 1ry metabolites more
imp. For plant growth]
 Low levels of expression of key enzymes at rate limiting
steps in a pathway [affected by age so juvenile more
producer]
 Uncoordinated fragmentary expression of genes, leading
to partial operation of the pathway

12. Screening of the culture for secondary
metabolites
 Cells cloning method
 Visual screening for colored products such as “shikonin” and
“naphthaquinone”
 Micro spectrophotometric and micro flurometric have been used to
identify clones in which products with absorbance or auto fluorescent
properties accumulate
 For colorless products, cells are squashed onto filter paper and the
filter paper was sprayed with a specific reagent to identify high
yielding clones
 Radioimmunoassays

13. Control of production of secondary
metabolite from PTC
A) Abiological control:
Related to Environment.
B) Biological control:
Related to the Plant itself.

14. Abiological control
Related to environment

15. A) Light
 Examples:
1- Volatile oils of Ruta graveolensis
2- Shikonin derivatives in Lithospermum culture
3- Anthocyanins in Aralia cordata

16. B) Exogenous plant growth regulators
 Examples:
1- Nicotine was inhibited by 2,4-D and promoted by
kinetin
2- Shikonin in Lithospemum cultures was inhibited by 2,4-
D and NAA and promoted by IAA.

17. C) Nutrients
 Examples:
1- Catechol tannins in Sycamore suspension cultures was promoted by
increasing the carbon to nitrogen ratio.
2- Shikonin production by Lithospermum culture was highly promoted
by:
i. increasing the sucrose content in the medium
ii. omission of ammonium salts and the inclusion of nitrate as the sole
nitrogen source.

18. D) Addition of precursors to the culture
medium
 Tropic acid in the media of Datura and Scopolia potentiate
tropane alkaloids
 0-succinylbenzoic acid to the cultures of Morinda resulted
in a two folds increase in the accumulation of
anthraquinones.
 Addition of phenylalanine to the culture of Taxus
cupsidata cultures potentiated the levels of Rosmarinic
acid and Taxol respectively.
 Trp. Is starting material for indole alkaloids [ergot
alkaloids, curare alkaloids]

19. E) Incorporation of inducers (stress
factors)
 Biotic in nature
 The abiotic elicitation
Both types of elicitation (alexin addition) act by stress mechanism(s)

20. F) Mutagenesis in the cultured cells
 Examples:
i. C. roseus produced more catharanthine
ii. Tobacco cell culture accumulated higher levels of phenolics
 Methods:
Chemicals Physical
• Ethylmethane sulfonate
• N-ethyl-N-nitrosourea
• X-rays

21. G) Immobilization of the culture or of
the key enzymes
 immobilization of cells occur by use of specific
types of gels.
ENTRAPMENT WITHIN A
MATRIX
ATTACHEMENT OR ADSORPTION TO
A PREFORMED CARRIER
SELF AGGREGATION OF
CELLS
CELLS CONTAINED BEHIND A
BARRIER

22. G) Immobilization of the culture or of
the key enzymes
 Calcium alginate entrapment in which the plant
cells are embedded within an alginate bead matrix
and jelled with calcium as divalent cation.
 Physiologically inactive intermediate is produced by the cultivated
plant cell in high amount, while the physiologically active metabolite
is present in rather small quantities i.e.
1. Papaver somniferum ,20-30 % of the codinone are converted to codeine
2. Catharanthus roseus culture immobilized on polyacrylamide gel enhanced the
production Ajmalicine; antitumor alkaloid.

23. G) Immobilization of the culture or of
the key enzymes
 Procedures:
1.gel sterilization
2.column loading with the gel
3.column loading with suspension culture
[make good distribution of cells]
4.cells will grown & fill voids volumes
between gels  this will apply pressure
[mechanical stress on the cells so will
lead to opening of 2ry metabolic
pathways & synthesis of new
metabolites as defense mechanisms]
ADSORBENT
BEADS
CELL
Particulate
containing process
fluid
A B

24. Biological control
Related to the plant itself

25. A) Study The growth of the culture
 Parallel with growth of cells, Example: nicotine
production by Nictiana
 Delayed until cell growth declines: polyphenols and
shikonin production by Lithospermum erythrorhizon.

26. A) Study The growth of the culture
 Occurs in the lag phase of
growth cycle, Example: as in
the production of diosgenin by
Dioscrea cultures.
Adapt the two-stage culture for
production of specific metabolite
[in which the composition of the
medium will changed by addition
of the precursors of the required
metabolite at the phase of
production].

27. A) Study The growth of the culture
 High cell density culture:
another method used in case of Coptis japonica
produce berberine [anti AD] , 3.5 g/L, of
fermenter At 55 g/L of the cell mass density.

28. B) Cell cloning and repeated selection of
high yielding strains
 Morphological appearance.
 Pigmentation: Visual screening for colored products such as shikonin
and naphthaquinone.
 Fluorescence under UV-light: Micro spectrophotometric and micro
flurometric have been used to identify clones in which products with
absorbance or auto-fluroscent properties accumulate.
 Induction of mutants by:
1. Chemicals, as nitrosurea or ethylmethylsulophonate (EMS).
2. Physical, as irradiation.

29. C) Morphological differentiation
(Organogenesis) or bio-differentiation
 Environmental stimuli.
 Genetic stimuli.
Examples:
1. Shoot differentiation in undifferentiated culture, example: tobacco induced more
nicotine biosynthesis.
2. Roots in suspension culture, example: Scopolia parviflora induced synthesis of
more tropane alkaloids.
3. The ordinary culture of Bupleururn falcatum was unable to produce saikasaponins
a, b, c, d [anticancer] but Root culture was high producer as that of the original
plant roots.
4. Cardenolide synthesis in shoot differentiated Digitalis culture
5. Harmane alkaloids in Peganum harmala culture.

30. D) Genetic control of secondary
metabolites production
 Definition
Application of genetic engineering (gene technology)
techniques to increase yield of 2ry plant metabolites.
 Axenic = main gene In the plant cells
 Transgenic genes = after integration of bacterial gene plasmid in the
plant DNA.

31. D) Genetic control of secondary
metabolites production
 Types of bacteria:
1. Agrobacterium tumifaciens: Cause crown gall tumor.
2. Agrobacterium rhisogenes: Cause Hairy Root (HR) tumor
or root tratoma.

32. Agrobacterium tumifaciens

33. Agrobacterium tumifaciens
 Entry:
enters plant via wound sites.
 Spread:
Widespread soil bacterium

34. Agrobacterium tumifaciens
 Disease:
 Crown gall disease
(causes cancerous-like
tumors).
 Gall grows and divides
(even if bacteria dies):
1. Gall tissues have high
[CK] and [IAA]
2. Opines (unusual a.a.)
made (C source)

35. Agrobacterium tumifaciens
 Plasmid transfer:
Agrobacterium stably transfers its plasmid into plant DNA by Interkingdom
horizontal DNA transfer.

36. Agrobacterium tumifaciens
 Plasmid types:
Specific plasmid known as Ti-plasmid [tumor-inducing plasmid] contain
T-DNA that integrated in the DNA of the plant cell leads to crown gall
tumor.
 Virulent native promoter:
 Constructed virulent A. tumefaciens (which is lysogenized with Cauliflower
mosaic virus 35 S):
1. Ca MV-35 S
2. Ca MV-19 S
So, we have 3 strains of A. tumefaciense.

37. Agrobacterium tumifaciens
 Response to plasmid strains:
is independent & give different results according to type
of the plant & no ideal but logic they are causing CGT but
some cause HRT as A. rhizogenes where:
 if constructed with Ca-MV-35  tumor morphology [shooty
tratoma] & tms plasmid formed
 if constructed with Ca-MV-19  tumor morphology [rooty
tratoma] & tmr plasmid formed.

38. Agrobacterium tumifaciens
 Morphological changes and effects on enzymes
Enzymes Responsible for synthesis of
cytokinines
Enzymes Responsible for synthesis of auxins
Isopentenyl Transferase 
enzyme encoded by IPT gene
Tryptophan Mono-oxygenase  encoded by TMS I
(IAAM)
Indole Acetamide Hydrolase  encoded by TMS II
(IAAH)
TMS = Tumor morphogenesis into shoots

39. Agrobacterium tumifaciens
 Results of bacteria
infection leads to:
1. Repression of
(IAAM) & (IAAH)
genes
2. Activation of IPT
gene so leads to
crown gall tumor

40. Agrobacterium rhisogenes

41. Agrobacterium rhisogenes
 Disease:
Hairy Root (HR) tumor; root tratoma.
 Plasmid and it's segment:
Ri-plasmid [root inducing plasmid].
Ri-DNA Plasmid
Left Segment
(TL-DNA)
B Locus A&C Loci
A&B
or C&B Loci
D Locus
Right Segment
(TR-DNA)
iaaM Gene iaaH Gene

42. Right segment Left segment
TR-DNA TL-DNA
Contain iaam & iaah genes Contain 4-genes A,B,C,D loci which also known as ORFS
10-11-12-15 [open reading frams]
When expressed leads to increased production of IAA &
increase production of auxins  leads to hairy root
growth
Integration of B locus [ORFs 11} causes Hair Root (HR)
formation i.e. Root Tratoma (RT)
Integration of A&C loci relatively induce RT depending on
plant genome
Integration of A & B or C & B loci has synergistic effect
on HR. induction
Locus D integration still unknown and it may cause
sensitization of cells to the endogenous IAA
Agrobacterium rhisogenes

43. Agrobacterium rhisogenes
Right segment Left segment
So finally Integration of TL - DNA loci and
appearance of HR may be attributed to:
a. Synthesis of non-transportal auxin
penylacetic acid (PAA)
b. Sensitization of cells to the action of
IAA
c. Synthesis of ethylenes which inhibits IAA
catabolism

44. Agrobacterium rhisogenes
 Integration of TL - DNA loci and appearance of
HR may be attributed to:
1. Synthesis of non-transportal auxin penylacetic acid
(PAA).
2. Sensitization of cells to the action of IAA.
3. Synthesis of ethylenes which inhibits IAA catabolism.

45. Agrobacterium rhisogenes
Detection of transformation
 Methods:
1. Morphological changes as [crown gall or HR]
2. Physiological changes as the transformed cells will
produce their own requirements of growth regulators
3. Biological changes as Rare amino acid derivatives "the
opines“ are formed in the transgenic tissues

46. Agrobacterium rhisogenes
Detection of transformation
 Types of amino acids:
 We can use either the Ti-plasmid or Ri-plasmid as vector for cloning of genes
responsible for production of 2ry metabolites in the plant cells for production
of transgenic plants
Octopine and Nopaline Agropine, Mannopine and Cucumopine
for Ti-plasmids for Ri-plasmids

47. Agrobacterium rhisogenes
Limitation of hairy root cultures
 Restricted to the production of secondary metabolites only produced in roots
such as
a. tropane alkaloids
b. steroidal precursors as solasodine
c. Catharanthus alkaloids
d. nicotine, quinoline alkaloids and the emetic alkaloids
 Some secondary metabolites occurring only in the aerial parts of the intact
plants were also found in the hairy roots of some species.
 Few hairy root cultures have been found capable of producing significant
levels of compounds synthesized by aerial parts of plants
 Rheological characteristics of heterogeneous system should also be taken into
consideration

48. Thank You!
SEMINAR DONE BY 4th YEAR CLASS OF CLINICAL PHARMACY, MANSOURA
UNIVERSITY