plant tissue culture as a tool for production of secondary metabolites.

1. PLANT
TISSUE
CULTURE
Ph. Islam Adel
Abdelhakim
1

2. Definition:
• Plant tissue culture is the science of growing plant
cells, tissues or organs isolated from the Mother plant,
on artificial media in vitro under controlled conditions.
2
Edwin F. George, M. A. H., Geert-Jan De Klerk (2008). "Plant Propagation by Tissue Culture
3rd Edition." Volume 1. The Background.

3. History
• HABERLANDT is considered to be the father of plant tissue
culture who conceived the concept of totipotency & cell culture
in 1902.
• Totipotency is the ability of a single cell to divide
and produce all of the differentiated cells in an
organism.
• He was the first to consider culturing cells aseptically in a
nutrient solution.
• However, he was unsuccesful in his culture as he failed to
recognize that cell differentiation requires plant growth
regulators
3
Krikorian, A. and D. L. Berquam (1969). "Plant cell and tissue cultures: the role of
Haberlandt." The Botanical Review 35(1): 59-67.

4. Types of Growth:
 Organized Growth:
• It occurs when plant cell or tissue (explant) are
transferred to culture media and continue to grow with
their structure preserved.
 Unorganized Growth:
• It occurs when pieces of whole plants are cultured in
vitro and the cells aggregate and typically lack any
recognizable structure.
e.g. Callus and Cell Suspension cultures.
4
Edwin F. George, M. A. H., Geert-Jan De Klerk (2008). "Plant Propagation by Tissue Culture
3rd Edition." Volume 1. The Background.

5. 5
Organized growth Unorganized growth
“Callus”
Unorganized growth
“Suspension”

6. Establishment of tissue culture systems
• Explant Selection:
May be from root, stem, leaves or buds.
• Isolation & Sterilization:
Prevention of contamination of
tissue culture media is important
for the whole process of plant
Propagation. So, all the work should
Be performed in special rooms or
Inside hoods or cabinets from which
Microorganisms are excluded.
6
Edwin F. George, M. A. H., Geert-Jan De Klerk (2008). "Plant Propagation by Tissue Culture
3rd Edition." Volume 1. The Background.

7. Suspension Culture System
7
Explant selection and surface sterilization
Callus culture induction on solid media
supplemented with sucrose, hormones and
agar
Callus is introduced to agitated liquid
media
Gaosheng, H. and J. Jingming (2012). Production of useful secondary metabolites
through regulation of biosynthetic pathway in cell and tissue suspension culture of
medicinal plants, INTECH Open Access Publisher.

8. Hairy root culture
• It is the culture produced after infection of explant and
culture by the gram negative soil bacterium Agrobacterium
rhizogenes.
• This process leads to formation of naturally occuring hairy
root disease.
8
Shanks, J. V. and J. Morgan (1999). "Plant ‘hairy root’culture." Current Opinion in
Biotechnology 10(2): 151-155.

9. The process…
9
Plant cellAgrobactrium cell

10. Induction of hairy root culture
10
Explants are wounded and inoculated with A.
rhizogenes
2-3 days later, explant transferred to a solid media
with antibiotic as Cefotaxim or Vancomycin.
Hairy roots will be induced within 1-4 weeks
depending on the plant species
Hairy roots separated and cultured on solid media
until enough biomass is obtained
Gaosheng, H. and J. Jingming (2012). Production of useful secondary metabolites
through regulation of biosynthetic pathway in cell and tissue suspension culture of
medicinal plants, INTECH Open Access Publisher.

11. Advantages of hairy root cultures:
• The hairy root system is genetically and
biosynthetically stable.
• High production of secondary metabolites.
• The culture can grow under phyto-hormone free
conditions.
• The culture shows fast growth which reduces the
culture time and easy the handling.
11
Shanks, J. V. and J. Morgan (1999). "Plant ‘hairy root’culture." Current Opinion in
Biotechnology 10(2): 151-155.

12. • Growth and development of plant cultures usually
also depends on the addition of plant growth
regulators to the medium.
• They are important in plant tissue culture since they
play vital roles in stem elongation, tropism, and
apical dominance.
• They are generally classified into the following
groups; auxins, cytokinins, gibberellins and abscisic
acid.
12
Plant growth regulators
Skoog, F. and C. Miller (1957). Chemical regularion of growth and organ formation in plant
fissue cultured, In vitro. Symp. Soc. Exp. Biol., v. 11, p. 118-131.

13. • Moreover, proportion of auxins to cytokinins
determines the type and extent of organogenesis in
plant cell cultures.
• ↑ Auxin ↓ Cytokinin = Root Development
• ↑ Cytokinin ↓ Auxin = Shoot Development
• Auxin = Cytokinin = Callus Development
13
Zeatin- natural cytokinin
Skoog, F. and C. Miller (1957). Chemical regularion of growth and organ formation in plant
fissue cultured, In vitro. Symp. Soc. Exp. Biol., v. 11, p. 118-131.

14. In Vitro Cultures and Production of
Important Secondary Metabolites
• Secondary metabolites play important role in defencing
insects, herbivores, microbial pathogens, and facilitating
pollination and reproduction.
• Based on the structures, the secondary metabolites can be
classified into alkaloids, flavonoids, phenylpropanoids,
terpenoids, steroids, tannins and proteins.
• These compounds are biosynthesized through series
enzyme catalyzed reactions using simple building blocks in
different ways.
14
Gaosheng, H. and J. Jingming (2012). Production of useful secondary metabolites
through regulation of biosynthetic pathway in cell and tissue suspension culture of
medicinal plants, INTECH Open Access Publisher.

15. The main biosynthetic pathways include:
• Shikimic acid pathway ………. (phenylpropanoids).
• Mevalonic acid pathway …….. (Sterols & triterpenes).
• Amino acid pathway ………….. (alkaloids).
• Acetate pathway ……………….(fatty acids).
• Combined pathways …………..(flavonoids).
15
Gaosheng, H. and J. Jingming (2012). Production of useful secondary metabolites
through regulation of biosynthetic pathway in cell and tissue suspension culture of
medicinal plants, INTECH Open Access Publisher.

16. 16
Strategies developed to maximize the production
of target compounds:
Over-expressing the key gene(s) involved in the
biosynthetic pathway.
Blocking the competitive branches of biosynthesizing
target compounds
increase the biomass of vagetation growth, and increase
the production of target compounds.
Introduce key genes into microbes and use combinatorial
biosynthesis to produce target compounds or important
intermediates.
Gaosheng, H. and J. Jingming (2012). Production of useful secondary metabolites
through regulation of biosynthetic pathway in cell and tissue suspension culture of
medicinal plants, INTECH Open Access Publisher.

17. • Elicitors are substances that can trigger the hypersensitive
reaction in treated plant cells. Due to the effective up-regulation
of genes expression, and activation of secondary metabolism.
• elicitors are used widely in medicinal plant cell and tissue
culture to maximize the production of target compounds.
• Biotic elicitors: include fungal polysaccharides, proteins, cell
debris and conidium.
• Abiotic elicitors: include heavy metals ions,, UV lights, osmotic
stress and even sonication have all been reported to have
positive effects towards improvement of secondary metabolites.
17
Elicitors and signaling pathways
Gaosheng, H. and J. Jingming (2012). Production of useful secondary metabolites
through regulation of biosynthetic pathway in cell and tissue suspension culture of
medicinal plants, INTECH Open Access Publisher.

18. • To improve the yield of secondary metabolites in plant
cell culture, precursor feeding is an effective approach.
• Precursors are compounds existed in upstream of
target compounds in biosynthetic pathway and most
intermediates can be used as precursors.
• The concentration of precursors determines the
reaction speed. At higher concentration, the reaction
speed is usually higher than that when precursor
concentration is lower.
18
Precursor feeding
Gaosheng, H. and J. Jingming (2012). Production of useful secondary metabolites
through regulation of biosynthetic pathway in cell and tissue suspension culture of
medicinal plants, INTECH Open Access Publisher.

19. Plant name Active ingredients Culture type Reference
Taxus spp. Taxol Suspension Malik, S., et al. (2011), "
Process Biochemistry
46(1): 23-34.
Capsicum annuum Capsaicin Suspension Johnson et al., 1990,
Plant Sci. 70: 223-229.
Catharanthus roseus Indole alkaloids Suspension Moreno et al., 1993,
Plant Cell Rep. 12: 702-
705.
Ephedra spp. L- Ephedrine Suspension O’Dowd et al., 1993,
Plant Cell Tiss. Org.
Cult. 34: 149-155.
Cassia acutifolia Anthraquinones Suspension Nazif et al., 2000,
Fitoterapia 71: 34-40.
Coffea arabica Caffeine Callus Waller et al., 1983, Plant
Cell Rep. 2: 109-112.
19
Bioactive secondary metabolites from plant tissue
cultures.
Hussain, M. S., et al. (2012). "Current approaches toward production of secondary plant
metabolites." Journal of pharmacy & bioallied sciences 4(1): 10.

20. • Taxol, a complex diterpene anticancer alkaloid drug
found in 1971, by Wani et al. from the Pacific yew tree,
Taxus brevifolia.
• At present the drug is approved
For clinical treatment of ovarian
and breast cancer by the FDA.
• However, the supply of taxol for clinical use is limited
from either its natural source or by total synthesis.
20
Production of Taxol from Taxus Sp.
Vanisree, M., et al. (2004). "Studies on the production of some important secondary
metabolites from medicinal plants by plant tissue cultures." Bot. Bull. Acad. Sin 45(1):
1-22.

21. Optimization of culture conditions:
• Dark conditions are suitable for the growth of cells and
taxol Production.
• A biotic elicitor from Rhyzopus stelonifera fungus
(25mg/L) used in combination with the abiotic elicitors
methyl jasmonate (10mg/L) and salicylic acid
(100mg/L)was shown to improve taxol production 16-fold
when added at day 25–30 of culture to a growth
medium.
21
Malik, S., et al. (2011). "Production of the anticancer drug taxol in Taxus baccata
suspension cultures: a review." Process Biochemistry 46(1): 23-34.

22. • Supplementation of the medium sucrose, phenylalanine
and ammonium citrate resulted in 5.6-fold higher taxol
production (13.75mg/L) compared with the control
(2.5mg/L).
• Currently, bioreactors of up to 75,000 L are being
employed for the commercial production of paclitaxel
from cell cultures by Phyton Biotech, ESCAgenetic,
Samyang Genex, Nattermann (Germany)
22
Malik, S., et al. (2011). "Production of the anticancer drug taxol in Taxus baccata
suspension cultures: a review." Process Biochemistry 46(1): 23-34.

23. • Capsaicin is an alkaloid obtained from Capsicum spp.
• It is used mainly as a pungent food additive in formulated
foods.
• Capsaicin is also used in pharmaceutical preparations as a
digestive stimulant and for rheumatic disorders.
23
Production of Capsaicin
Vanisree, M., et al. (2004). "Studies on the production of some important secondary
metabolites from medicinal plants by plant tissue cultures." Bot. Bull. Acad. Sin 45(1):
1-22.

24. • Suspension cultures of Capsicum frutescens
produce low levels of capsaicin, but immobilizing the
cells in reticulated polyurethane foam can increase
production approximately 100- fold.
• Supplying the medium with 2,4-D (2 mg/l), Kinetin
(0.5 mg/l) and Sucrose (3%) are the optimum
condition for capsaicin production.
• Further improvements can be brought about by
supplying precursors such as isocapric acid.
24
Vanisree, M., et al. (2004). "Studies on the production of some important secondary
metabolites from medicinal plants by plant tissue cultures." Bot. Bull. Acad. Sin 45(1):
1-22.

25. Thanks
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