This document provides an overview of basic techniques in plant tissue culture and plant cell immobilization. It discusses that tissue culture involves growing plant cells or tissues artificially in a controlled environment. The two main techniques are static culture (callus culture) and suspension culture. Static culture uses isolated plant tissue on a nutrient medium to form an unorganized callus mass, while suspension culture grows isolated cells or small cell aggregates in liquid medium. The document also describes various methods of plant cell immobilization including adsorption, covalent attachment, and entrapment in natural or synthetic polymers, which has advantages like continuous processing but also challenges like reduced biosynthetic capacity.

1. BASIC TECHNIQUES OF PLANT
TISSUE CULTURE AND PLANT
CELL IMMOBILIZATION
BY
NIVETHA.B
M .PHARMACY
(PHARMACOGNOSY) I YEAR1

2. WHAT IS TISSUE CULTURE ?
 Tissue culture is the term used for “the
process of growing cells artificially in the
laboratory”.
 Tissue culture is invitro cultivation of plant
cell or tissue under aseptic and controlled
environmental conditions,
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3. in liquid or on semisolid well defined nutrient
medium for the production of primary and
secondary metabolites or to regenerate plant.
 Tissue culture involves both plant and animal
cells.
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4. BASIC TECHNIQUES OT PLANT
TISSUE CULTURE
The technique of plant tissue and cell culture has
evolved over decades. This technique combine
with recent advances in developmental, cellular,
molecular genetic, metabolic engineering,
genetic transformation and using conventional
plant breeding have turned plant biotechnology
into an exciting research field with a significant
impact on pharmaceutical industries, agriculture,
horticulture and forestry.
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5. TYPES OF TECHNIQUES :
There are mainly two major techniques in
plant tissue culture. They are explained as
follows,
 STATIC CULTURE
 SUSPENSION CULTURE
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6. STATIC CULTURE
• Static culture is otherwise known as callus
culture or solid culture.
• In this technique isolated piece of plant tissue
is cultured on a nutrient medium.
• Plant growth regulators such as auxins ,
cytokinins, and gibberllins are supplemented
into the medium to initiate callus formation.
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7. • Finally, an unorganized mass of cell appears
and it is called as callus.
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8. 8
• This callus is transferred on to different
media to regenerate plants.
• This callus culture technique is easier and
convenient for initial maintanence of cell
lines and also for carrying out the
investigation studies related to
organogenesis.

9. CALLUS GROWTH
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10. SUSPENSION CULTURE
• Suspension culture is also known as liquid
culture.
• It is the culture of isolated cells or very small
cell aggregates dispersed in liquid medium.
The cell suspension is obtained by agitating
pieces of callus in liquid medium on gyrating
shaker.
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11. Growing cells in a liquid medium under
controlled conditions.
Transfer of an established callus culture into
the liquid medium in Erlenmeyer flask.
Medium with high auxin content, appropriate
concentration of auxin and yeast or auxin vs
kinetin helps to maintain suspension.
Incubated at 25˚ C in darkness or with less light
intensity fluorescent light.
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12. It contains a uniform suspension of separate cells in a
liquid medium
callus
liquid medium
agitated continuously
finally cells separated
sub-culture the cells
This can be achieved by rotary shaker attached within
the incubator at a rate of 50-150 rpm .
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13. TYPES OF SUSPENSION CULTURE
There are three types of suspension culture.
1.Batch suspension culture
2. Continuous culture
(I)Open type
(II)Close type
3.Semi-continuous culture
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14. BATCH CULTURE
• These cultures are maintained continously by
propagating a small aliquot of inoculum in the
moving liquid and transferring it to fresh
medium at regular intervals.
• Generally cell suspension are grown in
flasks(100-250 ml) containing 25-27 ml of the
culture medium.
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15. • When the cell number in suspension culture
is plotted against the time of incubation , a
growth curve is obtained.
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16. CONTINUOUS CULTURE
• In this system , the liquid medium is
continuously replaced by the fresh liquid
medium to stabilize the physiological stage of
growing cells.
• Cell proliferation takes place under constant
conditions
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17. • As a result ,the active growth phase of the cell
declines the depletion of nutrient.
• The cells passing through out flowing medium
are separated mechanically.
• The cells are always kept in exponential
growth phase.
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18. MEASUREMENT OF GROWTH OF
CELL CULTURES
Methods used to determine are..
CELL NUMBER
• By counting the cell number in
haemocytometer under a microscope.
• Suspension culture is preferable.
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19. 19

20. PACKED CELL VOLUME
Cell suspension is transfer to graduated
centrifuge.
• Centrifuged at 2000 rpm for 5mints.
• Cell will form pellets called biomass volume,
expressed by ml-1
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21. 21

22. 22

23. FRESH CELL WEIGHT
• When cells increase in number, the liquid will
be turbid.
• As a result optical density altered, detected by
colorimeter.
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24. 24

25. VIABLE CELL TEST
• The staining method such as fluorescein di-
acetate is used for accessing the cell viability.
• Dead cells appear as fluorescein red.
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26. 26

27. PLANT CELL IMMOBILIZATION
• Immobilization is the newest culture of plant
cell and considered has to be the most
‘natural’.
• It has been defined as a technique, which
confines to a catalytically active enzyme or to
a cell within a reactor system and prevents its
entry into mobile phase, which carries the
substrate and product.
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28. ADVANTAGES:
• Retention of biomass enables its continuous
reutilization as a product system, a definite
advantage with slow growing plant cells
e.g.Papaver somniferum have remained stable
and active for up to six months.
• Continuous process: Immobilization allows a
continuous process ,which increase volumetric
productivity and allows the removal of
metabolic inhibitors.
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29. • Separation of cells from medium:The
immobilization separates cells from medium
and the desired product in extra cellular,
which will simplify down stream processing
compared to extraction from tissue.
• Decoupling of growth and product formation:
Immobilization is compatible with non growth
associated product formation.
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30. • Reduces problems such as aggegrate,growth
and foaming.
• High biomass levels.
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31. DISADVANTAGES:
• Secretion of secondary metabolites requires
cellular transport or a artificially altered
membrane permeability’
• The efficiency of the production process
depends on the rate of release of products
rather than actual raye of biosynthesis.
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32. • The immobiliztion process may reduce
biosynthetic capacity.
• Products must be released from the cell into
medium.
• Relese of single cells from cell aggregate may
make processing of the product more
difficult.
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33. TYPES OF IMMOBILIZATION
• Direct intracellular binding due to natural
affinity(adsorption , adhesion and
agglutination).
• Covalent coupling
• Intracellular connection via bi or poly
functional reagent(cross-linking)
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34. • Mixing with suitable materials,changing their
consistency with temperature(embedding).
• Physical retention within the framework of
diverse pore size and permeability
(entrapment , micro encapsulation).
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35. METHOD OF IMMOBILIZATION
1. Adsorption
2. Covalent attachment
3. Entrapment
a) Natural polymer
Alginate
Agar
Agarose
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36. b)Synthetic polymer
Polyacrylamide
c)Porous structure
Polyurethane foam
d)Membranes
Hollow fiber
Flat plate
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37. ENTRAPMENT:
• GEL ENTRAPMENT BY POLYMERIZATION:
A monomer or a mixture of monomers is
polymerized in the presence of cell
suspension, which is entrapped inside the
lattice of the polymer.
The most common example is
polyacrylamide.
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38. GEL ENTRAPMENT FORMATION BY
PRECIPITATION:
• Gels may be formed by precipitation of some
natural and synthetic polymers by changing
one or more parameters in the solution, such
as temperature, salinity or pH of solvent .
• Several methods can be used for entrapment.
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39. Gel entrapment by precipitation
SPECIES GEL
Catharanthus roseus Agarose , Agrose, Agar
Silybum marianum Agar
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40. SURFACE IMMOBILIZATION:
• Surface immobilization may occur on both
natural and other matrices.
• Eg for natural matrices are deeper callus
layers and cellulose. For synthetic steel and
nylon are used.
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41. REFERENCE:
• Medicinal Plant Biotechnology by Ciddi
Veeresham.
• Pharmaceutical Biotechnology by S S Kori.
• Trease and evans pharmacognosy by W.C evans.
• A textbook of industrial pharmacognosy by
A.N.Kalia.
• Pharmacognosy and photochemistry-part 2 by
Vinod.D.Rangari. 41

42. THANK YOU
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