2. In plant cell culture plant cells, tissues and organ are grown on artificial
media
It is an important tool for basic studies on plant biochemistry and
molecular biology
These cultures represent a more versatile & powerful system than whole
plants to obtain different types of products.
Also used as Expression system with advantages over microbial Systems
Plant Cell Culture
3. These cultures are believed to possess the potential to produce useful
“secondary metabolites”.
Expression of foreign gene (s) in plant cell cultures by genetic
transformation, which enables production of recombinant proteins,
opened a new avenue for production of therapeutically valuable proteins
The technique depends mainly on the concept of totipotentiality.
Which is the ability of a single cell to express the full genome by cell
division.
4.
5. Maintenance of Plant Cell Culture
Tissue culture produces
clones, in which all product
cells have the same
genotype (unless affected
by mutation during culture)
6. In 1902, a German physiologist, Gottlieb Haberlandt for the first time
attempted to culture isolated single palisade cells
This technology is being widely used for large scale plant multiplication
Small pieces of tissue (explants) can be used to produce 100s and 1000s
of copies of plants
Endangered, threatened and rare species have conserved by this
technique
Plant Tissue Culture
7. Tissue culture has several critical requirements:
Appropriate tissue (some tissues culture better than others)
A suitable growth medium containing energy sources and inorganic salts
to supply cell growth needs. This can be liquid or semisolid
Aseptic (sterile) conditions, as microorganisms grow much more quickly
than plant and animal tissue and can overrun a culture
Growth regulators - in plants, both auxins & cytokinins
9. Preservation of plant biodiversity is essential for classical & modern (GE)
plant breeding programmes.
Provides a source of compounds to the pharmaceutical, food & crop
protection industries.
Storage of desiccated seeds at low temperature, is not applicable to
crops that do not produce seed (e.g., bananas) or with recalcitrant seed
as well as to plant species that are propagated vegetatively to preserve
the unique genomic constitution of cultivars
Preservation of Plant Cells
10. Minimize growth and development of the plant in vitro
Maintain the viability of the stored material at the highest possible level
Maintain the full developmental and functional potential of the stored
material
Make significant savings in labour input, materials and commitment of
specialized growing facilities
Properties of Storage System
11. Following methods for Plant Preservation are available
Minimal Growth at Reduced Temperature
Cryopreservation
Storage/Preservation Techniques
12. It is most obvious & simple way of influencing the growth rate of plant material
Suitable for differentiated plantlets, and entire cultures
Principle: The temperature is reduced for prolonged periods without significant injury to
the plant tissues
In conjunction with reduced temperature, growth might be further
limited by the addition of:
Inhibitory levels of growth regulators
OR Osmotic agents
Minimal Growth Storage
13. The term ‘cryopreservation’ (cryogenic preservation) refers to the
storage of cells, tissues and organs at the ultra-low temperature of
liquid nitrogen (-196°C).
At this temperature, the vegetative cells enters in a state of “absolute
quiescence”.
Application of cryogenics to the conservation of plant material, proposed
for the first time in the year 1968 for the maintenance of cell cultures.
Cryopreservation
14. Cryopreservation of biological tissues can be successful only if intra-
cellular ice crystal formation is avoided
Liquid Nitrogen is the most common medium for cryostorage as it is
relatively inexpensive and readily available
Following cultures can be cryopreserved
1. Cells 2. Hairy Root Cultures
3. Callus Cultures 4. Embryogenic Cultures
5. Genetically transformed Cultures
6. Somatic embryos 7. pollen & plant buds
15.
16. Once material has been successfully cooled to LN temperatures, it can
be conserved indefinitely
Once in storage, no risk of new contamination by fungus or bacteria
Low storage costs
Minimal space requirements
Reduced labor maintenance
Main Advantages of Cryostorage
19. Technique involves the simple dehydration of plant material before
cryogenic storage in LN
This can be done by slow cooling of plant tissue to a temp of -40°C
This forces the formation of extracellular ice ahead of intracellular ice
Results in an outflow of water from the cells due to osmotic imbalance
and, consequently, dehydration takes place
Classical slow-cooling
21. Vitrification refers to the physical process of transition of an aqueous
solution into an amorphous and glassy (i.e., non-crystalline) state
Two requirements must be met for a cell to vitrify:
(i) Rapid freezing rates and
(ii) A concentrated cellular solution
It involves the treatment of tissues in a mixture of highly concentrated
penetrating and non-penetrating CPAs applied at non-freezing
temperatures, followed by rapid cooling in LN
Vitrification
24. Involves encapsulating shoot tips, somatic embryos or callus cells within
alginate beads
Followed by incubation in media with high sugar concentrations in order
to raise intracellular solute concentrations
Silica gel or airflow is used to dehydrate the beads until the moisture
content drops to 20-30%
Immersion in LN
Encapsulation Dehydration
26. This technique is a modification of the basic vitrification protocol
It involves placing the sample within a droplet of of cryoprotective
solution on a piece of Aluminium foil before immersion in LN
This approach achieves higher cooling and re-warming rates
The droplet-vitrification protocol has been successfully applied in the
cryopreservation of garlic and chrysanthemum, yams, lily, potato and
other plants
Droplet Vitrification
28. Another modification of the vitrification approach
Combines elements of the encapsulation/dehydration method with the
vitrification method
Shoot tips or calluses are first encapsulated in alginate beads and then
the encapsulated materials incubated in a vitrification solution to
promote sufficient dehydration and vitrification rather than dehydration
Encapsulation-vitrification
30. Regeneration is an important criterion for the cryopreserved materials
It is done by putting the ampoule containing the frozen tips in a warm
water bath (35 to 40°C) with a vigorous swirling action up to the point of
ice disappearance.
After thawing quickly transfer the tubes to a water bath maintained at
room temperature
Sub-culturing of the thawed samples
Regeneration of plants after Cryopreservation
31.
32. Regrowth of the plants from stored tissues or cells is the only test of
survival of plant materials.
Viability of the explants/cells after cryopreservation can be assessed
by:
Flourescein diacetate (FDA) Test
Triphenyltetrazolium chloride (TTC) Test
Evan’s Blue Staining
Viability Cell Tests
33. The viable cells covert the FDA into fluorescin (Green) due to esterase
Cells with an intact plasma membrane fluoresce green in ultraviolet light
The larger molecules of fluorescin are unable to pass through the
membrane.
Flourescein diacetate (FDA) Test
34. The viable cells which contain the enzyme mitochondrial dehydrogenase
will give positive TTC test
TTC Mitochondrial Dehydrogenase Formazon (RED)
The mitochondrial dehydrogenase reduces the tetrazolium salt and
converts it into a red Formazon which can be assayed spectrometrically
Triphenyltetrazolium chloride (TTC) Test
35. One drop of 0.1% solution of Evan’s blue is added to cell suspension on a
microscope slide and observed under light microscope.
Only Non-viable cells (dead cells) stain with Evan’s blue
% of viable cells= No. of fluorescent cells Х 100
Total no. of cells (Viable + Dead)
Evan’s Blue Staining
36. Thorpe T (2007) History of plant tissue culture. J. Mol. Microbial
Biotechnol. 37: 169-180.
Anthony, P.; Jerodar, N.B., Lowe, K.C., Power, J.B. & Davey, M.R. (1996).
Pluronic F-68 increases the post-thaw growth of cryopreserved plant
cells. Cryobiology, Vol.33, 508-514.
Bajaj, Y.P.S. (1995). Biotecnology Agriculture and Forestry 32
Cryopreservation of Plant Germplasm, Springer-Verlag, ISBN 3-540-
57451-4.
References