This document discusses plant cell culture and preservation techniques. It provides information on: - Growing plant cells, tissues, and organs in artificial media for research and production of products. - Techniques for maintaining and preserving plant cells including minimal growth at reduced temperatures, cryopreservation at liquid nitrogen temperatures, and various cryopreservation protocols like slow cooling, vitrification, and encapsulation-dehydration. - Assessing viability of cryopreserved plant cells through tests like FDA, TTC, and Evan's blue staining. Regeneration of plants from stored tissues or cells is the ultimate test of survival.

1. Maintenance & Preservation of
Plant cells

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.

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

8. Preservation of Plant Cells

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

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

17. Osmotic Pre-treatment
Cryprotection
Freezing
Thawing
Subsequent Regeneration
Steps of Cryopreservation

18. Classical slow-cooling,
Vitrification,
Droplet vitrification,
Encapsulation/dehydration
Encapsulation/vitrification protocols
Plant Cryopreservation Methods

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

20. 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

22. Vitrification

23. Dehydration

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

25. 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

27. Droplet
Method

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

29. 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

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

37. Thanku………… Hope your minds are not frozen 