This presentation is about to the Tissue Culture Technique like Culture room, Sterilization, Autoclaving, Nutrients media.

1. Presented By
Vivek Kumar Singh
M.Sc. (Botany) 4th semester
2016-2017

2. • The term tissue culture is commonly used in a
very wide sense to include in vitro culture of
plant cells, tissues as well as organs.
• Tissue Culture denotes the in vitro cultivation
of plant cells in an unorganized mass, e.g.
Callus Culture.
• Another term, cell culture is used for in vitro
culture of single or relatively small group of
plant cells, e.g. suspension culture.
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3. The culture of plant cells,
tissue, and organs such as root,
shoot tips and leaves in
artificial nutrient media
aseptically under defined physical
and chemical condition is referred
to as “Plant Tissue Culture”.
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4. • “Tissue Culture” is commonly
used as a broad term to describe
all type of plant cultures,
namely callus, cell, protoplast,
anther, meristem, embryo and
organ cultures.
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5. • The in vitro technique were
developed initially to demonstrate
the totipotency of plant cells
predicted by Gottlieb Haberlandt in
1902.
• Totipotency- is the ability of a
plant cell to perform all the
function of development which are
characteristic of zygote.
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6. • The first embryo culture was carried out
by Hanning in 1904; he cultured nearly
mature embryo of certain crucifer and
grew them to maturity.
• Haploid plants from pollen grain was
first produced by Maheshwari and Guha in
1964 by culturing the anther of Datura.
This marked the beginning of anther and
pollen culture for production of haploid
plant.
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7. • Plant protoplast are naked cells from
which cell wall has been removed. In
1960, Cocking produced large
quantities of protoplast by using
cell wall degradation enzyme.
• In 1972 Carlson and coworkers
produced the first somatic hybrid
plant by fusion the protoplast of
Nicotiana glauca and Nicotiana
longsdorfii.
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8. • The first continuously growing callus
culture were established from cambium
tissue in 1939 independently by
Gautheret, White and Nobecart.
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9. • The technique of in vitro cultivation of
plant cells or organ is –
1. Keep the plant cell free from microbes.
2. Suitable nutrient media
3. Environmental condition
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10. • Laboratory Space-
In general space for the following is
needed.
1. Washing, Drying and Storage of
Vessels
2. Preparation, Sterilization and
Storage of Media
3. Aseptic handling of explant and
cultures
4. Maintains of culture
5. Observation of culture
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11. • The culture room should have the
following facilities:
1. Controlled temperature (usually 25°±
2°C).
2. Culture racks fitted with light.
3. A shaker for agitation of liquid
cultures.
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12. • Generally, glass culture vessels are
used as they are cheaper, reusable and
autoclavable.
• It is desirable to use only borosilicate
or pyrex glassware as ordinary soda
glass may be toxic to some tissue.
• Tissue are generally cultured in culture
tube, flasks and Petri plate but many
specially designed dishes are also used.
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13. • Culture vessels and other labware are
generally soaked in a suitable nontoxic
detergent solution overnight, washed with
a suitable brush, thoroughly rinsed clean
with tap water, followed by rinse with
distilled water and dried in a hot air
oven 70 - 80° C.
• Washed culture vessels should be stored
in a dust proof cabinet.
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14. • All the material used in culture work must
be freed from microbes. This is done by
one of the following method.
1. Dry heat
2. Autoclaving
3. Filter Sterilization
4. Wiping with 70% Ethanol
5. Surface Sterilization
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15. • Glassware and Teflon plasticware, and
instruments may be sterilized by dry
heat in an oven at 160 - 180°C for 3
hours.
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16. • Culture vessels, (both empty and
containing media) are generally
sterilized by heating in autoclave or a
pressure cooker to 121° C at 15 pound
per square inch for 15 to 40 minutes the
time being longer for larger medium
volume.
• Sterilization during autoclaving depends
mainly on temperature.
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17. • Some growth regulators, e.g. GA3, urea,
certain vitamins, and enzyme are heat
labile. Such compound are filter
sterilized by passing their solution
through a membrane filter of 0.45 micron
or lower pore size.
• Laminar air flow cabinets are used to
create an aseptic working space by
blowing filter – sterilized air through
an enclosed space.
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18. • The surface that can not be
sterilized by other techniques
are sterilized by wiping them
thoroughly with 70% ethyl alcohol
and the alcohol is allowed to
dry.
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19. • All plant materials to be used for culture
are treated with an appropriate
sterilizing agent to inactive the microbes
present on their surface, this is called
Surface Sterilization.
• It depends mainly on the source and type
of tissue of the explant which will
determine the contamination load and
tolerance of the sterilizing agent.
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20. • The sterilizing Agent used for surface
disinfection are-
• Calcium Hypochlorite (9-10%)
• Sodium Hypochlorite (2%)
• Mercuric Chloride (0.1-1%)
• Silver nitrate (1%)
• Bromine water (1-2%)
• H2O2 (10-12%)
• Antibiotic (4-50 mg/l)
commonly used
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21. • A synthetic medium consist of only
chemically defined compounds.
Inorganic Nutrients- In addition to C, H,
and O, all nutrient media provide the 12
element essential for plant growth, viz., N,
P, K, Ca, S, Mg ( These are macronutrients).
Fe, Zn, Mn, Cu, B, Mo (these are
micronutrients).
The different nutrients media provide
different concentration of the inorganic
nutrients.
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22. Vitamins- For the optimum callus
growth, The following Vitamins are
required-
Inositol (B-8), thiamine (B-1),
pyridoxine (B-6) and nicotinic acid
(B-3) of which thiamine is essential
and the rest are promotory.
Pantothenic acid is also known to be
promotory.
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23. Carbon Source- Sucrose (20-25 g/l) is
the most commonly used carbon source
for all culture material, including
even green shoots. Autoclaving
hydrolyses sucrose, which enhances
its availability to plants. In some
System such as in monocots Glucose
may be superior to sucrose.
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24. Growth Regulators- The following growth
regulators are used in plant tissue culture.
Auxin e.g. IAA, (indole-3-acetic acid)
IBA, (indole-3-butyric acid) NAA,
(naphthalene acetic acid) NOA, (naphthoxy
acetic acid),
2,4-D, (2,4- dichlorophenoxy acetic acid)
are commonly used to support cell division
and callus growth (especially 2,4-D)
,somatic embryo induction, rooting etc.
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25. • Cytokinins like kinetin (furfurylamino
purine), BAP (benzylamino purine), Zeatin,
2-ip (isopentenyl adenine), TZD (
Thidiazuron, a compound having cytokinin
activity) are employed to promote cell
division, regeneration of shoots, often
somatic embryo induction and to enhance
proliferation and growth of auxillary
buds.
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26. • Abscisic acid promotes somatic embryo and
shoot bud regeneration in many species and
markedly improves somatic embryo
maturation.
• Gibberellins (over 20 types known), GA3 is
almost exclusively used; it promote shoot
elongation and somatic embryo germination.
• Concentration are as follows- auxin, 0.1-3
mg/l; cytokinins, 0.1-3mg/l; ABA,
0.2mg/l; and GA3 , 0.1-1mg/l.
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27. Complex Organic Additives – Complex additive
like-
1. Yeast extract,
2. Coconut milk,
3. Casein hydrolysate,
4. Corn milk,
5. Malt extract and
6. Tomato juice
-were used to support plant tissue
growth.
Such additive should be used only when
synthetic media fail.
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28. • A simple process is to first test several
concentration of auxin and cytokinin to
identify a suitable combination of the two.
• Now check the ½, full, even higher salt
conc. of the selected medium as well as
different sucrose conc.
• Alternatively worker find the various conc.
Of low, medium and high conc. of four
solution viz; minerals, organics, auxin &
cytokinin.
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29. • BIOTECHNOLOGY- B. D. SINGH
• Plant Tissue Culture- M. K. Razdan
• Plant Tissue Culture –P. K.Gupta
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