The document discusses the key requirements and techniques for in vitro plant cell and organ cultivation. It explains that a suitable nutrient media, keeping cells free from microbes, and maintaining optimal environmental conditions are necessary. It also describes the laboratory space and equipment needed, including areas for vessel washing/storage, media preparation/storage, aseptic handling, and culture maintenance/observation. Sterilization methods like autoclaving and filtering are discussed. Nutritional requirements for plant tissue culture media are outlined, including inorganic salts, carbon sources, organic nutrients, growth regulators, and gelling agents.

1. • 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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2. • 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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3. • Generally, glass
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culture
used as they are cheaper,
vessels are
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.

4. 18/04/2017 4
generally soaked
• Culture vessels and other labware are
in a suitable nontoxic
detergent solution overnight, washed with
a suitable brush, thoroughly rinsed clean
with tap
distilled
water,
water and dried
followed by rinse with
in a hot air
oven 70 - 80° C.
• Washed culture vessels should be stored
in a dust proof cabinet.

5. • The culture
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room should have the
following facilities:
1. Controlled temperature (usually
2°C).
2. Culture racks fitted with light.
3. A shaker for agitation of
cultures.
25°±
liquid

6. • 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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7. • Glassware
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and Teflon
instruments
plasticware, and
by dry
heat in an
may be sterilized
oven at 160 - 180°C for 3
hours.

8. empty and
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vessels,
media)
(both
are generally
• Culture
containing
sterilized by heating in autoclave or a
pressure cooker to 121° C at 15 pound
per square inch for 15 to 40 minutes the
longer for larger medium
time being
volume.
• Sterilization during autoclaving depends
mainly on temperature.

9. • Some growth
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regulators, e.g.
vitamins, and enzyme
GA3, urea,
are heat
certain
labile.
sterilized
Such compound
by passing
are
their
filter
solution
through a membrane filter of 0.45 micron
or lower pore size.
• Laminar
create
air flow cabinets are used to
an aseptic working space by
blowing filter – sterilized air through
an enclosed space.

10. • The surface
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that can not be
sterilized by
are sterilized
other techniques
by wiping them
thoroughly with 70% ethyl alcohol
and the alcohol is allowed to
dry.

11. • 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.
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• It depends mainly
of tissue of the explant
determine the contamination
which
load
on the source and type
will
and
tolerance of the sterilizing agent.

12. • 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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13. NUTRITIONAL REQUIREMENTS FOR
PLANT TISSUE CULTURE

14. • Medium depends upon the type of plant tissue or
cell used for culture
• Generally nutrient consist of
 Inorganic salts (both micro & macro elements)
 a carbon source (usually sucrose)
 Organic nutrients: Eg: Vitamins (eg. nicotinic acid,
thiamine, pyridoxine) and amino acids (eg. arginine)
 Growth regulators (eg. Auxins, cytokinin)
 Gelling agent (eg: Agar 0.8-1.0%)
 An optimum pH (5.7) is also very important
Nutrient Medium

15. 1. Inorganic salts:
1. Micronutrients are those which are required in small
amount but essential for proper growth of plant cells.
Examples are Cu, Boron, Iron, Zinc, Mn
2. Macronutrients: are those which are required in higher
amount. Examples: N,P, K, Ca, Mg, S

16. 2. Carbon source: Sucrose (2-5%) is the
most commonly used carbon source for all
culture material, including even green
shoots. Autoclaving hydrolyses sucrose,
which enhances its availability to
plants..
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17. Vitamins- For the optimum callus
growth, The following Vitamins are
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required-
Inositol
pyridoxine
(B- 8) , thiamine (B- 1) ,
(B-6) and nicotinic acid
(B-3) of which thiamine is essential
and the rest are promotory.
3. Organic nutrients:

18. – Complex additive
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Complex Organic Additives
like-
1. Yeast extract,
2. Coconut milk,
3. Casein hydrolysate,
4. Corn milk,
5. Malt extract and
6. Tomato juice
-were used to support
growth.
Such additive should be used
synthetic media fail.
plant tissue
only when

19. 4. Growth regulators:
I. Auxins
used for cell division, cell elongation, formation of
adventitious roots generally. Examples are: Naphthalene
Acetic Acid [NAA], Indole Acetic Acid [IAA], and Indole
Butyric Acid [IBA].
II.Cytokinins : are used for shoot induction, cell
elongation. Generally, kinetin or Benzyl adenine is used.
III. Gibberellins
Gibberellins are used for plant regeneration, elongation of
internodes and induction of embryogenesis. Ex: Gibberellic
Acid

20. • Abscisic acid promotes somatic embryo and
shoot bud regeneration in many species and
markedly improves somatic embryo
maturation.
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21. 5. Gelling agents:
Most commonly agar (0.8-1.0%) which is obtained from
red algae i.e. Gellidium gracilaria. Others are silica gel,
starch, gelatin and alginate.