PLANT TISSUE CULTURE

PRESENTED BY :
 ASIMA DAS
 GAURI POPHALI
 MAHESHWARI AKULWAR
 RUCHA TAYADE
 SAURABH MANE
 SHIVANI JARULKAR
 SHIVANI NAMJOSHI
HISTORY
1902 The idea of totipotency of plant cell was given by
Haberlandt

1937 White first time established successful root culture of tomato

1941 Vabnoverbeek used coconut milk for growth and development of

young datura embroys

1957 Skoog ana Miller demonstrated the role og auxin and cytokinin on
root and shoot formation in tobacco culture

1962 Murashige and Skoog introduced the medium for tobacco

1987 Isolation of Bt form bacterium Bcillus thuringiensis

 What is Plant Tissue
Culture?
Plant tissue culture is defined
as culturing plant seeds, organs,
explants, tissues, cells, or
protoplasts on a chemically
defined synthetic nutrient
media under sterile and
controlled conditions of light,
temperature, and humidity.
PRINCIPLES
 Plant tissue culture relies on the fact that
many plant cells have the ability to
regenerate a whole plant (totipotency).
Single cells, plant cells without cell walls
(protoplasts), pieces of leaves, stems or
roots can often be used to generate a
new plant on culture media given the
required nutrients and plant hormones.
TECHNIQUE OF PLANT TISSUE
CULTURE

Selection of explant
Sterlization of explant
Plant transferred to solid culture
media
Addition of hormone
Placed in another media
Transplantation
SELECTION OF EXPLANT
 Explants are small pieces
of plant parts or tissues
that are aseptically cut and
used to initiate a culture in
a nutrient medium.
Explants can be taken from
different parts of a plant
such as shoots, leaves,
stems, flowers, roots, and
from many types of mature
cells provided they are able
to de-differentiate
into totipotent cells.
 The plant used as a explant
is Vigna mungo (Moong)
MS MEDIA PREPARATION
 Murashige and Skoog
medium (or MSO or MS0 (MS-zero)) is
a plant growth medium used in the laboratories
for cultivation of plant cell culture. MSO was
invented by plant scientists Toshio
Murashige and Folke K. Skoog in 1962 during
Murashige's search for a new plant growth
regulator. A number behind the letters MS is used
to indicate the sucrose concentration of the
medium. For example, MS0 contains no sucrose
and MS20 contains 20 g/l sucrose. Along with its
modifications, it is the most commonly used
medium in plant tissue culture experiments in the
laboratory
 Ingredients
Major salts (macronutrients)
Vitamins and organics
Ammonium nitrate (NH4NO3) 1,650 mg/l
Myo-Inositol 100 mg/l
Calcium chloride (CaCl2 · 2H2O) 440 mg/l
Nicotinic Acid 0.5 mg/l
Magnesium sulphate (MgSO4 ·
Pyridoxine· HCl 0.5 mg/l
7H2O) 370 mg/l
Thiamine· HCl 0.1 mg/l
Monopotassium
Glycine 2 mg/l
phosphate (KH2PO4) 170 mg/l
Lactalbumin
Potassium nitrate (KNO3) 1,900 mg/
Hydrolysate (optional) 1 g/
Minor salts (micronutrients) Indole Acetic Acid 1-30 mg/l
Boric acid (H3BO3) 6. 54 mg/l Kinetin 0.04-10 mg/l
Cobalt chloride (CoCl2 · 6H2O) 0.025 mg/l
Ferrous sulphate (FeSO4 · 7H2O) 27.8 mg/l
Manganese(II) sulphate (MnSO4 ·
4H2O) 22.3 mg/l
Potassium iodide (KI) 0.83 mg/l
Sodium molybdate (Na2MoO4 ·
2H2O) 0.25 mg/l
Zinc sulphate (ZnSO4·7H2O) 8.6 mg/l
Ethylenediaminetetraacetic acid ferric sodium
STERLIZATION OF EXPLANT
 Sterlization technique
is a very important
aspect of plant tissue
culture, as tissue
culture aims at in
vitropropagated of a
desired plant material ,
which should be free
from contamination of
any other
microorganisms.
STERLIZATION TECHNIQUES
 Sterlization Methods Used In Tissue
Culture are :
• Dry heat treatment
• Flame sterlization
• Autoclaving
• Filter sterlization
• Wiping with 70% ethanol
• Surface sterlization
INOCULATION
 It is done to prevent the
entry of micro organims
at the time of
transferring the sterlised
explant on nutrient
medium.
 Dust, hair, hands and
clothes are the potential
source of contamination.
 The inoculating chamber
should be dust free the
operator should wear
sterile haedgear and
aprons before entering
the culture area.
INCUBATION
 Cultures are incubated in a
culture room where light,
temperature and humidity are
controlled.
 For some tissues dark is
essential while for some both
dark and light conditions are
required.
 The cultures are incubated on
culture rack at 25-28 oC
temperature. Culture tubes
are placed at 35-40 o inclined
position.
 Intensity of light given to a
culture was about 16hrs
CULTURING
 Essential elements are
mineral ions, supplied
as a complex mixture
of salts.
 An organic supplement
supplying vitamins and
amino acids
 A source of fixed
carbon usually, supplied
as a sugar sucrose.
HORMONES FOR Ptc
CALLUS FORMATION
 It makes a big blob of
tissues called callus.
 It may directly lead
to formation of new
shoots or roots from
explant.
 The callus starts
formation of new
plantlets.
SUBCULTURING
 The growing plant is
transferred to new
culture media so it
can multiply their
and produce more
roots or shoots
without deprivation
of nutrients.
TRANSPLANTATION
 When plantlets
become larger they
are transferred to
the pots.
 The young plants are
grown in Green
House.
APPLICATIONS
 Can conserve  Exact copy of the
endangered species. plant is obtained.
 Production of
 Can obtain viral free
plant. multiple plants
without pollinators.
 Hybrids can be
 Whole plant can be
regenerated by
protoplasm fusion. genetically modified.
 Totipotency can be
 Plants can be grown
in less time. used that is whole
plant can be grown
by single cell.