Tissue Culture

PLANT BIOTECHNOLOGY
UNIT – IV
TISSUE CULTURE
Dr.E.Gayathiri
1
CONTENTS
1. INTRODUCTION
2. TISSUE CULTURE?
3. MURASHIGE & SKOOG MEDIUM
4. MILE STONES IN PLANT TISSUE CULTURE
5. ADVANTAGES & DISADVANTAGES
6. TYPES OF TISSUE CULTURE
7. CHOICE OF EXPLANT
8. TECHNIQUES
9. REGENERATION PATHWAYS
10. APPLICATIONS
11. HAIRY ROOT CULTURE
12. RECOGNITION OF TISSUE CULTURE FACILITIES
13. CONCLUSION
2
INTRODUCTION
 Conservation of medicinal plants deals with the
controlled utilization & official supervision in order to
preserve or protect them.
 Acc to WHO, as many as 80% of the world’s population
depends on traditional herbal medicine for their primary
health care needs.
 Today many medicinal plants face extinction or severe
genetic loss.
 Tissue culture is one of the many techniques in
biotechnology which can be used for the conservation of
such medicinal plants.
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 Gottlieb Haberlandt, pioneer of plant tissue culture.
 Murashige & Skoog medium, an important plant growth

medium.
5
WHAT DO WE MEAN BY TISSUE
CULTURE ???
Plant tissue culture is a collection of techniques

used to maintain or grow plant cells, tissues or
organs under sterile conditions on a nutrient culture
medium of known composition.
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 It is widely used to produce clones of a plant in
a method known as Micropropagation.
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MURASHIGE & SKOOG MEDIUM
 Murashige & Skoog medium(MSO/MS0) is a plant

growth medium used in laboratories for the cultivation
of plant cell culture.
 Invented by Plant scientists Toshio Murashige & Folke
K.Skoog in 1962 during Murashige’s search for new
plant growth regulator.
 A number behind the letters MS is used to indicate the
sucrose concentration of the medium.
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INGREDIENTS
Major salts (Macronutrients)

 Ammonium nitrate(NH4NO3)- 1650mg/l
 Calcium chloride(CaCl2.2H2O)- 440mg/l
 Magnesium sulphate(MgSo4.7H2O)- 370mg/l
 Potassium phosphate(KH2PO4)- 170mg/l
 Potassium nitrate(KNO3)- 1900mmg/l
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Minor salts(Micronutrients)
 Boric acid(H3BO3)- 6.2mg/l
 Cobalt chloride(CoCl2.6H2O)- 0.025mg/l
 Cupric sulphate (CuSO4.5H2O)- .025mg/l
 Ferrous sulphate(FeSO4.7H2O)- 27.8mg/l
 Manganese sulphate (MnSO4.4H2O)- 22.3mg/l
 Potassium iodide(KI)- .83mg/l
 Sodium molybdate (Na2MoO4.2H2O)- .25mg/l
 Zinc sulphate(ZnSO4.7H2O)- 8.6mg/l
 Na2EDTA.2H2O- 37.2mg/l
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Vitamins & Organics
 i-Inositol – 100mg/l
 Niacin - 0.5mg/l
 Pyridoxine.HCl - 0.5mg/l
 Thiamine.HCl – 0.1mg/l
 Glycine – 2mg/l
 Edamine (optional) – 1g/l
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DISADVANTAGES
 It is labour intensive & expensive process.

 All plants cannot be successfully tissue cultured.
 It is usually because the medium of growth is not known.
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TYPES OF TISSUE CULTURE
Plant tissue culture includes two major methods
A. Type of in vitro growth- Callus & Suspension
cultures.
B. Type of Explant-
 Single cell culture
 Shoot & root culture
 Somatic embryo culture
 Meristem culture
 Anther culture & haploid production
 Protoplast culture & somatic hybridization
 Embryo culture, Ovule culture, Ovary culture
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CHOICE OF EXPLANT
 The tissue obtained from a plant to be cultured is called

an Explant.
 In a totipotent, explant can be collected from any part of
the plant.
 In many plants, explants of various organs vary in their
rate of growth & regeneration.
 The choice of explant material also determines if the
plantlets developed via tissue culture are haploid/diploid.
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TECHNIQUES
PERFORMED UNDER ASEPTIC CONDITIONS UNDER HEPA
FILTERED AIR PROVIDED BY ALAMINAR FLOW CABINET
STERILIZATION OF EXPLANTS
EXPLANTS ARE PLACED OVER SOLID/LIQUID MEDIUM
PROFOUND EFFECT ON THE MORPHOLOGY OF TISSUES
CULTURES GROW
PIECES ARE TYPICALLY SLICED OFF &TRANSFERRED TO NEW MEDIA
SHOOTS EMERGE FROM CULTURE
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MAY BE SLICED OFF
MATURED ONE ARE TRANSFERRED TO POTTING SOIL FOR

FURTHER GROWTH IN THE GREEN HOUSE AS NORMAL PLANTS
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REGENERATION PATHWAYS
 Propagation from pre-existing meristems(shoot
culture/nodal culture)
 Organogenesis
 Non-zygotic (somatic) embryogenesis
26
 The specific differences in the regeneration potential
include:
*Differences in the stage of the cells in the cell cycle.
*Availability or ability to transport endogenous growth
regulators.
*Metabolic capabilities of the cells
 The most commonly used tissue explants are the
meristematic ends of the plants like the stem tip, auxillary
bud tip & root tip.
 These tissues have high rates of cell division & produce
required growth regulating substances including auxins &
cytokinins.
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 Shoot culture : Performed in 4 stages for mass
production of plantlets through in vitro vegetative
multiplication
 Organogenesis : Common method of
Micropropagation that involves tissue regeneration of
adventitious organs/axillary buds directly or indirectly
from the explants.
 Non-zygotic embryogenesis: Important pathway for
producing somaclonal variants, developing artificial
seeds & synthesizing metabolites.
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APPLICATIONS
 The commercial production of plants which uses

meristem & shoot culture to produce large numbers of
identical individuals.
 To conserve rare or endangered plant species.
 A plant breeder may use tissue culture to screen cells
rather than plants for advantageous characters.
 Large scale growth of plants in liquid culture in
bioreactors for the production of valuable compounds.
 To cross distantly related species by protoplast fusion &
regeneration of the novel hybrid.
29
 To rapidly study the molecular basis for physiological,
biochemical & reproductive mechanisms in plants.
 To cross pollinate distantly related species & then tissue
culture the resulting embryo which would otherwise
normally die (Embryo Rescue)
 For chromosome doubling & induction of polyploidy.
 As a tissue for transformation, followed by either short
term testing of genetic constructs or regeneration of
transgenic plants.
 Certain techniques such as meristem tip culture can be
used to produce clean plant material from virused stock.
30
HAIRY ROOT CULTURE
 It is also called Transformed root culture.

 It is used to study plant metabolic processes or to
produce valuable secondary metabolites or recombinant
proteins, often with plant genetic engineering.
 A naturally occurring soil bacterium that contains root
inducing plasmids can infect plant roots & cause them
to produce a food source for the bacterium & to grow
abnormally.
31
 The abnormal roots are particularly easy to culture in
artificial media because hormones are not needed.
 These roots will be having a high growth rate as well
as genetic & biochemical stability.
 It is also used for regeneration of whole plants & for the
production of artificial seeds.
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CONCLUSION
 It is important for a researcher to be ethical while
performing Tissue Culture, as this technique comes
with great responsibility
 Plant tissue Culture is meant to produce products that
are useful to the human kind or the ecosystem.
 Plant tissue culture is our hope to end world hunger.
 However when it comes to manipulating a living
organism many ethical issues will arise.
 Hence, this technique must be performed with caution
to minimize the risks while capitalizing on the benefits.
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PLANT BIOTECHNOLOGY

 Murashige & Skoog medium, an important plant growth

Plant tissue culture is a collection of techniques

 Murashige & Skoog medium(MSO/MS0) is a plant

Major salts (Macronutrients)

 It is labour intensive & expensive process.

 The tissue obtained from a plant to be cultured is called

EXPLANTS ARE PLACED OVER SOLID/LIQUID MEDIUM

PROFOUND EFFECT ON THE MORPHOLOGY OF TISSUES

PIECES ARE TYPICALLY SLICED OFF &TRANSFERRED TO NEW MEDIA

SHOOTS EMERGE FROM CULTURE

MATURED ONE ARE TRANSFERRED TO POTTING SOIL FOR

 Non-zygotic (somatic) embryogenesis

 The commercial production of plants which uses

 It is also called Transformed root culture.

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