Nutrient mrdia

1. PLANT BIOTECHNOLOGY
UNIT – IV
TISSUE CULTURE
Dr.E.Gayathiri
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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

3. 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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4.  Gottlieb Haberlandt, pioneer of plant tissue culture.
 Murashige & Skoog medium, an important plant growth
medium.
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5. WHATDOWE MEAN BYTISSUE
CULTURE ???
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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.

6.  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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17. TECHNIQUES
STERILIZATION OF EXPLANTS
EXPLANTS ARE PLACED OVER SOLID/LIQUID MEDIUM
PROFOUND EFFECT ON THE MORPHOLOGY OFTISSUES
CULTURES GROW
PIECES ARE TYPICALLY SLICED OFF &TRANSFERRED TO NEWMEDIA
SHOOTS EMERGE FROM CULTURE
PERFORMED UNDER ASEPTIC CONDITIONS UNDER HEPA
FILTERED AIR PROVIDED BY ALAMINAR FLOW CABINET
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18. 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

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potential
 The specific differences in the regeneration
include:
*Differences in the stage of the cells in the cell cycle.
endogenous growth
*Availability or ability to transport
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.

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 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.

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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.

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 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.