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Dept.of Dravyaguna 1
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TISSUE CULTURE TECHNIQUE:AN OVERVIEWPresented by : Guided by:
Dr.Nayana Raj Dr.Priya.S2nd year PG Scholar Dr.Vimala.K.SDepartment of Dravyaguna Dr.Priyalatha.B Dr.Raiby.P.Paul
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CONTENTS1. INTRODUCTION 2. TISSUE CULTURE?3. MURASHIGE & SKOOG MEDIUM4. MILE STONES IN PLANT TISSUE CULTURE5. ADVANTAGES & DISADVANTAGES6. TYPES OF TISSUE CULTURE7. CHOICE OF EXPLANT8. TECHNIQUES 9. REGENERATION PATHWAYS10. APPLICATIONS 11. HAIRY ROOT CULTURE12. RECOGNITION OF TISSUE CULTURE FACILITIES13. CONCLUSION
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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.
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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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MILE STONES IN PLANT TISSUE CULTURE
1902 Haberlandt proposed concept of invitro cell culture
1922 Kolte & Robbins successfully cultured root & stem tips respectively
1926 Went discovered first plant growth hormone- Indole acetic acid
1941 Overbeek was first to add coconut milk for cell division in Datura
1955
1957
Skoog & Miller discovered Kinetin as cell division hormone
Skoog & Miller gave concept of hormonal control of organ formation
1960
1962
Kanta & Maheswari developed test tube fertilization technique
Murashige & Skoog developed MS medium with higher salt concentration
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1977
1974 Reinhard introduced biotransformation in plant tissue cultures
Chilton et al. successfully integrated Ti plasmid DNA from Agrobacterium tumefaciens in plants
1978 Melchers et al. carried out somatic hybridization of tomato & potato resulting in Pomato
1981 Larkin & Scowcroft introduced the term somaclonal variation.
2005 Rice genome sequenced under International Rice Genome Sequencing Project
ADVANTAGES
PRODUCTION OF EXACT
COPIES
QUICK PRODUCTION OF MATURE
PLANTS
PRODUCTION OF MULTIPLES
IN THE ABSENCE OF
POLLINATORS
REDUCED CHANCES OF
TRANSMITTING DISEASES
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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 CULTUREPlant tissue culture includes two major methodsA. 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
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
PERFORMED UNDER ASEPTIC CONDITIONS UNDER HEPA FILTERED AIR PROVIDED BY A LAMINAR FLOW CABINET
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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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LAMINAR FLOW CABINET
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MICROPROPAGATION VIDEO
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REGENERATION PATHWAYS
Propagation from pre-existing meristems(shoot culture/nodal culture)
Organogenesis
Non-zygotic (somatic) embryogenesis
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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.
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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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RECOGNITION OF TISSUE CULTURE PRODUCTION FACILITIES
The need for a certification programmes for the plant tissue culture is imperative since inadvertent Micropropagation of virus infected plants will not only result in its poor performance, but also in undesired spread of viruses wherever such plants are grown.
Also, failure to use prescribed standard protocols will result in variation in the plants produced.
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The National certification system for tissue culture raised plants(NCS-TCP) has been developed for the first time to provide support to Plant Tissue Culture Industry to facilitate production of quality planting material through tissue culture/ Micropropagation.
The Department of Biotechnology, Ministry of Science & Technology, Government of India as authorised under section 8 of seeds act 1966,Vide Gazette Notification dated 10th march 2006 is the Certification Agency for the purpose for certification of the Tissue culture raised propagules upto laboratory level & to regulate its genetic fidelity as prescribed.
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Tissue culture production facilities can get their material certified under this programme from accredited Test laboratories as per prescribed criteria.
Only recognised tissue culture production facilities are eligible to register for certification of their material.
The Project management unit(PMU) has been set up at Biotech consortium India Ltd, New Delhi for the implementation of this programme & in undertaking and recognition of Tissue culture production facilities.
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PREVIOUS RESEARCHES Invitro propagation of Garlic by shoot
proliferation,S.S.Bhojwani-Scientia Horticulturae,1980 Invitro propagation of Potato (Solanum tuberosum . L),
G. Hussey, N.J. Stacey-Annals of Botany 1981 Invitro propagation and low temperature storage of
Saussurea lappa CB Clarke – An Endangered , Medicinal plant, R. Arora , S. S. Bhojwani - Plant Cell Reports 1989
Invitro propagation of Gymnema sylvestre-A multipurpose medicinal plant, N.Komalavalli,M.V.Rao-Plant cell, Tissue & organ culture 2000
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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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THANK YOU