tissue culture techniques

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Dept.of Dravyaguna 1

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Page 1: Tissue culture techniques

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.

Dept.of Dravyaguna

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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

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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