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Dept.of Dravyaguna 1
TISSUE CULTURE
TECHNIQUE:AN OVERVIEW
Presented by : Guided by:
Dr.Nayana Raj Dr.Priya.S
2nd year PG Scholar Dr.Vimala.K.S
...
CONTENTS
1. INTRODUCTION
2. TISSUE CULTURE?
3. MURASHIGE & SKOOG MEDIUM
4. MILE STONES IN PLANT TISSUE CULTURE
5. ADVANTAG...
INTRODUCTION
 Conservation of medicinal plants deals with the
controlled utilization & official supervision in order to
p...
 Gottlieb Haberlandt, pioneer of plant tissue culture.
 Murashige & Skoog medium, an important plant growth
medium.
Dept...
WHAT DO WE MEAN BY TISSUE
CULTURE ???
Plant tissue culture is a collection of techniques
used to maintain or grow plant ce...
 It is widely used to produce clones of a plant in
a method known as Micropropagation.
Dept.of Dravyaguna 7
MURASHIGE & SKOOG MEDIUM
 Murashige & Skoog medium(MSO/MS0) is a plant
growth medium used in laboratories for the cultiva...
INGREDIENTS
Major salts (Macronutrients)
 Ammonium nitrate(NH4NO3)- 1650mg/l
 Calcium chloride(CaCl2.2H2O)- 440mg/l
 Ma...
Minor salts(Micronutrients)
 Boric acid(H3BO3)- 6.2mg/l
 Cobalt chloride(CoCl2.6H2O)- 0.025mg/l
 Cupric sulphate (CuSO4...
Vitamins & Organics
 i-Inositol – 100mg/l
 Niacin - 0.5mg/l
 Pyridoxine.HCl - 0.5mg/l
 Thiamine.HCl – 0.1mg/l
 Glycin...
MILE STONES IN PLANT TISSUE
CULTURE
Dept.of Dravyaguna 12
1902 Haberlandt proposed concept of invitro cell culture
1922 Ko...
Dept.of Dravyaguna 13
1977
1974 Reinhard introduced biotransformation in plant tissue cultures
Chilton et al. successfully...
ADVANTAGES
PRODUCTION
OF EXACT
COPIES
QUICK
PRODUCTION
OF MATURE
PLANTS
PRODUCTION
OF MULTIPLES
IN THE
ABSENCE OF
POLLINAT...
DISADVANTAGES
 It is labour intensive & expensive process.
 All plants cannot be successfully tissue cultured.
 It is u...
TYPES OF TISSUE CULTURE
Plant tissue culture includes two major methods
A. Type of in vitro growth- Callus & Suspension
cu...
Dept.of Dravyaguna 17
Dept.of Dravyaguna 18
CHOICE OF EXPLANT
 The tissue obtained from a plant to be cultured is called
an Explant.
 In a totipotent, explant can b...
Dept.of Dravyaguna 21
TECHNIQUES
STERILIZATION OF EXPLANTS
EXPLANTS ARE PLACED OVER SOLID/LIQUID MEDIUM
PROFOUND EFFECT ON THE MORPHOLOGY OF TIS...
MAY BE SLICED OFF
MATURED ONE ARE TRANSFERRED TO POTTING SOIL FOR
FURTHER GROWTH IN THE GREEN HOUSE AS NORMAL PLANTS
Dept....
Dept.of Dravyaguna 24
LAMINAR FLOW CABINET
Dept.of Dravyaguna 25
MICROPROPAGATION VIDEO
REGENERATION PATHWAYS
 Propagation from pre-existing meristems(shoot
culture/nodal culture)
 Organogenesis
 Non-zygotic...
 The specific differences in the regeneration potential
include:
*Differences in the stage of the cells in the cell cycle...
 Shoot culture : Performed in 4 stages for mass
production of plantlets through in vitro vegetative
multiplication
 Orga...
APPLICATIONS
 The commercial production of plants which uses
meristem & shoot culture to produce large numbers of
identic...
 To rapidly study the molecular basis for physiological,
biochemical & reproductive mechanisms in plants.
 To cross poll...
HAIRY ROOT CULTURE
 It is also called Transformed root culture.
 It is used to study plant metabolic processes or to
pro...
 The abnormal roots are particularly easy to culture in
artificial media because hormones are not needed.
 These roots w...
Dept.of Dravyaguna 33
Dept.of Dravyaguna 34
Dept.of Dravyaguna 35
RECOGNITION OF TISSUE CULTURE
PRODUCTION FACILITIES
 The need for a certification programmes for the plant
tissue culture...
 The National certification system for tissue culture
raised plants(NCS-TCP) has been developed for the
first time to pro...
 Tissue culture production facilities can get their
material certified under this programme from
accredited Test laborato...
PREVIOUS RESEARCHES
 Invitro propagation of Garlic by shoot
proliferation,S.S.Bhojwani-Scientia Horticulturae,1980
 Invi...
CONCLUSION
 It is important for a researcher to be ethical while
performing Tissue Culture, as this technique comes
with ...
Dept.of Dravyaguna 41
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Tissue culture techniques

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an overview of tissue culture techniques

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Tissue culture techniques

  1. 1. Dept.of Dravyaguna 1
  2. 2. TISSUE CULTURE TECHNIQUE:AN OVERVIEW Presented by : Guided by: Dr.Nayana Raj Dr.Priya.S 2nd year PG Scholar Dr.Vimala.K.S Department of Dravyaguna Dr.Priyalatha.B Dr.Raiby.P.Paul Dept.of Dravyaguna 2
  3. 3. 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 Dept.of Dravyaguna 3
  4. 4. 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 4
  5. 5.  Gottlieb Haberlandt, pioneer of plant tissue culture.  Murashige & Skoog medium, an important plant growth medium. Dept.of Dravyaguna 5
  6. 6. 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. Dept.of Dravyaguna 6
  7. 7.  It is widely used to produce clones of a plant in a method known as Micropropagation. Dept.of Dravyaguna 7
  8. 8. 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. Dept.of Dravyaguna 8
  9. 9. 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 Dept.of Dravyaguna 9
  10. 10. 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 Dept.of Dravyaguna 10
  11. 11. 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 Dept.of Dravyaguna 11
  12. 12. MILE STONES IN PLANT TISSUE CULTURE Dept.of Dravyaguna 12 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
  13. 13. Dept.of Dravyaguna 13 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
  14. 14. ADVANTAGES PRODUCTION OF EXACT COPIES QUICK PRODUCTION OF MATURE PLANTS PRODUCTION OF MULTIPLES IN THE ABSENCE OF POLLINATORS REDUCED CHANCES OF TRANSMITTING DISEASES Dept.of Dravyaguna 14
  15. 15. 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. Dept.of Dravyaguna 15
  16. 16. 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 Dept.of Dravyaguna 16
  17. 17. Dept.of Dravyaguna 17
  18. 18. Dept.of Dravyaguna 18
  19. 19. 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. Dept.of Dravyaguna 20
  20. 20. Dept.of Dravyaguna 21
  21. 21. 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 Dept.of Dravyaguna 22 PERFORMED UNDER ASEPTIC CONDITIONS UNDER HEPA FILTERED AIR PROVIDED BY A LAMINAR FLOW CABINET
  22. 22. MAY BE SLICED OFF MATURED ONE ARE TRANSFERRED TO POTTING SOIL FOR FURTHER GROWTH IN THE GREEN HOUSE AS NORMAL PLANTS Dept.of Dravyaguna 23
  23. 23. Dept.of Dravyaguna 24 LAMINAR FLOW CABINET
  24. 24. Dept.of Dravyaguna 25 MICROPROPAGATION VIDEO
  25. 25. REGENERATION PATHWAYS  Propagation from pre-existing meristems(shoot culture/nodal culture)  Organogenesis  Non-zygotic (somatic) embryogenesis Dept.of Dravyaguna 26
  26. 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. Dept.of Dravyaguna 27
  27. 27.  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. Dept.of Dravyaguna 28
  28. 28. 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. Dept.of Dravyaguna 29
  29. 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. Dept.of Dravyaguna 30
  30. 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. Dept.of Dravyaguna 31
  31. 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. Dept.of Dravyaguna 32
  32. 32. Dept.of Dravyaguna 33
  33. 33. Dept.of Dravyaguna 34
  34. 34. Dept.of Dravyaguna 35
  35. 35. 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. Dept.of Dravyaguna 36
  36. 36.  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. Dept.of Dravyaguna 37
  37. 37.  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. Dept.of Dravyaguna 38
  38. 38. 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 Dept.of Dravyaguna 39
  39. 39. 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. Dept.of Dravyaguna 40
  40. 40. Dept.of Dravyaguna 41

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