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PLANT TISSUE CULTURE
BY NANDAKISHOR B DESHMUKH.
Asst Prof At Shraddha Institute Of Pharmacy (B.Pharm), Kondala
Zambre, Washim
1
Content Table
SR NO Topic
01 Introduction to Plant Tissue Culture
02 Historical development
03 Laboratory requirement
04 Types of culture
05 Nutritional requirements
06 Maintenance of callus and suspension culture Maintenance
07 Applications
08 Edible vaccines
2
Introduction
Tissue
Definition
'Tissue culture is in vitro cultivation of plant cells or tissue
under aseptic and controlled environmental conditions, in
liquid or on semisolid well-defined nutrient medium to
produce primary and secondary metabolites or to
regenerate plant.
In other words it is an experimental technique through
which a mass of
cells (callus) is produced from an explant
tissue.
3
 The technique of in-vitro cultivation of plant cells or
organs isprimarily devoted to solve two basic problems:
1. To keep the plant cells or organs free from microbes
1. To ensure the desired development in cells and organs by
providing suitable nutrient media and other
environmental condition.
4
Advantages of tissue culture
 Availability of raw material
 Fluctuation in supplies and quality
 New methods for isolation
 Disease free and desired propagule
 Immobilization of cells
 Continuous, uniform biomass is obtained.
 Medicinally important compound can be synthesized, which can’t
be synthesized chemically.
Improvement of medicinal plant species
5
Disadvantages of tissue culture
1. High level of expertise is required.
2. A small error may lead to complete collapse of product/plant.
3. Lots of chemicals are required for plant tissue culture which must
contain high purity.
4. There is no chance for evaluation of mutation.
5. Culture on artificial medium may lead to the depression of unusual
metabolic pathways, which may not be beneficial to biotechnologist.
6. In majority cases amount of secondary metabolites produced is
negligible.
7. The protocols for individual plants differ very widely and Change in
the medium constitution & environmental parameters affect the rate of
cell growth & accumulation of secondary metabolites.
6
• To maximize on the cell mass produced the cell suspension
culture eventually becomes very dense and these presents
problems of even aeration.
• Instability
• Slow growth
• Expensive process
• Aseptic conditions are to be maintained through out the growth
of plant.
7
Historical development of Plant Tissue Culture-
The principles of tissue culture were involved 1838-1839 38-1839 in cell theory advanced by Schleiden and
Schwann. The important historical events of PTC are the following
1902 The idea of the totipotency of plant cells was given by Haberlandt
1937 White first time established successful root culture of tomato
1941 Vanoverbeek used coconut milk for the growth and development of young Datura embryos
1957 Skoog and Miller demonstrated the role of auxin and
1962 cytokinin on root and shoot formation in tobacco tissue Murashige and Skoog introduced the medium for
tobacco culture
1987 Isolation of Bt. gene form bacterium Bacillus thuringiensis
8
Basic requirements of Plant Tissue Culture
 1.Plant material
 2. Equipment and Glasswares
 3.Aseptic Condition
 4.Washing and storage facilities
 5.Media preparation room
 6.Sterilization room
 7.Nutrient medium
 8.Transfer room Culture room or incubators
 9.Proper and optimum aeration
 10.Well-equipped observation or recording area
9
Types of Cultures
Types of Cultures
1. Depending upon the type of medium
2. Depending on the part used for culture
10
1. Depending upon the type of medium- Two types
a. Callus culture (Static culture)
b. Suspension culture- three types-
i. Batch culture
ii. Semi-continuous culture
iii. Continuous culture- Two type- Open and Closed. Open
system is of two types- Turbidostat and Chemostat
11
2. Depending on the part used for culture
a) Organ Culture-
i. Root tip culture (Meristem root tip culture) ii. Shoot tip culture (Meristem
shoot tip culture)
iii. Leaves or leaf primordia culture iv. Flower culture (Meristem - floral
culture)
v. Anther and pollens culture vi. Ovule and embryo culture
vii. Ovaries culture viii. Nucellus culture ix. Seed
culture
x. Cotyledon culture xi. Endosperm culture
xii. Fruit culture xiii. Plant cell culture
12
b) Hairy Root Culture
c)Protoplast Culture and Somatic Hybridization
13
b) Hairy Root Culture
Certain soil bacteria of the genus Agrobacterium (Gram
negative bacteria) infects a wide range of plant species
and causes the infection in plant termed as "Hairy root"
disease.
 The disease is transformed bye their genome t-DNA from
a bacterial plasmid to plant hairy root cells
14
Protoplast culture-
Protoplast culture-
Protoplast are plant cells with a plasma membrane but without cell wall,
because of this the protoplast provide the starting point for many of the
technique of genetic manipulator of plants, in particular the induction of
somaclonal variation, somatic hybridization and genetic transfer.
Isolation of Protoplasts is by two methods. Protoplasts can be isolated
from almost all plant parts i.e., roots, leaves, fruits, tubers, root nodules,
endosperm, pollen cells, and cells of callus tissue.
15
 1. Mechanical method
 2. Enzymatic method
 Enzymatic method of protoplast isolation can be classified into two groups.
 A) Sequential enzymatic- This involves two steps where first macerated plant tissues
are incubated with pectinase to get single cells followed by cellulase treatment to get
protoplast.
 B) Mixed enzymatic This involves simultaneous separation of cells and degradation of
their walls to convert protoplast by immersing plant tissues in mixture of pectinases and
cellulases.
16
Applications of Plant Tissue Culture
 1. Clonal Propagation and Micro-Propagation
 2. Biomass Energy
 3. Secondary Metabolites
 4. Genetic Variability
 5. Somatic Embryogenesis and Synthetic Seed
 6. Breaking Dormancy
 7. Haploid Plants
 8. Somatic Hybrids
 9. Transgenic Plants
 10. Germplasm Conservation.
17
Application-1. Clonal Propagation and Micro-
Propagation:
Plant population derived from a single donor plant is
called a clone and the multiplication of genetically
identical copies of that cultivar is called clonal
propagation which may be an useful tool to get a large
population of plant species having desirable traits.
18
Application- 2. Biomass Energy :
In recent years, the interest has aroused in commercializing the
in vitro propagation of forest trees. Micro-propagation has been
successfully done in many trees of economic importance like
Acacia .
Development of automated procedure, plant delivery systems
using somatic embryos and artificial seeds are also in progress.
19
Application-3. Secondary Metabolites:
Production of many useful compounds like alkaloids (Codeine,
Vincristine, Quinine, etc.). Steroids (Diosgenin), Glycosidic
compounds (Digoxin) and many other essential oils (Jasmine),
flavouring and colouring agents (saffron) can be done by plant
cell culture.
This aim can be achieved by selection of specific cells
producing high amount of desired compounds and development
of a suitable medium.
20
Application- 4. Genetic Variability:
The variability generated by the use of a tissue culture cycle has been
termed as somaclonal variation by Larkin and Scowcroft. This genetic
variability is due to cells of various ploidy levels and genetic constitution
conditions.
The chromosomal instability in the cultured cells play an important role
in polyploidization of cells and genetically variable plants can be raised.
Such kind of variations may show some useful characters such as
resistance to a particular disease, herbicide resistance, stress tolerance,
etc.
21
Application-5. Somatic Embryogenesis and Synthetic
Seed:
Direct or indirect somatic embryogenesis may be achieved
from pro-embryonic cell of the direct explant or the embryoids
developed within the callus tissue from induced embryogenic
cells.
The potential application of this technique is the mass
production of adventitious embryos which ultimately develop
into complete plantlet in maturing media.
22
Application-6. Breaking Dormancy:
Using embryo (zygotic) culture technique the seed dormancy
period can be reduced or eliminated and the breeding cycle can
be shortened in many of the plants like Malus sp, Ilex sp. and
Telia americana etc.
The life cycle of Iris was reduced from 2-3 years to less than
one year.
23
Application-7. Haploid Plants:
Haploid plants can be obtained through anther or pollen
culture (androgenesis) or through ovaries or ovule
culture (gynogenesis).
The triploid or polyploid can also be produced by using
protoplast fusion technique of this kind of androgenic
haploids which may be used for different breeding
programmes.
24
Application-8. Somatic Hybrids:
Isolation and regeneration of plant from the protoplasts in vitro
has opened up a new avenue in various fields of plant breeding
and in plant biotechnology.
Somatic hybridisation, i.e., the asexual hybridisation using
isolated somatic protoplasts is a new tool to make the wide
hybridisation successful.
25
Application-9. Transgenic Plants:
Application-9. Transgenic Plants:
The genetically modified (GM) plants, in which a functional
foreign gene has been incorporated by biotechnological
method, are called transgenic plants.
A number of transgenic plants have been produced carrying
genes for different traits like insect resistance, herbicide
tolerance, delayed ripening, increased amino acid and vitamin
content, improved oil quality etc.
26
Application-10. Germplasm Conservation
Many of the important crop species produce recalcitrant seeds
with early embryo degeneration. Also many of the plants are
vulnerable to insects, pathogens and various climatic hazards.
Cryopreservation involves storage of cells, tissues, etc. at a
very low temperature using liquid nitrogen.
27
EDIBLE VACCINES
Edible vaccines are nothing but transgenic plant and animal
based production of or those that contain agents that trigger an
animal’s immune response.
In simple terms, edible vaccines are plant or animal made
pharmaceuticals. This essay highlights the importance of edible
vaccines produced in plants.
28
Plants used for edible vaccine
• Tobacco
• Potato
• Banana
• Tomato
• Rice
• Lettuce
• Soybean
• Alfalfa
• Carrot
• Peanuts
• Wheat
• corn
29
INITIALDEVELOPMENTS IN DESIGNING THE EDIBLE VACCINES
The concept of edible vaccines was developed by
Arntzen in the 1990s.
He currently heads the department of plant
biology at the Arizona State University.
He fell upon the idea after he attended a
conference in New York, organized by the WHO.
30
CURRENT STATUS
Several plant derived vaccines for human use are approaching the
market but it is likely that the first commercial Plant derived vaccine
will be a veterinary vaccine.
At least 30 such products have been expressed in plants, some
providing protection against challenges with disease causing agents.
31
HOW DO EDIBLE VACCINES WORK?
Edible vaccines contain DNA fragments from the original
pathogen. These fragments code for a protein that is usually a
surface protein of the pathogen.
 This is responsible for eliciting the body’s immune response.
32
SOME EXAMPLES OF EDIBLE VACCINES
SOME EXAMPLES OF EDIBLE VACCINES
•Transgenic Potatoes For Diarrhea
The first human trial for an edible vaccine took place in 1997
33
ADVANTAGES OF EDIBLE VACCINES
1. They are cheap; therefore they can be mass-produced.
2. They can be ingested by eating the plant/part of the plant. So, the need to process and purify
doesnot arise.
3. Extensive storage facilities like cold storage are not required.
4. If the local/native crop of a particular area is engineered to produce the vaccine, then the need
fortransportation and distribution can be eliminated.
5. Most importantly, they trigger the immunity at the mucosal surfaces such as those that line the
mouth (mucosal immunity) which is the body’s first line of defense.
34
DISADVANTAGES OF EDIBLE VACCINES
 Development of immunotolerance to vaccine peptide or protein.
 Consistency of dosage form fruit to fruit, plant-to-plant, and generation- to-
generation is not similar.
 Stability of vaccine in fruit is not known.
 Dosage of vaccines would be variable.
 Selection of best plant is difficult.
 Certain foods like potato are not eaten raw, and cooking the food might
 weakens the medicine present in it.
 Not convenient for infants
35
FACTORS AFFECTING EDIBLE VACCINES
Antigen selection
Efficacy in model systems Choice of plant species
Delivery and dosing issues Safety issues
Public perceptions and attitudes to genetic modification
Quality control and licensing.
36
37

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Plant tissue culture pharmacongosy-1 Semester 4

  • 1. PLANT TISSUE CULTURE BY NANDAKISHOR B DESHMUKH. Asst Prof At Shraddha Institute Of Pharmacy (B.Pharm), Kondala Zambre, Washim 1
  • 2. Content Table SR NO Topic 01 Introduction to Plant Tissue Culture 02 Historical development 03 Laboratory requirement 04 Types of culture 05 Nutritional requirements 06 Maintenance of callus and suspension culture Maintenance 07 Applications 08 Edible vaccines 2
  • 3. Introduction Tissue Definition 'Tissue culture is in vitro cultivation of plant cells or tissue under aseptic and controlled environmental conditions, in liquid or on semisolid well-defined nutrient medium to produce primary and secondary metabolites or to regenerate plant. In other words it is an experimental technique through which a mass of cells (callus) is produced from an explant tissue. 3
  • 4.  The technique of in-vitro cultivation of plant cells or organs isprimarily devoted to solve two basic problems: 1. To keep the plant cells or organs free from microbes 1. To ensure the desired development in cells and organs by providing suitable nutrient media and other environmental condition. 4
  • 5. Advantages of tissue culture  Availability of raw material  Fluctuation in supplies and quality  New methods for isolation  Disease free and desired propagule  Immobilization of cells  Continuous, uniform biomass is obtained.  Medicinally important compound can be synthesized, which can’t be synthesized chemically. Improvement of medicinal plant species 5
  • 6. Disadvantages of tissue culture 1. High level of expertise is required. 2. A small error may lead to complete collapse of product/plant. 3. Lots of chemicals are required for plant tissue culture which must contain high purity. 4. There is no chance for evaluation of mutation. 5. Culture on artificial medium may lead to the depression of unusual metabolic pathways, which may not be beneficial to biotechnologist. 6. In majority cases amount of secondary metabolites produced is negligible. 7. The protocols for individual plants differ very widely and Change in the medium constitution & environmental parameters affect the rate of cell growth & accumulation of secondary metabolites. 6
  • 7. • To maximize on the cell mass produced the cell suspension culture eventually becomes very dense and these presents problems of even aeration. • Instability • Slow growth • Expensive process • Aseptic conditions are to be maintained through out the growth of plant. 7
  • 8. Historical development of Plant Tissue Culture- The principles of tissue culture were involved 1838-1839 38-1839 in cell theory advanced by Schleiden and Schwann. The important historical events of PTC are the following 1902 The idea of the totipotency of plant cells was given by Haberlandt 1937 White first time established successful root culture of tomato 1941 Vanoverbeek used coconut milk for the growth and development of young Datura embryos 1957 Skoog and Miller demonstrated the role of auxin and 1962 cytokinin on root and shoot formation in tobacco tissue Murashige and Skoog introduced the medium for tobacco culture 1987 Isolation of Bt. gene form bacterium Bacillus thuringiensis 8
  • 9. Basic requirements of Plant Tissue Culture  1.Plant material  2. Equipment and Glasswares  3.Aseptic Condition  4.Washing and storage facilities  5.Media preparation room  6.Sterilization room  7.Nutrient medium  8.Transfer room Culture room or incubators  9.Proper and optimum aeration  10.Well-equipped observation or recording area 9
  • 10. Types of Cultures Types of Cultures 1. Depending upon the type of medium 2. Depending on the part used for culture 10
  • 11. 1. Depending upon the type of medium- Two types a. Callus culture (Static culture) b. Suspension culture- three types- i. Batch culture ii. Semi-continuous culture iii. Continuous culture- Two type- Open and Closed. Open system is of two types- Turbidostat and Chemostat 11
  • 12. 2. Depending on the part used for culture a) Organ Culture- i. Root tip culture (Meristem root tip culture) ii. Shoot tip culture (Meristem shoot tip culture) iii. Leaves or leaf primordia culture iv. Flower culture (Meristem - floral culture) v. Anther and pollens culture vi. Ovule and embryo culture vii. Ovaries culture viii. Nucellus culture ix. Seed culture x. Cotyledon culture xi. Endosperm culture xii. Fruit culture xiii. Plant cell culture 12
  • 13. b) Hairy Root Culture c)Protoplast Culture and Somatic Hybridization 13
  • 14. b) Hairy Root Culture Certain soil bacteria of the genus Agrobacterium (Gram negative bacteria) infects a wide range of plant species and causes the infection in plant termed as "Hairy root" disease.  The disease is transformed bye their genome t-DNA from a bacterial plasmid to plant hairy root cells 14
  • 15. Protoplast culture- Protoplast culture- Protoplast are plant cells with a plasma membrane but without cell wall, because of this the protoplast provide the starting point for many of the technique of genetic manipulator of plants, in particular the induction of somaclonal variation, somatic hybridization and genetic transfer. Isolation of Protoplasts is by two methods. Protoplasts can be isolated from almost all plant parts i.e., roots, leaves, fruits, tubers, root nodules, endosperm, pollen cells, and cells of callus tissue. 15
  • 16.  1. Mechanical method  2. Enzymatic method  Enzymatic method of protoplast isolation can be classified into two groups.  A) Sequential enzymatic- This involves two steps where first macerated plant tissues are incubated with pectinase to get single cells followed by cellulase treatment to get protoplast.  B) Mixed enzymatic This involves simultaneous separation of cells and degradation of their walls to convert protoplast by immersing plant tissues in mixture of pectinases and cellulases. 16
  • 17. Applications of Plant Tissue Culture  1. Clonal Propagation and Micro-Propagation  2. Biomass Energy  3. Secondary Metabolites  4. Genetic Variability  5. Somatic Embryogenesis and Synthetic Seed  6. Breaking Dormancy  7. Haploid Plants  8. Somatic Hybrids  9. Transgenic Plants  10. Germplasm Conservation. 17
  • 18. Application-1. Clonal Propagation and Micro- Propagation: Plant population derived from a single donor plant is called a clone and the multiplication of genetically identical copies of that cultivar is called clonal propagation which may be an useful tool to get a large population of plant species having desirable traits. 18
  • 19. Application- 2. Biomass Energy : In recent years, the interest has aroused in commercializing the in vitro propagation of forest trees. Micro-propagation has been successfully done in many trees of economic importance like Acacia . Development of automated procedure, plant delivery systems using somatic embryos and artificial seeds are also in progress. 19
  • 20. Application-3. Secondary Metabolites: Production of many useful compounds like alkaloids (Codeine, Vincristine, Quinine, etc.). Steroids (Diosgenin), Glycosidic compounds (Digoxin) and many other essential oils (Jasmine), flavouring and colouring agents (saffron) can be done by plant cell culture. This aim can be achieved by selection of specific cells producing high amount of desired compounds and development of a suitable medium. 20
  • 21. Application- 4. Genetic Variability: The variability generated by the use of a tissue culture cycle has been termed as somaclonal variation by Larkin and Scowcroft. This genetic variability is due to cells of various ploidy levels and genetic constitution conditions. The chromosomal instability in the cultured cells play an important role in polyploidization of cells and genetically variable plants can be raised. Such kind of variations may show some useful characters such as resistance to a particular disease, herbicide resistance, stress tolerance, etc. 21
  • 22. Application-5. Somatic Embryogenesis and Synthetic Seed: Direct or indirect somatic embryogenesis may be achieved from pro-embryonic cell of the direct explant or the embryoids developed within the callus tissue from induced embryogenic cells. The potential application of this technique is the mass production of adventitious embryos which ultimately develop into complete plantlet in maturing media. 22
  • 23. Application-6. Breaking Dormancy: Using embryo (zygotic) culture technique the seed dormancy period can be reduced or eliminated and the breeding cycle can be shortened in many of the plants like Malus sp, Ilex sp. and Telia americana etc. The life cycle of Iris was reduced from 2-3 years to less than one year. 23
  • 24. Application-7. Haploid Plants: Haploid plants can be obtained through anther or pollen culture (androgenesis) or through ovaries or ovule culture (gynogenesis). The triploid or polyploid can also be produced by using protoplast fusion technique of this kind of androgenic haploids which may be used for different breeding programmes. 24
  • 25. Application-8. Somatic Hybrids: Isolation and regeneration of plant from the protoplasts in vitro has opened up a new avenue in various fields of plant breeding and in plant biotechnology. Somatic hybridisation, i.e., the asexual hybridisation using isolated somatic protoplasts is a new tool to make the wide hybridisation successful. 25
  • 26. Application-9. Transgenic Plants: Application-9. Transgenic Plants: The genetically modified (GM) plants, in which a functional foreign gene has been incorporated by biotechnological method, are called transgenic plants. A number of transgenic plants have been produced carrying genes for different traits like insect resistance, herbicide tolerance, delayed ripening, increased amino acid and vitamin content, improved oil quality etc. 26
  • 27. Application-10. Germplasm Conservation Many of the important crop species produce recalcitrant seeds with early embryo degeneration. Also many of the plants are vulnerable to insects, pathogens and various climatic hazards. Cryopreservation involves storage of cells, tissues, etc. at a very low temperature using liquid nitrogen. 27
  • 28. EDIBLE VACCINES Edible vaccines are nothing but transgenic plant and animal based production of or those that contain agents that trigger an animal’s immune response. In simple terms, edible vaccines are plant or animal made pharmaceuticals. This essay highlights the importance of edible vaccines produced in plants. 28
  • 29. Plants used for edible vaccine • Tobacco • Potato • Banana • Tomato • Rice • Lettuce • Soybean • Alfalfa • Carrot • Peanuts • Wheat • corn 29
  • 30. INITIALDEVELOPMENTS IN DESIGNING THE EDIBLE VACCINES The concept of edible vaccines was developed by Arntzen in the 1990s. He currently heads the department of plant biology at the Arizona State University. He fell upon the idea after he attended a conference in New York, organized by the WHO. 30
  • 31. CURRENT STATUS Several plant derived vaccines for human use are approaching the market but it is likely that the first commercial Plant derived vaccine will be a veterinary vaccine. At least 30 such products have been expressed in plants, some providing protection against challenges with disease causing agents. 31
  • 32. HOW DO EDIBLE VACCINES WORK? Edible vaccines contain DNA fragments from the original pathogen. These fragments code for a protein that is usually a surface protein of the pathogen.  This is responsible for eliciting the body’s immune response. 32
  • 33. SOME EXAMPLES OF EDIBLE VACCINES SOME EXAMPLES OF EDIBLE VACCINES •Transgenic Potatoes For Diarrhea The first human trial for an edible vaccine took place in 1997 33
  • 34. ADVANTAGES OF EDIBLE VACCINES 1. They are cheap; therefore they can be mass-produced. 2. They can be ingested by eating the plant/part of the plant. So, the need to process and purify doesnot arise. 3. Extensive storage facilities like cold storage are not required. 4. If the local/native crop of a particular area is engineered to produce the vaccine, then the need fortransportation and distribution can be eliminated. 5. Most importantly, they trigger the immunity at the mucosal surfaces such as those that line the mouth (mucosal immunity) which is the body’s first line of defense. 34
  • 35. DISADVANTAGES OF EDIBLE VACCINES  Development of immunotolerance to vaccine peptide or protein.  Consistency of dosage form fruit to fruit, plant-to-plant, and generation- to- generation is not similar.  Stability of vaccine in fruit is not known.  Dosage of vaccines would be variable.  Selection of best plant is difficult.  Certain foods like potato are not eaten raw, and cooking the food might  weakens the medicine present in it.  Not convenient for infants 35
  • 36. FACTORS AFFECTING EDIBLE VACCINES Antigen selection Efficacy in model systems Choice of plant species Delivery and dosing issues Safety issues Public perceptions and attitudes to genetic modification Quality control and licensing. 36
  • 37. 37