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PLANT
BIOTECHNOLOGY
VII Semester
MODULE – 1
PLANT TISSUE CULTURE &
GENETIC ENGINNERING OF
PLANTS
Dr. Pavan K. J
M.Sc.., Ph.D.,
1
 Plant tissue culture refers to the culture of any part of a plant
(cells, tissues, or organs) in artificial media, in aseptic
conditions, and under controlled environments.
 The major techniques of biotechnology are genetic
engineering, cell culture, tissue culture, bioprocessing,
protein engineering etc.
 Plant Tissue Culture, Cell Culture or Micropropagation is
the technique of producing selected plants of known desirable
agriculture qualities, in large numbers of plants from small
pieces of plant in relatively short period times.
2
Steps in Tissue Culture Protocol
a) Initial cut on 4 day old seedling.
b) Bisected shoot starting material
(cotyledons cut off ).
c) Shoot bisection starting
material.
d) Co-cultivation with filter paper
on media.
e) Inserting half of explant into
agar so the meristem contacts the
PPT medium.
f) Untransformed controls
g) Explants transformed with
construct after 2 rounds of
selection of 15 days each.
h) Transfer to shoot development
media.
Intact plants
Regenerated plants ready for
transfer into soil
3
Meet to spread awareness about plant tissue culture ...
www.business-standard.com › ... › National › News
Jul 4, 2016 - Plant tissue culture has emerged as an
important biotechnology and commercially viable tool to
multiply elite varieties of high-quality, disease-free and high-
yielding plants rapidly in the laboratory irrespective of the
season of the year. ... The plant tissue culture market in
India is estimated at Rs 500 crore and by 2025 it reaches
2000 crore Market.
4
Cambium Biotechnologies
5.0 (3) · Biotechnology company
Jigani, Karnataka
⋅ Opens 9AM · 099002 15767
"... plant tissue culture, extraction of phyto-chemicals, and many more."
Hafi Biotech
4.0 (8) · Biotechnology company
Kerala
0484 268 6065
"Bought a few tissue culture banana ..."
Labland Biotech Private Limited
4.7 (15) · Biotechnology company
Mysuru, Karnataka
⋅ Opens 9AM · 080500 06777
"One of the best applications of Biotechnology, “Tissue culture” , It ..."
Jagadamba Bio Plants
5.0 (1) · Plant nursery
Bengaluru, Karnataka
⋅ Opens 9AM · 080 4114 1151
APEX BIOTECHNOLOGY TRAINING AND RESEARCH INSTITUTE
4.6 (96) · Biotechnology company
Chennai, Tamil Nadu
· 099625 22686
Jagadamba Bio Plants
4.4 (17) · Plant nursery
Tharalu, Karnataka
⋅ Opens 8AM · 080 4114 1151
"Best place to order tissue culture Banana plants ." 5
Plant Tissue Culture Laboratory
4.5 (48) · Laboratory
Bengaluru, Karnataka
Opens 9AM · 080 2333 0153
MOTHER AGRI BIOTECH LABORATORIES INDIA PVT LTD
4.5 (19) · Biotechnology company
Bengaluru, Karnataka
Opens 9AM · 087904 31313
Their website mentions tissue culture plants
GROWMORE BIO-TECH LTD.
3.9 (29) · Biotechnology company
Hosur, Tamil Nadu
Opens 9AM · 04344 260 564
"Tissue culture plants ..."
Genewin Biotech
4.0 (49) · Research foundation
Hosur, Tamil Nadu
Opens 8AM · 089404 44777
Their website mentions tissue culture plants
Chlorbios -Plant tissue culture lab
4.4 (12) · Laboratory
Ramagondanahalli, Karnataka
Opens 8AM
Sunglow Biotech
4.1 (8) · Biotechnology company
Coimbatore, Tamil Nadu
⋅ Opens 10AM
6
7
Plant Tissue Culture Laboratory
4.5 (48) · Laboratory
Bengaluru, Karnataka
Opens 9AM · 080 2333 0153
Krishikuta Tissue Culture Lab
And Farm
4.5 (2) · Training centre
Karnataka
089511 52864
Genelon Institute Of Life
Sciences
3.0 (7) · Biotechnology
company
Bengaluru, Karnataka
· 080 4094 2696
8
9
ADVANTAGES OF TISSUE CULTURE
helping developing countries to increase food production
 The new plantlets can be grown in a short amount of time.
 Only a small amount of initial plant tissue is required.
 The new plantlets and plants are more likely to be free of viruses
and diseases.
 The process is not dependent on the seasons and can be done
throughout the year.
 You need only a relatively small space to perform the process (ten
times the plants in one-tenth of the space).
 On a larger scale, the tissue culture process helps to supply the
consumer market with new subspecies and variety.
 People looking to cultivate challenging plants such as specific
breeds of orchid find more success with the tissue culture process
than traditional soil. 10
Milestones in Plant Tissue Culture
Haberlandt (1902)
cultured plant cells in artificial condition called in vitro (inside glass) in culture
medium (Knop’s salt solution) containing glucose and peptone and developed
callus (unorganized growth of cells and tissue) and proposed the concept
Totipotency, it means the development of whole plant from isolated cells or
tissue in in vitro condition.
P.R.White (1934)
developed root cultures, used Knop’s solution along with three vitamins like
pyridoxine, thiamine and nicotinic acid
F.C. Steward (1948)
used coconut water in plant tissue culture work and obtained cell proliferation
from carrot explants (Cellular totipotency ). 11
Morel and Martin (1952, 1955)
developed virus-free Dahlia and potato plants using shoot
meristem culture.
Murashige and Skoog (1962)
formulated tissue culture medium, a land mark in plant tissue
culture and it is the most frequently used medium for all kinds
of tissue culture work.
Kanta et al. (1962)
produced test-tube fertilization in flowering plants.
Yamada et al. (1963)
produced calli and free cells in tissue culture of Tradescantia
reflexa.
12
Guha and Maheshwari (1964)
developed in vitro production of haploid embryos from anthers
of Datura.
Vasil and Hildbrandt (1965)
achieved differentiation of tobacco plants from single, isolated
cells in micro propagation.
Takebe et al. (1971)
regenerated tobacco plants from isolated mesophyll protoplasts.
Carlson
and co-workers obtained protoplast fusion between Nicotiana
glauca and Nicotiana longsdorffii and developed first
interspecific somatic hybrid in 1971. 13
Melchers and co-workers in 1978
developed intergenic hybrid between potato and tomato called
pomato.
Chilton (1983)
produced transformed tobacco plants from single cell
transformation and gene insertion
Horsh et al. (1984)
developed transgenic tobacco by Agrobacterium mediated gene
transfer.
14
Basic concepts
of
Plant Tissue
Culture
15
Basic concepts of Tissue Culture
Basic concepts of plant tissue culture are totipotency, differentiation,
dedifferentiation and redifferentiation.
Totipotency
The property of live plant cells that they have the genetic potential when
cultured in nutrient medium to give rise to a complete individual plant.
Differentiation
The process of biochemical and structural changes by which cells become
specialized in form and function.
16
Redifferentiation:-
The further differentiation of already differentiated cell into another type of
cell. For example, when the component cells of callus have the ability to
form a whole plant in a nutrient medium, the phenomenon is called
redifferentiation.
Dedifferentiation:-
The phenomenon of the reversion of mature cells to the meristematic state
leading to the formation of callus is called dedifferentiation.
These two phenomena of redifferentiation and dedifferentiation are the
inherent capacities of living plant cells or tissue. This is described as
totipotency.
17
Plant Tissue Culture Media:
Types, Constituents, Preparation and Selection
18
Culture media are largely responsible for the in vitro growth and morphogenesis of
plant tissues.
The success of the plant tissue culture depends on the choice of the nutrient
medium. In fact, the cells of most plant cells can be grown in culture media.
 It may be noted that plants in nature can synthesize their own food material.
However, plants growing in vitro are mainly heterotrophic i.e. they cannot
synthesize their own food.
Composition of Media:
The composition of the culture media is primarily dependent on two parameters:
 The particular species of the plant.
The type of material used for culture i.e. cells, tissues, organs, protoplasts.
19
Major Types of Media:
 White’s medium
 MS medium
 B5 medium
 N6 medium
 Nitsch’s medium
20
White’s medium:
This is one of the earliest plant tissue culture media developed for root culture.
MS medium:
Murashige and Skoog (MS) originally formulated a medium to induce
organogenesis, and regeneration of plants in cultured tissues. These days, MS
medium is widely used for many types of culture systems.
B5 medium:
Developed by Gamborg, B5 medium was originally designed for cell
suspension and callus cultures.
At present with certain modifications, this medium is used for protoplast
culture.
N6 medium:
Chu formulated this medium and it is used for cereal anther culture, besides
other tissue cultures.
21
Nitsch’s medium:
This medium was developed by Nitsch and Nitsch and frequently used for
anther cultures.
Among the media referred above, MS medium is most frequently used in
plant tissue culture work due to its success with several plant species and
culture systems.
Synthetic and natural media:
When a medium is composed of chemically defined components, it is
referred to as a synthetic medium.
If a medium contains chemically undefined compounds (e.g., vegetable
extract, fruit juice, plant extract), it is regarded as a natural medium.
Synthetic media have almost replaced the natural media for tissue culture.
22
Expression of concentrations in media:
The concentrations of inorganic and organic constituents in culture media are
usually expressed as mass values (mg/l or ppm).
The concentrations of macronutrients should be expressed as mmol/l– and
micronutrients as µmol/l–.
Constituents of Media:
Many elements are needed for plant nutrition and their physiological functions.
Thus, these elements have to be supplied in the culture medium to support
adequate growth of cultures in vitro.
.
23
Constituents of Media:
Many elements are needed for
plant nutrition and their
physiological functions. Thus,
these elements have to be
supplied in the culture medium
to support adequate growth of
cultures in vitro.
24
The culture media usually contain the following
constituents:
1. Inorganic nutrients
2. Carbon and energy sources
3. Organic supplements
4. Growth regulators
5. Solidifying agents
6. pH of medium
25
Inorganic Nutrients:
The inorganic nutrients consist of macronutrients (concentration >0.5 mmol/l–)
and micronutrients (concentration <0.5 mmol/l–).
A wide range of mineral salts (elements) supply the macro- and micronutrients. The
inorganic salts in water undergo dissociation and ionization.
Macronutrient elements:
The six elements namely nitrogen, phosphorus, potassium, calcium, magnesium
and sulfur are the essential macronutrients for tissue culture. The ideal
concentration of nitrogen and potassium is around 25 mmol /l while for calcium,
phosphorus, sulfur and magnesium, it is in the range of 1-3 mmol / l.
For the supply of nitrogen in the medium, nitrates and ammonium salts are
together used.
Micronutrients:
Although their requirement is in minute quantities, micronutrients are essential for
plant cells and tissues. These include iron, manganese, zinc, boron, copper and
molybdenum. Among the microelements, iron requirement is very critical. Chelated
forms of iron and copper are commonly used in culture media.
26
Carbon and Energy Sources:
Plant cells and tissues in the culture medium are heterotrophic and therefore, are
dependent on the external carbon for energy.
Besides sucrose and glucose, other carbohydrates such as lactose, maltose, galactose,
raffinose, trehalose and cellobiose have been used in culture media but with a very
limited success.
Organic Supplements:
The organic supplements include vitamins, amino acids, organic acids, organic extracts,
activated charcoal and antibiotics.
Vitamins:
Plant cells and tissues in culture (like the natural plants) are capable of synthesizing
vitamins but in suboptimal quantities, inadequate to support growth.
Medium should be supplemented with vitamins to achieve good growth of cells.
The vitamins added to the media include thiamine, riboflavin, niacin, pyridoxine, folic
acid, pantothenic acid, biotin, ascorbic acid, myoinositol, Para amino benzoic acid
and vitamin E. 27
Amino acids:
Although the cultured plant cells can synthesize amino acids to a
certain extent.
Media supplemented with amino acids stimulate cell growth and
help in establishment of cells lines. Further, organic nitrogen (in the
form of amino acids such as L-glutamine, L-asparagine, L-
arginine, L-cysteine) is more readily taken up than inorganic
nitrogen by the plant cells.
Organic acids:
Addition of Krebs cycle intermediates such as citrate, malate,
succinate or fumarate allow the growth of plant cells. Pyruvate also
enhances the growth of cultured cells.
28
Organic extracts:
• It has been a practice to supplement culture media with organic extracts such as yeast,
casein hydrolysate, coconut milk, orange juice, tomato juice and potato extract.
• It is however, preferable to avoid the use of natural extracts due to high variations in the
quality and quantity of growth promoting factors in them.
• In recent years, natural extracts have been replaced by specific organic compounds e.g.,
replacement of yeast extract by L-asparagine; replacement of fruit extracts by L-
glutamine.
Activated charcoal:
• Supplementation of the medium with activated charcoal stimulates the growth and
differentiation of certain plant cells (carrot, tomato, orchids).
• Some toxic/inhibitory compounds (e.g. phenols) produced by cultured plants are
removed (by adsorption) by activated charcoal, and this facilitates efficient cell growth in
cultures.
• Addition of activated charcoal to certain cultures (tobacco, soybean) is found to be
inhibitory, probably due to adsorption of growth stimulants such as phytohormones.
29
Antibiotics:
It is sometimes necessary to add antibiotics to the medium to
prevent the growth of microorganisms. For this purpose, low
concentrations of streptomycin or kanamycin are used. As far as
possible, addition of antibiotics to the medium is avoided as they
have an inhibitory influence on the cell growth.
Growth Regulators:
Plant hormones or phytohormones are a group of natural organic
compounds that promote growth, development and differentiation
of plants. Four broad classes of growth regulators or hormones are
used for culture of plant cells-auxins, cytokinins, gibberellins (Fig.
43.1) and abscisic acid. They promote growth, differentiation and
organogenesis of plant tissues in cultures.
30
Auxins:
Auxins induce cell division, cell elongation, and formation of callus in
cultures. At a low concentration, auxins promote root formation while at
a high concentration callus formation occurs.
Cytokinins:
Chemically, cytokinins are derivatives of a purine namely adenine.
These adenine derivatives are involved in cell division, shoot
differentiation and somatic embryo formation.
Cytokinins promote RNA synthesis and thus stimulate protein and
enzyme activities in tissues.
31
Gibberellins:
About 20 different gibberellins have been identified as growth
regulators. Of these, gibberellin A3 (GA3) is the most commonly
used for tissue culture. GA3 promotes growth of cultured cells,
enhances callus growth and induces dwarf plantlets to elongate.
Gibberellins are capable of promoting or inhibiting tissue cultures,
depending on the plant species. They usually inhibit adventitious
root and shoot formation.
Abscisic acid (ABA):
The callus growth of cultures may be stimulated or inhibited by
ABA. This largely depends on the nature of the plant species.
Abscisic acid is an important growth regulation for induction of
embryogenesis.
32
Solidifying Agents:
For the preparation of semisolid or solid tissue culture media,
solidifying or gelling agents are required. In fact, solidifying agents
extend support to tissues growing in the static conditions.
Agar:
Agar, a polysaccharide obtained from seaweeds, is most
commonly used as a gelling agent for the following reasons.
1. It does not react with media constituents.
2. It is not digested by plant enzymes and is stable at culture
temperature.
Agar at a concentration of 0.5 to 1% in the medium can form a gel.
33
Gelatin:
It is used at a high concentration (10%) with a limited success. This is
mainly because gelatin melts at low temperature (25°C), and
consequently the gelling property is lost.
Other gelling agents:
Bio-gel (polyacrylamide pellets), phytagel, gelrite and purified
agarose are other solidifying agents, although less frequently used. It
is in fact advantageous to use synthetic gelling compounds, since
they can form gels at a relatively low concentration (1.0 to 2.5 g l-1).
34
pH of medium:
The optimal pH for most tissue cultures is in the range of 5.0-6.0. The pH
generally falls by 0.3-0.5 units after autoclaving. Before sterilization, pH can
be adjusted to the required optimal level while preparing the medium. It is
usually not necessary to use buffers for the pH maintenance of culture
media.
At a pH higher than 7.0 and lower than 4.5, the plant cells stop growing in
cultures. If the pH falls during the plant tissue culture, then fresh medium
should be prepared. In general, pH above 6.0 gives the medium hard
appearance, while pH below 5.0 does not allow gelling of the medium.
35
Preparation of Media:
The general methodology for a medium preparation
involves preparation of stock solutions (in the range
of 10x to 100x concentrations) using high purity
chemicals and demineralized water.
The stock solutions can be stored (in glass or plastic
containers) frozen and used as and when required.
Most of the growth regulators are not soluble in
water. They have to be dissolved in NaOH or alcohol.
36
Sterilization of Media:
The culture medium is usually sterilized in an autoclave at 121°C and 15
psi for 20 minutes.
Hormones and other heat sensitive organic compounds are filter-
sterilized and added to the autoclaved medium.
37
Selection of a Suitable Medium:
In order to select a suitable medium for a particular plant culture system, it is customary to
start with a known medium (e.g. MS medium, B5 medium) and then develop a new medium
with the desired characteristics. Among the constituents of a medium, growth regulators
(auxins, cytokinins) are highly variable depending on the culture system.
In practice, 3-5 different concentrations of growth regulators in different combinations are
used and the best among them are selected. For the selection of appropriate concentrations of
minerals and organic constituents in the medium, similar approach referred above, can be
employed.
Medium-utmost Important for Culture:
For tissue culture techniques, it is absolutely essential that the medium preparation and
composition are carefully followed. Any mistake in the preparation of the medium is likely to
do a great harm to the culture system as a whole.
38
39

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Plant tissue culture

  • 1. PLANT BIOTECHNOLOGY VII Semester MODULE – 1 PLANT TISSUE CULTURE & GENETIC ENGINNERING OF PLANTS Dr. Pavan K. J M.Sc.., Ph.D., 1
  • 2.  Plant tissue culture refers to the culture of any part of a plant (cells, tissues, or organs) in artificial media, in aseptic conditions, and under controlled environments.  The major techniques of biotechnology are genetic engineering, cell culture, tissue culture, bioprocessing, protein engineering etc.  Plant Tissue Culture, Cell Culture or Micropropagation is the technique of producing selected plants of known desirable agriculture qualities, in large numbers of plants from small pieces of plant in relatively short period times. 2
  • 3. Steps in Tissue Culture Protocol a) Initial cut on 4 day old seedling. b) Bisected shoot starting material (cotyledons cut off ). c) Shoot bisection starting material. d) Co-cultivation with filter paper on media. e) Inserting half of explant into agar so the meristem contacts the PPT medium. f) Untransformed controls g) Explants transformed with construct after 2 rounds of selection of 15 days each. h) Transfer to shoot development media. Intact plants Regenerated plants ready for transfer into soil 3
  • 4. Meet to spread awareness about plant tissue culture ... www.business-standard.com › ... › National › News Jul 4, 2016 - Plant tissue culture has emerged as an important biotechnology and commercially viable tool to multiply elite varieties of high-quality, disease-free and high- yielding plants rapidly in the laboratory irrespective of the season of the year. ... The plant tissue culture market in India is estimated at Rs 500 crore and by 2025 it reaches 2000 crore Market. 4
  • 5. Cambium Biotechnologies 5.0 (3) · Biotechnology company Jigani, Karnataka ⋅ Opens 9AM · 099002 15767 "... plant tissue culture, extraction of phyto-chemicals, and many more." Hafi Biotech 4.0 (8) · Biotechnology company Kerala 0484 268 6065 "Bought a few tissue culture banana ..." Labland Biotech Private Limited 4.7 (15) · Biotechnology company Mysuru, Karnataka ⋅ Opens 9AM · 080500 06777 "One of the best applications of Biotechnology, “Tissue culture” , It ..." Jagadamba Bio Plants 5.0 (1) · Plant nursery Bengaluru, Karnataka ⋅ Opens 9AM · 080 4114 1151 APEX BIOTECHNOLOGY TRAINING AND RESEARCH INSTITUTE 4.6 (96) · Biotechnology company Chennai, Tamil Nadu · 099625 22686 Jagadamba Bio Plants 4.4 (17) · Plant nursery Tharalu, Karnataka ⋅ Opens 8AM · 080 4114 1151 "Best place to order tissue culture Banana plants ." 5
  • 6. Plant Tissue Culture Laboratory 4.5 (48) · Laboratory Bengaluru, Karnataka Opens 9AM · 080 2333 0153 MOTHER AGRI BIOTECH LABORATORIES INDIA PVT LTD 4.5 (19) · Biotechnology company Bengaluru, Karnataka Opens 9AM · 087904 31313 Their website mentions tissue culture plants GROWMORE BIO-TECH LTD. 3.9 (29) · Biotechnology company Hosur, Tamil Nadu Opens 9AM · 04344 260 564 "Tissue culture plants ..." Genewin Biotech 4.0 (49) · Research foundation Hosur, Tamil Nadu Opens 8AM · 089404 44777 Their website mentions tissue culture plants Chlorbios -Plant tissue culture lab 4.4 (12) · Laboratory Ramagondanahalli, Karnataka Opens 8AM Sunglow Biotech 4.1 (8) · Biotechnology company Coimbatore, Tamil Nadu ⋅ Opens 10AM 6
  • 7. 7
  • 8. Plant Tissue Culture Laboratory 4.5 (48) · Laboratory Bengaluru, Karnataka Opens 9AM · 080 2333 0153 Krishikuta Tissue Culture Lab And Farm 4.5 (2) · Training centre Karnataka 089511 52864 Genelon Institute Of Life Sciences 3.0 (7) · Biotechnology company Bengaluru, Karnataka · 080 4094 2696 8
  • 9. 9
  • 10. ADVANTAGES OF TISSUE CULTURE helping developing countries to increase food production  The new plantlets can be grown in a short amount of time.  Only a small amount of initial plant tissue is required.  The new plantlets and plants are more likely to be free of viruses and diseases.  The process is not dependent on the seasons and can be done throughout the year.  You need only a relatively small space to perform the process (ten times the plants in one-tenth of the space).  On a larger scale, the tissue culture process helps to supply the consumer market with new subspecies and variety.  People looking to cultivate challenging plants such as specific breeds of orchid find more success with the tissue culture process than traditional soil. 10
  • 11. Milestones in Plant Tissue Culture Haberlandt (1902) cultured plant cells in artificial condition called in vitro (inside glass) in culture medium (Knop’s salt solution) containing glucose and peptone and developed callus (unorganized growth of cells and tissue) and proposed the concept Totipotency, it means the development of whole plant from isolated cells or tissue in in vitro condition. P.R.White (1934) developed root cultures, used Knop’s solution along with three vitamins like pyridoxine, thiamine and nicotinic acid F.C. Steward (1948) used coconut water in plant tissue culture work and obtained cell proliferation from carrot explants (Cellular totipotency ). 11
  • 12. Morel and Martin (1952, 1955) developed virus-free Dahlia and potato plants using shoot meristem culture. Murashige and Skoog (1962) formulated tissue culture medium, a land mark in plant tissue culture and it is the most frequently used medium for all kinds of tissue culture work. Kanta et al. (1962) produced test-tube fertilization in flowering plants. Yamada et al. (1963) produced calli and free cells in tissue culture of Tradescantia reflexa. 12
  • 13. Guha and Maheshwari (1964) developed in vitro production of haploid embryos from anthers of Datura. Vasil and Hildbrandt (1965) achieved differentiation of tobacco plants from single, isolated cells in micro propagation. Takebe et al. (1971) regenerated tobacco plants from isolated mesophyll protoplasts. Carlson and co-workers obtained protoplast fusion between Nicotiana glauca and Nicotiana longsdorffii and developed first interspecific somatic hybrid in 1971. 13
  • 14. Melchers and co-workers in 1978 developed intergenic hybrid between potato and tomato called pomato. Chilton (1983) produced transformed tobacco plants from single cell transformation and gene insertion Horsh et al. (1984) developed transgenic tobacco by Agrobacterium mediated gene transfer. 14
  • 16. Basic concepts of Tissue Culture Basic concepts of plant tissue culture are totipotency, differentiation, dedifferentiation and redifferentiation. Totipotency The property of live plant cells that they have the genetic potential when cultured in nutrient medium to give rise to a complete individual plant. Differentiation The process of biochemical and structural changes by which cells become specialized in form and function. 16
  • 17. Redifferentiation:- The further differentiation of already differentiated cell into another type of cell. For example, when the component cells of callus have the ability to form a whole plant in a nutrient medium, the phenomenon is called redifferentiation. Dedifferentiation:- The phenomenon of the reversion of mature cells to the meristematic state leading to the formation of callus is called dedifferentiation. These two phenomena of redifferentiation and dedifferentiation are the inherent capacities of living plant cells or tissue. This is described as totipotency. 17
  • 18. Plant Tissue Culture Media: Types, Constituents, Preparation and Selection 18
  • 19. Culture media are largely responsible for the in vitro growth and morphogenesis of plant tissues. The success of the plant tissue culture depends on the choice of the nutrient medium. In fact, the cells of most plant cells can be grown in culture media.  It may be noted that plants in nature can synthesize their own food material. However, plants growing in vitro are mainly heterotrophic i.e. they cannot synthesize their own food. Composition of Media: The composition of the culture media is primarily dependent on two parameters:  The particular species of the plant. The type of material used for culture i.e. cells, tissues, organs, protoplasts. 19
  • 20. Major Types of Media:  White’s medium  MS medium  B5 medium  N6 medium  Nitsch’s medium 20
  • 21. White’s medium: This is one of the earliest plant tissue culture media developed for root culture. MS medium: Murashige and Skoog (MS) originally formulated a medium to induce organogenesis, and regeneration of plants in cultured tissues. These days, MS medium is widely used for many types of culture systems. B5 medium: Developed by Gamborg, B5 medium was originally designed for cell suspension and callus cultures. At present with certain modifications, this medium is used for protoplast culture. N6 medium: Chu formulated this medium and it is used for cereal anther culture, besides other tissue cultures. 21
  • 22. Nitsch’s medium: This medium was developed by Nitsch and Nitsch and frequently used for anther cultures. Among the media referred above, MS medium is most frequently used in plant tissue culture work due to its success with several plant species and culture systems. Synthetic and natural media: When a medium is composed of chemically defined components, it is referred to as a synthetic medium. If a medium contains chemically undefined compounds (e.g., vegetable extract, fruit juice, plant extract), it is regarded as a natural medium. Synthetic media have almost replaced the natural media for tissue culture. 22
  • 23. Expression of concentrations in media: The concentrations of inorganic and organic constituents in culture media are usually expressed as mass values (mg/l or ppm). The concentrations of macronutrients should be expressed as mmol/l– and micronutrients as µmol/l–. Constituents of Media: Many elements are needed for plant nutrition and their physiological functions. Thus, these elements have to be supplied in the culture medium to support adequate growth of cultures in vitro. . 23
  • 24. Constituents of Media: Many elements are needed for plant nutrition and their physiological functions. Thus, these elements have to be supplied in the culture medium to support adequate growth of cultures in vitro. 24
  • 25. The culture media usually contain the following constituents: 1. Inorganic nutrients 2. Carbon and energy sources 3. Organic supplements 4. Growth regulators 5. Solidifying agents 6. pH of medium 25
  • 26. Inorganic Nutrients: The inorganic nutrients consist of macronutrients (concentration >0.5 mmol/l–) and micronutrients (concentration <0.5 mmol/l–). A wide range of mineral salts (elements) supply the macro- and micronutrients. The inorganic salts in water undergo dissociation and ionization. Macronutrient elements: The six elements namely nitrogen, phosphorus, potassium, calcium, magnesium and sulfur are the essential macronutrients for tissue culture. The ideal concentration of nitrogen and potassium is around 25 mmol /l while for calcium, phosphorus, sulfur and magnesium, it is in the range of 1-3 mmol / l. For the supply of nitrogen in the medium, nitrates and ammonium salts are together used. Micronutrients: Although their requirement is in minute quantities, micronutrients are essential for plant cells and tissues. These include iron, manganese, zinc, boron, copper and molybdenum. Among the microelements, iron requirement is very critical. Chelated forms of iron and copper are commonly used in culture media. 26
  • 27. Carbon and Energy Sources: Plant cells and tissues in the culture medium are heterotrophic and therefore, are dependent on the external carbon for energy. Besides sucrose and glucose, other carbohydrates such as lactose, maltose, galactose, raffinose, trehalose and cellobiose have been used in culture media but with a very limited success. Organic Supplements: The organic supplements include vitamins, amino acids, organic acids, organic extracts, activated charcoal and antibiotics. Vitamins: Plant cells and tissues in culture (like the natural plants) are capable of synthesizing vitamins but in suboptimal quantities, inadequate to support growth. Medium should be supplemented with vitamins to achieve good growth of cells. The vitamins added to the media include thiamine, riboflavin, niacin, pyridoxine, folic acid, pantothenic acid, biotin, ascorbic acid, myoinositol, Para amino benzoic acid and vitamin E. 27
  • 28. Amino acids: Although the cultured plant cells can synthesize amino acids to a certain extent. Media supplemented with amino acids stimulate cell growth and help in establishment of cells lines. Further, organic nitrogen (in the form of amino acids such as L-glutamine, L-asparagine, L- arginine, L-cysteine) is more readily taken up than inorganic nitrogen by the plant cells. Organic acids: Addition of Krebs cycle intermediates such as citrate, malate, succinate or fumarate allow the growth of plant cells. Pyruvate also enhances the growth of cultured cells. 28
  • 29. Organic extracts: • It has been a practice to supplement culture media with organic extracts such as yeast, casein hydrolysate, coconut milk, orange juice, tomato juice and potato extract. • It is however, preferable to avoid the use of natural extracts due to high variations in the quality and quantity of growth promoting factors in them. • In recent years, natural extracts have been replaced by specific organic compounds e.g., replacement of yeast extract by L-asparagine; replacement of fruit extracts by L- glutamine. Activated charcoal: • Supplementation of the medium with activated charcoal stimulates the growth and differentiation of certain plant cells (carrot, tomato, orchids). • Some toxic/inhibitory compounds (e.g. phenols) produced by cultured plants are removed (by adsorption) by activated charcoal, and this facilitates efficient cell growth in cultures. • Addition of activated charcoal to certain cultures (tobacco, soybean) is found to be inhibitory, probably due to adsorption of growth stimulants such as phytohormones. 29
  • 30. Antibiotics: It is sometimes necessary to add antibiotics to the medium to prevent the growth of microorganisms. For this purpose, low concentrations of streptomycin or kanamycin are used. As far as possible, addition of antibiotics to the medium is avoided as they have an inhibitory influence on the cell growth. Growth Regulators: Plant hormones or phytohormones are a group of natural organic compounds that promote growth, development and differentiation of plants. Four broad classes of growth regulators or hormones are used for culture of plant cells-auxins, cytokinins, gibberellins (Fig. 43.1) and abscisic acid. They promote growth, differentiation and organogenesis of plant tissues in cultures. 30
  • 31. Auxins: Auxins induce cell division, cell elongation, and formation of callus in cultures. At a low concentration, auxins promote root formation while at a high concentration callus formation occurs. Cytokinins: Chemically, cytokinins are derivatives of a purine namely adenine. These adenine derivatives are involved in cell division, shoot differentiation and somatic embryo formation. Cytokinins promote RNA synthesis and thus stimulate protein and enzyme activities in tissues. 31
  • 32. Gibberellins: About 20 different gibberellins have been identified as growth regulators. Of these, gibberellin A3 (GA3) is the most commonly used for tissue culture. GA3 promotes growth of cultured cells, enhances callus growth and induces dwarf plantlets to elongate. Gibberellins are capable of promoting or inhibiting tissue cultures, depending on the plant species. They usually inhibit adventitious root and shoot formation. Abscisic acid (ABA): The callus growth of cultures may be stimulated or inhibited by ABA. This largely depends on the nature of the plant species. Abscisic acid is an important growth regulation for induction of embryogenesis. 32
  • 33. Solidifying Agents: For the preparation of semisolid or solid tissue culture media, solidifying or gelling agents are required. In fact, solidifying agents extend support to tissues growing in the static conditions. Agar: Agar, a polysaccharide obtained from seaweeds, is most commonly used as a gelling agent for the following reasons. 1. It does not react with media constituents. 2. It is not digested by plant enzymes and is stable at culture temperature. Agar at a concentration of 0.5 to 1% in the medium can form a gel. 33
  • 34. Gelatin: It is used at a high concentration (10%) with a limited success. This is mainly because gelatin melts at low temperature (25°C), and consequently the gelling property is lost. Other gelling agents: Bio-gel (polyacrylamide pellets), phytagel, gelrite and purified agarose are other solidifying agents, although less frequently used. It is in fact advantageous to use synthetic gelling compounds, since they can form gels at a relatively low concentration (1.0 to 2.5 g l-1). 34
  • 35. pH of medium: The optimal pH for most tissue cultures is in the range of 5.0-6.0. The pH generally falls by 0.3-0.5 units after autoclaving. Before sterilization, pH can be adjusted to the required optimal level while preparing the medium. It is usually not necessary to use buffers for the pH maintenance of culture media. At a pH higher than 7.0 and lower than 4.5, the plant cells stop growing in cultures. If the pH falls during the plant tissue culture, then fresh medium should be prepared. In general, pH above 6.0 gives the medium hard appearance, while pH below 5.0 does not allow gelling of the medium. 35
  • 36. Preparation of Media: The general methodology for a medium preparation involves preparation of stock solutions (in the range of 10x to 100x concentrations) using high purity chemicals and demineralized water. The stock solutions can be stored (in glass or plastic containers) frozen and used as and when required. Most of the growth regulators are not soluble in water. They have to be dissolved in NaOH or alcohol. 36
  • 37. Sterilization of Media: The culture medium is usually sterilized in an autoclave at 121°C and 15 psi for 20 minutes. Hormones and other heat sensitive organic compounds are filter- sterilized and added to the autoclaved medium. 37
  • 38. Selection of a Suitable Medium: In order to select a suitable medium for a particular plant culture system, it is customary to start with a known medium (e.g. MS medium, B5 medium) and then develop a new medium with the desired characteristics. Among the constituents of a medium, growth regulators (auxins, cytokinins) are highly variable depending on the culture system. In practice, 3-5 different concentrations of growth regulators in different combinations are used and the best among them are selected. For the selection of appropriate concentrations of minerals and organic constituents in the medium, similar approach referred above, can be employed. Medium-utmost Important for Culture: For tissue culture techniques, it is absolutely essential that the medium preparation and composition are carefully followed. Any mistake in the preparation of the medium is likely to do a great harm to the culture system as a whole. 38
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