Plant tissue culture

PRESENTED BY
Gourav singh
B-pharmacy
Presented by- Gourav singh 1

PLANT TISSUE CULTURE
 A method practiced for plant propagation under sterile condition to produce
clones of a plant is called Plant tissue culture.
Advantage Disadvantage
It can be used to study biogenesis of
secondary metabolite.
This procedure are very variable
The cells of any plants can be
multiplied to obtain the specific
metabolite they produce
The procedure need special
attention and diligently done
observation
Cultured cell are maintained such
that they remain free from
microbial contamination and insect
attack
There may be error in the identity
of the organism after culture

HISTORY
Year Author Plant species Result
1902 Haberlandt Tradeseantia First cultivation experiment with
isolated plant cell, cell growth, but
no cell division obtained
1904 Hannig Cochleria
Saphanus
Establishment of embryo culture
from several cruciferous species.
1892 Klercher ------------- First attempt to isolate protoplast
1922 Kolte, Robbins Zea In vitro-cultivation of root tips, no
permanent culture obtained
1924 Dieterich luium Embryo rescue –”artificial
premature birth”
1934 White Lycopersicum First permanent root culture which
terminated in 1988
1934 Gautheret Naucus, Nicotina First permanent callus culture using
B-vitamins

HISTORY
1946 Ball (Father of
micro-
propagation)
Lupinus and
Trapaeolum
Development of transplantable
whole plants from leaf primoda
1954 Muiretal Jagetes Nicotiana First suspension culture of single
cell or cell aggregates nurse
culture
1957 Skoog, Miller Nicotiana Demonstrated the role of auxin
and cytokinin on root and shoot
formation
1959 Julecke,
Nickess
lolium First report of large scale (1341)
culture of plant cell, carboy
system
1960 Bergmann Nicotiana Cells clone obtained from single
culture cell placed in an agar
medium

HISTORY
1962 Murashige,
skoog
Nicotiana Introduced the medium
1965 Morel lycopersicon Clonal multiplication of horticulture
through tissue culture, protocorn
formation
1966 Kohlenbach Macleaya First cell division and culture of
differentiated mesophyll cells
1970 Carlson Nicotiana Isolation of auicotrophic mutant from
cultured cells
1971 Nagata,
Takabe
Nicotiana Regeneration of plant cells from
cultured protoplast
1974 Reinhard _________ Biotransformation in plant tissue culture

History
1978 Meichers ___________ Production of somatic hybrid pomato
1983 Mitsui
petrochemical
Industry Limited
Lithosperman First industrial production of
secondary plant products by
suspension culture

TERMS USED IN TISSUE CULTURE
Terms Description
Explants An excised piece of differentiated tissue or organ is regarded as
an explant.
The explant may be taken from any part of the plant body .
e.g. leaf, stem, root etc.
Callus The unorganized, autonomus, uncontrolled and undifferentiated
mass of plant cell is known as callus
Differentiation It is the process by which meristem cells are converted into two
or more types of cells or tissues that are different from each
other.
Dedifferentiation The phenomenon of mature cells reverting to meristematic state
to produce callus, means the process of formation of unorganized
tissues from the highly organized tissues.
Re-
Differentiation
It means that the dedifferentiated cells lose the capacity of
division and become mature to form specific functions

TERMS USED IN TISSUE CULTURE
Terms Description
Totipotency The ability of the callus cells to differentiate into a plant
organ or a whole plant is referred to as cellular totipotency.
Plasticity It is the condition of the adaptability of a plant species to
change in its environments or differences between its
various habitats.
Organogenesis The development of adventitious organ or primordia from
undifferentiated cell mass in tissue culture by the process of
differentiation.
Synthetic seed Where artificial encapsulation of somatic embryos shoot bud
or aggregates of cell of any tissues done by a hydrogel which
have the ability to form a plant in In-Vitro
Somaclonal
variation
The genetic variations found in the In-vitro cultured cells
are known as somaclonal variation
Micropropagation The production of a large number of individual plants from a
small piece of plant tissue cultured without formation of
callus in an aseptic nutrient medium
Presented by- Gourav singh
8

BAISC REQUIREMENT FOR TISSUES CULTURE
LABORATORY
 A tissues culture laboratory should have the general basic facilities:-
Sr. no Requirement Description
01 Apparatus For growing culture different kind of vessel are used.
Callus culture can be grown successfully in large test
tube (25 x 150mm) or wide mouth flask. Glassware such
as graduated pipettes, measuring cylinder, beaker,
filters, funnel and petridishes are also required for
making preparation. All the glassware should be
cleaning
02 Equipment Sprit burner, An Autoclave, Hot air oven, Scissors,
Seallpels and forceps, pH meter, shaker, weighing
balance, laminar air flow, and BOD incubator.

Sr.
no
Requirement Description
03 Washing and storage
facilities
First requirement of tissue culture laboratory is
provision for fresh water supply and disposal of the
waste water. Acid and Alkali resistant wash basin for
apparatus washing, It is compulsory to maintain
cleanliness in the area of washing, drying and storage
04 Media preparation
room
It should have sufficient space to accommodate,
chemical, glassware, culture vessel and equipment
required for weighing and mixing, pH meter, hot plate.
Burner with gas supply, autoclave, microwave oven and
freezer for storage for prepared media and stock
solution.
05 Aseptic chamber for
culture
Aseptic chamber requires an ordinary type of small
wooden hood, have a glass door, fitted with U.V. tubes.
The air is coming out of the filter is ultra clean and
having adequate velocity to prevent micro
contamination of working area by worker sitling in the
front of the cabinet. Inside the cabinet, there is
arranged for bunsen burner and a UV tube fitted on the
ceiling of the cabinet.

Sr no. Requirement Description
06 Culture room Environmental have a great effect on the growth and
differentiation of cultured tissues. Therefore it is very
much essential to incubate all types of culture in well
controlled. Environmental condition like temperature,
air circulation and humidity.
07 Data collection and
recording of the
observation
The growth and maintenance of the tissue culture in
the incubator should be observed and recorded at
regular interval. All the observation should be done in
the laminar air flow. For microscopic examination
separate dust free space should be marked for
microscopic work. All the recorded data shou;d be
feeded in computer.

GENERAL PROCEDURE USED FOR PLANT
TISSUE CULTURE
Sterilization of
glassware
vessels
Preparation
and
sterilization of
explant
Production of
callus from
explant
Proliferation of
cultured callus
Sub culturing
of cells
Suspension
culture
12

TYPES OF TISSUE CULTURE
Culture of Organized
Structures
Meristem Cultures (shoot apices)
Shoot Culture (shoot tips, or buds)
Node Culture (lateral buds)
Isolated root Culture
Embryo Culture (Zygotic (seed)
embryos)
Culture of Unorganized
Structure
Callus (or tissue) cultures
Suspension (or cell) cultures
Protoplast cultures
Anther cultures

 MERISTEM CULTURE:
1) Shoot apex (apical meristematic dome with or without one or two leaf
primordia)
2) Give one single shoot.
3) Generally, meristem tips, between 0.2-0.5 mm.
4) It help in production of virus free plants.
5) It help in rapid clonal multiplication.
6) The method is successful in case of herbaceous plants than woody plants.
7) It help in a culture of potato, Banana, Cardamom, Sugarcane, Sweet potato
etc.

 SHOOT CULTURE:
1) Shoot tips, or buds (larger than the shoot apices having several leaf
primordia).
2) Usually produce multiple shoots.
3) In which the terminal end (0.1 -1.0 mm) of a shoot carrying the meristem
(0.05 -0.1 mm)
4) Primordial and developing leaf and adjacent stem tissues is cultured.

 NODE CULTURE:
1) Stem piece carrying either single or multiple nodes.
2) Each bud is grown to provide a single shoot.

 ROOT CULTURE
1) Can be established from root tips taken from primary or lateral roots of
many plants.
2) It is a process in which the radical tips of seed germinated aseptically are
cut out and cultured in a liquid medium under controlled condition to
facilitates their growth.

 EMBRYO AND OVULE CULTURES:
1) Embryos are dissected from seeds, individually isolated and germinated in
vitro to provide one plant per explant.
2) In some plant, It has been possible to excise and culture pollinated ovaries
and immature ovules.

 CALLUS CULTURE:
1) An amorphous mass of loosely arranged thin walled parenchyma cells arising
from the proliferating cells of the parents tissues cultured on agar medium
under aseptic condition is known as callus culture.
2) This method is the source of Tissue for cell Suspension Culture.
3) Several biochemical assays are performed from callus culture.
4) Chromosomal variation occurs genetically in the cells of callus tissue.

 SUSPENSION CULTURES:
1) Tissue and cells cultured in a agitated liquid medium produce a suspension of
single cells and cells clumps of few to may cell, these are called suspension
cultures
2) Shorter duration and continuous process.
3) It help for induction in somatic embryos and shoots.
4) No toxic products are formed with this culture techniques.
 Batch culture:
• A batch culture is a cells suspension culture growth in a fixed volume of
nutrient culture medium.
 Continuous cultures:
1. Open: Both cells and the used medium are taken out from open continuously
culture and replaced by equal volume of fresh medium.
2. Closed: The cells separated from used medium taken out for replacement and
added back to the suspension culture, so that the cell biomass keeps on
increasing.

 PROTOPLAST CULTURES:
1) Isolated protoplasts have been described as “naked” cells because the cell
wall has been removed by either a mechanical or an enzymatic process.
2) Protoplast can be induced to reform a cell and divide if placed in a suitable
nutrient than form callus.
3) It develop novel hybrid plants through protoplast fusion
4) Protoplast cells also can regenerate into whole plants.
5) It help in gene transfer.

 EMBRYO CULTURE:
1) Embryo culture is usually done from the need to rescue embryo from wide
crosses where fertilization occurred, but not the embryo developments.
2) Production of haploid plants.
3) A common explant for the initiation of somatic embryogenetic cultures.
4) Overcoming abortion of embryos of wide hybrids at very early stages of
development due to incompatibility barriers.
5) In vitro fertilization for the production of distant hybrids avoiding style and
stigmatic incompatibility that inhibits pollen germination and pollen tube
growth.
6) Fig:-different step in ovule culture of spathiphyllum: flower(leaf), ovary
(middle left),ovule (middle right), secondary embryogenesis

 ANTHER CULTURE
1) Anther culture is a technique by which the developing anthers from
unopened flower bud are cultured on a nutrient medium where the
microspores within the cultured anther develop into callus tissue or
embryoids that give rise to haploid plants.
2) Production of haploid plants
3) Uncovering mutations or recessive phenotypes.
4) It is used for mutation studies
5) It is used for formation of double haploid that are homozygous and fertile.
6) It is used to study of factor controlling pollen embryogenesis of higher
plants.
7) It is used to study genetic recombination in higher plants.

ESTABLISHMENT AND MAINTENANCE OF
VARIOUS CULTURE
 There are three main culture system, selected on the basic of the objective.
I. Growth of callus masses on solidified media.
II. Growth in lipid media consist of mixture of single cell or cell aggregates.
III. Protoplast culture.

A.CALLUS CULTURE
 Callus is an amorphous aggregates of loosely arranged parenchyma cell, which
proliferate from mother cell. Cultivation of callus usually on a solidified
nutrient medium under as condition is known as callus culture.
 Maintenance of callus culture:-
After sufficient time of callus growth of same medium following change will
occur such as:-
1) Depletion of nutrient in the medium
2) Gradually losses of water
3) Accumulation of metabolic toxins
4) Callus tissues is transferred under aseptic condition to fresh medium.
5) Sub-culturing should be repeated after of 4 -5 weeks.
6) Many callus culture remain healthy and continue to grow at slow rate for
much longer period without sub-culturing.

A.CALLUS CULTURE
7) If the incubation to be carried out at low temperature 5 – 10 degree Celsius
below the normal temperature (16 – 18 0C).
8) Normally, total depletion take about 28 days.
 Principal:-
1) Aseptic preparation of plant material.
2) Incubation of culture under controlled physical condition.
3) Selection of suitable nutrient medium supplemented with appropriate ratio of
plant growth regulators such as auxin and cytokinins or only appropriate
auxin.

 Step involved:-
Steps of callus culture
Initiation
Selection and
preparation of
explants
Selected of
culture
medium
Transfer of
explant
Incubation
of culture
Maintenance
Sub -
Culture

B. SUSPENSION CULTURE
 Maintenance of suspension culture:-
Suspension culture
Batch
culture
Semi
continuous
culture
Continuous
culture
Open type
Chemostats Turbidostats
Closed type

 Suspension culture contains a uniform suspension of separate cells in liquid
medium
 Flow diagram :- Illustrating the method of cell suspension culture and
regeneration of plant through embryogenesis.
Callus tissue
in liquid
medium
Liquid medium
shaking on
shaker
Cell
suspension
Filtration
Centrifugation
decrease
resuspension in
fresh medium
Haemocytome-
ter
Shaking on
shaker
Plating on cell
suspension
Single cell Embryoids
Regenerated
plants
33

CULTURE MEDIUM
 Defined medium for the growth of cell cultures consists of the following
components.
1. Inorganic cells Concentration of potassium and nitrate at least 50-25mM
and phosphate, sulphate 1-3mM and magnesium appear to
be adequate, Ammonium is essential 8Mm, and
micronutrient like iodide, boric acid, zinc, manganese,
copper, cobalt, iron.
2. Vitamins Thaimine, pyridoxine, myo-inositol, nicotinic acid improves
cell growth.
3. Carbon source Sucrose, 2-4%
4. Growth
regulators
It induce cell division most commonly used regulator is NAA
naphthalene acetic acid, and 2,4-dichlrophenoxy acetic
acid(2,4-D)
5 Organic
supplements
Protein hydrolyzates, yeast, malt, extract and coconut milk
used for enhancement in growth rate.
34

06 Whites medium used for root culture
07 MS medium used for organogenesis
08 B5 medium used for callus culture, cell suspension and Protoplast
culture
09 N6 medium used for cereal anther culture
10 Nitsch’s medium used for anther culture

MEDIA PREPARATION
1) Chemical are dissolved in distilled water. The stock solution of vitamins,
micro-nutrient and growth regulators and hormones are added and pH
adjusted to 5.5-6.5.
2) The solution is made to volume 50 to 100 ml quantities distributed in to 250
ml Erlenmeyer flask.
3) Flasks are supported with cotton plug and autoclaved at 120 0C 15 min, all
media are stored at 10 0C.

COMPOSITION OF PLANT TISSUE CULTURE
MEDIUM
1) Macronutrients
2) Micronutrients
3) Iron source
4) Carbon and energy source
5) Vitamin
6) Amino acid
7) Other complex organic supplement
8) Plant growth regulators
9) Gelling or solidifying agents
10) pH regulators.

Macroelements
1. Potassium (K) 20-30 mM
2. Phosphorous (P) 1-3 mM
3. Calcium (Ca) 1-3 mM
4. Magnesium (Mg)1-3 mM
5. Sulfur (S) 1-3 mM
Micronutrients
1. Iron (Fe) 1 mM
2. Manganese (Mn) 5-30 mM
3. Zinc (Zn)
4. Boron (B)
5. Copper (Cu) 0.1 mM
6. Molybdenum (Mo) 1 mM
7. Cobalt (Co) 0.1 mM
CULTURE MEDIUM
Vitamins
1. Thiamine (vitamin B1)
2. Nicotinic acid
3. Pyridoxine (B6)
4. Myo-inosital
Sugar
1. Sucrose
2. Others
3. 20 to 40
g/l
Support system
1. Agar
2. Agarose
3. Gelrite (Phytagel)
Plant growth regulator
Amino acid
1. Tyrosine
2. Proline
3. Glutamic acid
4. Alanine
5. Aspartic acid
6. Casein hydrolysate
Plant growth regulators
1. Auxins 4. Ethylene
2. Cytokinins 5. Activated charcoal
3. Gibberellins 6. Abscisic acid
38

Other complex organic
supplement
1. Coconut milk
2. Peptone
3. Yeast extract
4. Ground banana
5. Watermelon juice
6. Potato extract
CULTURE MEDIUM
Iron Source
1. Ferric sulphate
(FeSO4, 7H2O)
2. Chelate irons
(Fe-EDTA)
Carbon and energy source
1. Fructose
2. Maltose
3. Galactose
4. Raffinose
5. Sorbitol
Gelling agent
1. Gelrite
2. Gellan
3. Phytagel
4. Agargel

Element Function
Nitrogen (N) Compound of proteins, nucleic acid and some coenzymes
Element required in greatest amount.
Potassium (P) Regulates osmotic potential, principal inorganic cation.
Calcium (Ca) Cell wall synthesis, membrane function, cell signaling.
Magnesium (Mg) Enzyme cofactor, component of chlorophyll.
Phosphorous (P) Component of nucleic acids, energy transfer, component of
intermediates in respiration and photosynthesis.
Sulphur (S) Component of some amino acid (methionine, cysteine) and
some cofactors.
Chlorine (Cl) Required for photosynthesis.
Iron (Fe) Electron transfer as a component of cytochromes.
Manganese (Mn) Enzyme cofactor.
Cobalt (Co) Component of some vitamins.
Copper (Cu) Enzyme cofactor, electron-transfer reaction.
Zinc (Zn) Enzyme cofactor, component of nitrate reductase.
Molybdenum (Mo) Enzyme cofactor, component of nitrate reductase.
40

GENERAL STEP INVOLVES IN PLANT TISSUE
CULTURE
Selection
of plant
Isolation of
explants
Sterilization
of explants
Inoculation
of Explants
Incubation
Sub-
culturing
Regeneration
Hardening
Green field

APPLICATION OF PLANT TISSUE CULTURE
1) It help in rapid multiplication of plants.
2) A large number of plantlets are obtained within a short period.
3) To study Respiration and Metabolism.
4) Production of secondary Metabolites
5) Production of haploids
6) Micropropagation
7) Cloning
8) Development of Transgenic Plants.
9) Genetically similar plants are formed by this method.
10) It is an easy, safe and economical method for plant propagation.

APPLICATION OF PLANT TISSUE CULTURE
11) Production of artificial seeds.
12) Single cell culture of higher plants.
13) Germplasm Storage.
14) Plant breeding
15) Plant physiology

EDIBLE VACCINES
 In the edible vaccine, Transgenic plants are used as vaccine production
system.
 Definition:
1. Edible vaccine is define as the vaccination is a disease preventive measure,
where the immune system of a person is boosted against a particular disease.
2. Edible vaccine is a new approach to oral immunization certain food under
investigation for use in edible vaccine such as banana, potato, tomato, spice
etc.
3. Edible vaccine are act by Stimulating the mucosal as well as systemic
immunity
 Transformation:
Edible vaccine are develop by introducing the selected desirable genes into the
plants, and then allowing the production of encoded protein by these altered
plants the process is called transformation

 Mechanism of Action of plants Based Edible vaccines:-
Intake of edible vaccine
Peyer’s patches-rich source of Ig A Producing plasma cell.
Edible vaccine break down at Peyer’s patches(contain 30-40 lymphoid nodules
containing follicles for development of germinal center.)
Antigen penetrate follicles accumulating antigen in lymphoid structure.
Antigen contact M-cells which express MHC II molecule.
Pocket formation occur which is filled with β cells, T cells & Macrophage.
Mastication and
degradation in intestine

M-cell with antigen activates β cell with in the lymphoid follicles.
Activated β cell leaves lymphoid follicles and reaches Mucosal Associated
lymphoid tissue (MALT)
Plasma cells are differentiated from β cell and IgA are produced.
IgA are secreted into lumen where they interact with antigen.

 Advantage:
1) Edible vaccine have efficient mode of action for immunization.
2) They are comparatively cost effective, as they do not require cold chain
storage.
3) They are safe as they do not contain heat-killed pathogens.
4) The production process is scaled up rapidly by breeding.
5) They are affordable.
6) They cause mucosal immunity.
7) They have greater stability products.
8) They do not need sophisticated equipment and machine.
9) As they are easily grown on rich soils.
10) They are widely accepted as they are orally administered unlike traditional
vaccine they are injectable.

 Importance:
1) It is used for cancer therapies like colon cancer and cervical cancer.
2) It is used for autoimmune disease like Type-I diabetes and multiple sclerosis.
3) It is applied for many infectious diseases like AIDS, tetanus, small pox,
measles, plague, foot and mouth disease, tuberculosis, influenza etc.
 Limitation:
1) Immune tolerance may develop in the individual for particular vaccine
protein.
2) Edible vaccine are dependent on plant stability.
3) Edible vaccine are prone to get microbial infestation.

IMPORTANT QUESTION
1) What is the plant tissue culture technique and its type?
2) What are the nutritional requirement for the development of plant tissue
culture?
3) Write a brief note on application of plant tissue culture in Pharmacognosy?
4) Explain the detail history of plant tissue culture?
5) Definition of PTC, it’s advantage and disadvantages
6) Explain basic techniques of plant tissue culture
7) Write in detailed about edible vaccine

MCQ
1. Who is known as the Father of tissue culture?
(a) Bonner (b) Laibach
(c) Haberlandt (d) Gautheret
Sol: (c) Haberlandt.
2. The production of secondary metabolites requires the use of ________.
(a) Meristem (b) Protoplast
(c) Axillary buds (d) Cell suspension
Sol:(d) Cell suspension.
3. The pair of hormones required for a callus to differentiate are________.
(a) Ethylene and Auxin (b) Auxin and cytokinin
(c) Auxin and Abscisic acid (d) Cytokinin and gibberellin
Sol: (b) Auxin and cytokinin.

4. Which of the following is the main application of embryo culture?
(a) Clonal propagation (b) Production of embryoids
(c) Induction of somaclonal variations (d) Overcoming hybridization barriers
Sol: (d) Overcoming hybridization barriers.
5. Haploid plants can be obtained from________.
(a) Anther culture (b) Bud culture
(c) Leaf culture (d) Root culture
Sol: (a) Anther culture
6.Which of the following growth hormones produces apical dominance?
(a) Ethylene (b) Cytokinin
(c) Auxin (d) Gibberellin
Sol: (c) Auxin.

7. What is Callus?
(a) Tissues that grow to form an embryoid
(b) An unorganized actively dividing the mass of cells maintained in a culture
(c) An insoluble carbohydrate
(d) A tissue that grows from an embryo
Sol: (b) An unorganized actively dividing mass of cells maintained in culture.
8. Growth of plant tissues in artificial media is called _______.
(a) cell hybridization (b) plant tissue culture
(c) Transgenesis (d) gene expression
Sol: (b) Plant tissue culture
9. ___________ is the advantage of plant tissue culture over animal tissue culture
a) Plant culture require less time
b) Plant tissues are easily available
c) Totipotency of the plant cell
d) Plant tissue culture can be easily maintained with minimum requirements
Sol:(C) Totipotency of the plant cell

10.___________is the type of Cell culture
a) Organ culture b) Protoplast culture
c) Callus culture d) Explant culture
Sol: (b) Protoplast culture

Plant tissue culture

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