Bio-Botany - Lesson - 5 - Plant Tissue Culture

HIGHER SECONDARY SECOND YEAR
BIOLOGY – BOTANY
CHAPTER – 5
PLANT TISSUE CULTURE
A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)
GOTTLIEB HABERLANDT
The father of Plant tissue culture

Growing plant protoplasts, cells,
tissues or organs away from their natural
environment, under artificial condition is
known as Plant tissue culture

Plant tissue culturing is usually carried out under controlled environment inside the laboratory using test tubes or glass
vessels. Hence it is also called as In-vitro culture

BASIC CONCEPTS OF TISSUE CULTURE
Totipotency
Differentiation
Dedifferentiation
Redifferentiation
The genetic potentiality
TOTIPOTENCY of any live plant cell to give
rise to a complete individual plant when cultured in a
nutrient medium is called TOTIPOTENCY

Leaf cells
The process in which an undifferentiated

cell undergoes structural, biological and Xylem cell
Xylem cell
chemical changes and become specialized
in its form and function is called
DIFFERENTIATION
A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247) phloem cell
PARENCHYMA CELLS
DEDIFFERENTIATION MERISTEMATIC CELLS
Reversion of mature permanent tissue into

meristematic tissue leading to the formation of callus
PARENCHYMA CELLS The differentiation of already differentiated cell into another type
of cell is known as REDIFFERENTIATION
DEDIFFERENTIATION
REDIFFERENTIATION
CALLUS CELLS XYLEM CELLS DIFFEERNTIATIED

WITH MERISTEMATIC CAPACITY
A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247) FROM CALLUS
LABORATORY FACILITIES FOR PLANT TISSUE CULTURE
• A sterile room
• Laminar air flow chamber with HEPA filter • Sterile room
• A sterile culture room • Laminar air flow chamber
• Glass instruments
• Autoclave
• Autoclave
• Electronic balance • Electronic balance
• pH meter • pH meter
• Glass wares and other accessories
• proper illumination & temperature
• Washing room
• fd
Plant tissue culture lab
-
Transfer room

LAMINAR AIR FLOW CHAMBER
WITH
HIGH EFFICIENCY PARTICULATE FILTER
A
SCIENTIST
WORKING WITH
LAMINAR AIR FLOW CHAMBER

A STERILE CULTURE ROOM
FOR
MAINTAINING THE CULTURE

AUTOCLAVE

LABORATORY OVEN
&
THERMOMETERS

pH METER & ELECTRONIC BALANCE

GLASS WARES USED IN PLANT TISSUE CULTURING

EQUIPMENTS USED IN PLANT TISSUE CULTURE

NUTRIENT MEDIA
IN
• Murashige & Skoog medium
• Gamborg medium
• White medium
• Nitsch’s medium

NUTRIENT MEDIUM & SOLIDIFIER
USED IN

1 STERILIZATION STEPS INVOLVED
IN
2 MEDIA PREPERATION PLANT TISSUE CULTURE
3 CULTURE CONDITION
4 CALLUS FORMATION
5 EMBRYOGENESIS

6 HARDENING
STERILIZATION – THE PROCESS TO GET RID OF MICROBES
S MATERIALS TO BE STERILIZED MODE OF MATERIALS /
N STERILIZATION PROCESS
1. Glassware, forceps, Scalpels Steam sterilization Autoclaving at 15 psi,
121°C for 30 minutes
2. Floor of culture room Mopping & Chemical Detergents, 95%
sterilization Ethanol
Radiation 2% Sodium
hypochlorite
3 Laminar air flow chamber Chemical sterilization & 95% Ethanol & UV
Radiation rays
4. Culture media Steam sterilization Autoclaving at 15 psi,
121°C for 30 minutes
5. Plant extracts, vitamins, Amino acids, Filtration Millipore filter with
Hormones 0.2 mm pore diameter
PREPARATION OF MEDIUM
•A nutrient medium is a proper
composition of essential
macronutrients, micronutrients,
vitamins, hormones, carbon sources
etc. which is utilized by the tissues
for their growth in culturing process
•A medium may be solid,
semisolid(broth) or liquid
COMPOSITION
OF
MURASHIGE & SKOOG MEDIUM

OPTIMAL CULTURE CONDITION FOR PLANT TISSUE CULTURE
Sno PARAMETERS OPTIMUM CONDITION
1 pH of medium 5.6 to 6.0
2 Temperature 25°C ± 2°C
3 Relative humidity 50% to 60%
4 Light intensity 1000 lux to 2400 lux
5 Photoperiod 8 to16 hours
6 Aeration Through rotary shaker

INNOCULATION OF EXPLANT & INDUCTION OF CALLUS
•Transferring the explant to the nutrient medium is called inoculation
•Under optimal conditions, the explant develops callus over its surface
INNOCULATION CALLUS FORMATION

EMBRYOGENESIS
Differentiation of callus & the formation of embryos
from the callus
is called as
Embryogenesis

• The morphological changes occurring in callus to
form root and shoot is called organogenesis
ORGANOGENESIS • Auxin and cytokinin are the essential growth
hormones to induce organogenesis
CAULOGENESIS
CALLUS
RHIZOGENESIS
HARDENING
The gradual exposure of in-vitro developed plantlets
into humid chambers in diffused light for
acclimatization so as to enable them to grow under
normal field

SOMATIC EMBRYOGENESIS
• The development of embryo from the callus tissue grown from the somatic cells of plant in
in-vitro conditions is called as somatic embryogenesis
• The embryos are called as Embryoids
• This process is done in many plants including rice, onion , maize etc.
APPLICATIONS OF SOMATIC EMBRYOGENESIS

1. To produce synthetic seed
2. To provide potential plantlets
ARTIFICIAL SEEDS
• The somatic embryos (Embryoids) developed by in-vitro
culture are encapsulated with Agarose gel or Calcium
alginate or Sodium alginate to produce the artificial seeds.
• It is also called as Synthetic seeds or Synseeds

ADVANTAGES OF ARTIFICIAL SEEDS
• More seeds in quick time at low cost
• Easy way to produce genetically engineered plants
• To test the genotype of a plant
• Easy to cryopreserve for long time
• To produce identical plants
• Growth of plants from synseeds are faster due to reduced dormancy
TYPES OF PLANT TISSUE CULTURE
(based on the explant used)
ORGAN
CULTURE
MERISTEM PROTOPLAST
CULTURE PTC CULTURE
CELL
SUSPENSION
CULTURE

ORGAN
CULTURE
CULTURING
OF ANY ORGANS OF
A PLANT BODY

MERISTEM CULTURE
Culturing of plant meristematic tissue on a culture medium
SHOOT TIP MERISTEM

(microscope image)
PROTOPLAST CULTURE
•Culturing the protoplast to generate a whole plant is the principle of Protoplast culture
• Protoplast are the cells without a cell wall, but bounded by cell membrane
STEPS IN PROTOPLAST CULTURE
a) Isolation of protoplast
b) Fusion of protoplast
c) Culturing of protoplast
d) Induction of callus
e) Embryogenesis
ENTIRE PROCESS
OF
PROTOPLAST CULTURE

CELL SUSPENSION CULTURE
Growing of cells including the culture of single cells or small
aggregates of cells in-vitro in liquid medium is known as cell
suspension culture

STEPS IN THE CELL SUSPENSION CULUTRE
Development of Transfer of small Callus in medium Cells are Separated cells are
Callus by callus portion to is agitated by rotary separated due to cultured to form
PTC liquid medium shaker agitation callus

BENEFITS OF CELL SUSPENSION CULTURE
•Cell suspension culture is used in the production of secondary

metabolites like alkaloids, flavonoids, terpenoids, phenolic
compounds, recombinant proteins etc.
•Secondary metabolites are of high commercial importance and
used in various fields especially in medicine.

Some important secondary metabolites & their plant source
SECONDARY METABOLITES PLANT SOURCE MEDICINAL APPLICATIONS
DIGOXIN Digitalis purpuria Cardiac tonic
CODEINE Papaver sominiferum Analgesic
CAPSAICIN Capsicum annum Rheumatism
VINCRISTINE Catharanthus roseus Anti-carcinogenic
QUININE Cinchona officinalis Anti-malarial

PLANT
REGENERATION
PATHWAYS
FROM
EXPLANTS

MICROPROPOGATION PROTOCOL
OF
BANANA PLANT

STAGES
IN
MICROPROPAGATION
OF
BANANA
THROUGH
FLORAL BUD

SHOOT TIP MERISTEM CULTURE
(to obtain Virus – free plants)

Germplasm conservation
refers to the conservation of
GERMPLASM living genetic resources like
pollen, seeds or tissue of plant
CONSERVATION material maintained in live
condition and used for
selective plant breeding and
other research works
It is carried out to maintain

biological diversity and for
food security
CRYOPRESERVATION
• Cryopreservation is a process by which
various biological materials like
protoplasts, enzymes, tissues etc. are
preserved by cooling in a very low
temperature of -196℃ using liquid
nitrogen
• Before cryopreserving the materials are

treated with cryoprotectants like
sucrose, glycerol or dimethyl sulphoxide
to protect them from freezing stress
INTELLECTUAL PROPERTY RIGHTS
• Intellectual property rights is a type of property that includes intangible creation of human intellect
• It includes
a) copyrights
b) patents
c) Trade marks
d) Trade secrets
e) Publicity rights
f) Moral rights
g) Right against unfair competition
• IPR are legal rights given to persons over the creations of their minds
PATENT
• Patent is a special right to the discoverer/inventor that
has been granted by the Government by legislation for
trading new articles
• It is a personal property belonging to inventor/discoverer
• Patent terms gives the inventor the rights to exclude
others from making/using/selling his invention

IMPORTANT PARTS OF A PATENT
 REQUEST FOR GRANT OF PATENT
 SPECIFICATIONS
 CLAIMS
 ABSTRACT
 DRAWINGS
 GRANT
/
PATENT
REGISTRA
TION
PROCESS
BIOSAFE
TY
• Bio-safety is the prevention of large-scale loss of biological
integrity, focusing both on ecology and human health.
• It includes regular reviews on laboratory safety and guideline to be

followed
• It is used to protect the humans and ecology from harmful activities

like exposure to radiations, hazardous substances or disease
causing pathogens or genetically modified harmful microbes
CENTRAL GOVERNMENT COMMITTEES
INVOLVED IN
FRAMING BIOSAFETY GUIDELINES
•Institutional Bio-safety Committee

•Review Committee on Genetic Manipulation
•Genetic Engineering Approval committee

• GEAC is an apex body under Ministry of Environment
• It is established to accord approval of activities involving large scale use of hazardous
microbes and recombinants in research & industrial production
• It regulates the manufacturing, use, import, export and storage of hazardous microbes in the
country
• It is also responsible for the approval of proposals related to GMO’s and its products into the
environment including experimental field trials.

BIO-ETHICS
• Bio-ethics refers to the study of ethical issues caused due to advances in biology
and medicine field.
• It is a moral discernment
• It includes the study of values relating to primary care and other medicinal fields
• The scope of bio-ethics is related to technologies like cloning, gene therapy,
human genetic engineering, life extension, astro-ethics, alternations in DNA,
RNA and proteins etc.
• It aims to maintain the values of life and its basic biological characters and
structures
Ethical, Legal and Social Implications program
• ELSI program was founded in 1990
• It is founded as a integral part of Human Genome Project
• The goal was to identify and address issues raised due to genomic research that would affect
individuals, families and society.
Ethical issues in Genomic research
a) Privacy and fairness in using genetic informations
b) Integration of new genetic technologies into the clinical medicine
c) Issues related to design and conduct of genetic research with people
Future Bio-technology
leads to a modernized world

By
A. Sivakumaran
Post Graduate Teacher in Biology

Bio-Botany - Lesson - 5 - Plant Tissue Culture

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HIGHER SECONDARY SECOND YEAR

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

The process in which an undifferentiated

DEDIFFERENTIATION MERISTEMATIC CELLS

Reversion of mature permanent tissue into

CALLUS CELLS XYLEM CELLS DIFFEERNTIATIED

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

2 Temperature 25°C ± 2°C

3 Relative humidity 50% to 60%

4 Light intensity 1000 lux to 2400 lux

5 Photoperiod 8 to16 hours

6 Aeration Through rotary shaker

INNOCULATION CALLUS FORMATION

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

APPLICATIONS OF SOMATIC EMBRYOGENESIS

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

SHOOT TIP MERISTEM

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

•Cell suspension culture is used in the production of secondary

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

DIGOXIN Digitalis purpuria Cardiac tonic

CODEINE Papaver sominiferum Analgesic

CAPSAICIN Capsicum annum Rheumatism

VINCRISTINE Catharanthus roseus Anti-carcinogenic

QUININE Cinchona officinalis Anti-malarial

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

It is carried out to maintain

• Before cryopreserving the materials are

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

• It includes regular reviews on laboratory safety and guideline to be

• It is used to protect the humans and ecology from harmful activities

•Institutional Bio-safety Committee

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

A.SIVAKUMARAN, Post Graduate Teacher in Biology. (Contact no. 8939029247)

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