UNIT 4

Tissue Culture
Introduc on:
Tissue Culture: Removal of cells, ssues, or organs from an animal or plant and their
subsequent placement into an ar ficial environment for growth. This environment usually
consists of a glass or plas c culture vessel containing a liquid or semi-solid medium that
supplies the nutrients essen al for survival and growth.
Animal vs Plant Tissue Culture:

Animal Tissue Culture Plant Tissue Culture

Meaning Growth of animal ssues in vitro, Growth of plant cells or ssues,

in laboratory environment. under controlled condi ons in a
specialized nutrient medium.

Purpose Vaccine development, study cell Plant propaga on, disease free
behaviour, disease research, drug plants
development

Source of Animal organs, ssues or cells Plant ssues or cells

Cells

Growth Strict requirements for Less strict requirements

requirements temperature, CO2, and humidity compared to animal cultures

Nutrient Complex media containing animal Synthe c media containing

Media serum, hormones, and other sugars, salts, vitamins, and plant
nutrients hormones

Types • Organ culture: Whole organs or • Meristem Culture ( ssue at

intact organ fragments are shoots and roots)
cul vated to study their • Embryo Culture (immature and
con nued func on or mature embryos)
development. • Callus Culture (unorganized
• Cell culture: When the cells are mass of cells from plant
removed from the organ explants)
fragments prior to, or • Organ Culture (shoots, roots or
during cul va on. leaves)
• Anther Culture (help in
pollina on)
 • Protoplast Culture (plant cell
with no cell wall)
• Suspension Culture (liquid
media)

Examples Culturing kidney cells for drug Micropropaga on of orchids,

tes ng, growing nerve cells for soma c embryogenesis for
studying neurodegenera ve disease-free citrus plants,
diseases. cryopreserva on of endangered
tree species.

History of Tissue Culture:

 An early a empt at ssue culture was made in 1885 by German zoologist Wilhelm
Roux, who cul vated ssue from a chick embryo in a warm salt solu on.
 In 1902, Go lieb Haberlandt proposed the theore cal basis of plant ssue culture.
He is known as the father of plant ssue culture. He experimented with isolated
photosynthe c leaf cells but was unsuccessful in inducing any growth. However, he
predicted that one can obtain ar ficial embryos from vegeta ve cells using this
culturing technique and established the concept of to potency.
 In 1904, Henning isolated embryos of some crucifers and successfully grew on
mineral salts and sugar solu ons
 The first real success came in 1907, however, when American zoologist Ross G.
Harrison demonstrated the growth of frog nerve cell processes in a medium of clo ed
lymph.
 French surgeon Alexis Carrel and his assistant Montrose Burrows subsequently
improved upon Harrison’s technique, repor ng their ini al advances in a series of
papers published in 1910–11.
 Carrel and Burrows coined the term ssue culture and defined the concept.
 In 1934, cells from willow and poplar trees were cul vated from lab.
 In 1939, first con nuously growing plant cell cultures were derived from carrot root.
 In 1940s, the first mammalian cell was cultured. It was the con nuous cell line called
HeLa cells derived from the cervical cancer cells.
 By the mid-1950s, the availability of two cell lines, mouse L cells (1943) and human
HeLa cells (1951), as well as the ini al use of trypsin for subculturing cells into single
cell suspensions led to most cells being grown as monolayer cultures in glass flasks and
dishes.
 In 1959, plant was regenerated completely from a single carrot cell.
 In 1950s, serum-based media was also developed.
  In 1954, researchers at Connaught Medical Research Laboratories in Toronto cultured
monkey kidney cells in flasks to grow the virus for Jonas Salk’s successful polio virus
vaccine. This was the first use of cell culture to manufacture commercial product.
 In 1956 George Gey used rat tail collagen to coat glass surfaces to improve cell
a achment and growth.
 By the 1960s, plas c flasks, dishes, and 96 well plates were all available commercially.
Human fibroblast cells were isolated and it was seen to show finite life span.
 By the mid-1970s most researchers were growing their cell cultures in treated
polystyrene vessels rather than glass. Hybridoma technology for produc on of
monoclonal an bodies was developed in the 1970s.
 From 1970’s to present, in plant ssue culture there has been a lot of improvements
in terms of culture media, growth regulators and asep c techniques. A few plant ssue
culture techniques like micropropaga on, soma c embryogenesis and
cryopreserva on techniques were developed.
 In the 1980s, serum-free culture media was developed.
 In 1990s, 3D cell culture systems were inves gated for studying cell-cell interac ons.
 Advanced bioreactor systems (par cularly in the field of biopharmaceu cal
produc on) were developed in the 2000s.
 Organoids and stem cells were developed in 2010s. In 2020s, 3D prin ng of ssues &
organs and CRISPR technology (for gene edi ng) was developed.
ANIMAL TISSUE CULTURE:
Instruments used in Animal Tissue Culture:
 Tissue Culture Hood (Laminar Flow Hood: It provides a sterile environment for working
with cell cultures by maintaining a con nuous flow of filtered air. It is equipped with
HEPA filters to remove airborne contaminants. It protects the culture from microbial
contamina on.
 Incubator: It maintains a controlled environment for cell cultures by regula ng
temperature, humidity, and CO2 levels. It is typically set to 37°C (body temperature)
and 5% CO2, mimicking physiological condi ons for cell growth.
 Inverted Microscope: It allows observa on of cells from the bo om of the culture dish
or flask, making it suitable for monitoring cell growth. The inverted design allows for
the examina on of cells in a more natural orienta on.
 Centrifuge: It separates cellular components based on density, such as isola ng cells
from culture medium or obtaining subcellular frac ons. Has variable speed and rotor
op ons to accommodate different sample types and sizes.
 Water Bath: It maintains a constant temperature for thawing frozen cells, warming
media, or conduc ng specific temperature-sensi ve procedures. It has digital
temperature control and a water circula on system for uniform hea ng.
  Fridge & Freezer: It is used for storage of cell culture media, reagents, and biological
samples. Fridge is typically set at 4°C for short-term storage, while the freezer is set at
-20°C or -80°C for long-term storage of cells and biological materials.
 Liquid Nitrogen Storage: It preserves cells for long periods by freezing them in liquid
nitrogen. The cryogenic storage vessels is designed to store cells at ultra-low
temperatures (-196°C).
 Haemocytometer: It is used for manual cell coun ng and assessing cell concentra on.
It is a special coun ng chamber with a grid pa ern that allows for accurate
determina on of cell density.
Steps in Animal Tissue Culture:
Selection of Tissue

Sterilization

Preparation of Growth Media

Harvesting Cells

Isolating Cells

Cell Countig and Quality Control

Experimentation and Storage

1. Selec on of Tissue: The selec on of ssue for animal ssue culture is a pivotal step in
biological research. Researchers must define clear objec ves, considering the source
organism, ethical considera ons, and ssue characteris cs. Accessibility and poten al
contamina on risks should be weighed, and ssue collec on should be performed
me culously under sterile condi ons.
2. Steriliza on: Sterile environment is crucial in animal cell culture to prevent
contamina on and ensure the success of experiments.
Steriliza on methods used in animal cell culture are:
a. Autoclaving: Autoclaving is a steriliza on method that involves the use of an
autoclave, which is a sealed chamber that uses high pressure and high
temperature to eliminate microorganisms, including bacteria, viruses, and
spores, from equipment, instruments, or media.
b. Filtra on: It is a process of using filters to remove microorganisms and
par culate ma er from liquids, such as culture media or reagents, to create a
sterile environment for cell culture. This method is par cularly important to
prevent contamina on and maintain the integrity of cell cultures. Filtra on can
be accomplished using filters with pore sizes small enough to trap bacteria,
fungi, and other unwanted par cles.
c. Chemical Steriliza on: Chemical steriliza on in animal ssue culture involves
the use of chemical agents to eliminate or inhibit the growth of
microorganisms, including bacteria, viruses, and fungi, from surfaces,
equipment, or solu ons used in ssue culture. Ex: Ethanol and Isopropanol,
Hydrogen Peroxide, Chlorine-Based Compounds ec.
d. UV Irradia on: UV (ultraviolet) irradia on steriliza on in animal ssue culture
involves using ultraviolet light to disinfect surfaces, equipment, and air in order
to create a sterile environment for cell culture experiments. UV irradia on is
effec ve against bacteria, viruses, and fungi by causing damage to their DNA
or RNA, preven ng replica on and rendering them non-viable.
e. Gaseous Steriliza on: Gaseous steriliza on in animal ssue culture involves the
use of chemical gases, such as ethylene oxide or hydrogen peroxide vapor, to
achieve microbial inac va on on surfaces, equipment, or enclosed spaces in
order to create a sterile environment for cell culture experiments. This method
is par cularly useful for items that cannot withstand heat or moisture, and it is
employed to ensure the elimina on of bacteria, viruses, and spores.
f. Gamma Irradia on: Gamma irradia on steriliza on in animal ssue culture
involves the use of ionizing radia on, specifically gamma rays, to eliminate or
reduce the microbial load on surfaces, equipment, or materials used in cell
culture experiments. Gamma irradia on is effec ve against a broad spectrum
of microorganisms, including bacteria, viruses, and spores.
g. Alcohol Wipe/Spray: Alcohol wipe or spray steriliza on in animal ssue culture
involves the use of alcohol-based solu ons, typically isopropyl alcohol (IPA) or
ethanol, to disinfect surfaces, equipment, and tools in order to create a sterile
environment for cell culture experiments. Alcohol is a widely used disinfectant
in laboratories due to its broad-spectrum an microbial proper es.
h. Flame Steriliza on: This involves the use of an open flame to disinfect and
sterilize tools, such as metal forceps or inocula on loops, by exposing them to
high temperatures. This method is commonly employed in laboratory se ngs
to create a sterile environment and prevent contamina on during cell culture
experiments.
i. Heat Treatment: It involves the use of elevated temperatures to eliminate or
reduce the microbial load on surfaces, equipment, media, or other materials
used in cell culture experiments. This method relies on the principle that high
 temperatures can denature proteins and destroy the structural integrity of
microorganisms, rendering them non-viable.
j. Detergents and Disinfectants: Detergents are used to clean laboratory surfaces,
equipment, and glassware before steriliza on or disinfec on procedures.
Disinfectants are crucial for maintaining asep c condi ons in ssue culture
work. They are applied to surfaces, such as laminar flow hoods, biosafety
cabinets, and countertops, to eliminate or reduce the presence of bacteria,
viruses, and other contaminants.
3. Prepara on of Growth Media
Prepara on of animal ssue culture growth media combines specific components to
create a nutrient rich environment that supports growth and maintenance of cells.
Prepara on of media:
1. Media components are sterilized using filtra on or autoclaving. This keeps the
media away from contaminants.
2. Combining the sterilized components in a sterile environment ( ssue culture
cabinet)
3. pH adjusted to desired level using acids (HCl) or bases (NaCl).
4. A er mixing and pH stabiliza on, medium is filtered to remove any par culate
ma er and ensure sterility.
5. Prepared medium is stored at appropriate temp. Some at 4°C and others frozen at
–20 to -80°C
4. Harves ng:
Process of collec ng or obtaining ssues or organs from a living organism. There are
mul ple cell harves ng methods that one might choose depending on the context of
their experiment.
 Surgical excision used in organ transplant, organ or part of ssue taken.
 Biopsy: ssue removed for diagnosis or research
 Punch biopsy: remove a small cylindrical core of ssue.
 Needle aspira on: needle for fluid or ssue
 Cure age: spoon shaped instrument cure e used to remove ssue
 Endoscopy: obtain ssue collec on from internal organs.
 Laparoscopy: obtain ssue samples from the abdominal cavity.
 Bone marrow: withdraw liquid bone marrow from the hip or sternum.
 Amputa on: Surgical removal of a specific ssue or organ from an animal for the
purpose of isola ng cells for in vitro culture.
 Organ Harves ng for Transplanta on: Surgical removal of an en re organ from a
donor organism for the purpose of transplan ng it into a recipient to replace a
damaged or malfunc oning organ.
5. Isola ng cells
1. Primary Culture:
When harvested cells from the organism is placed into a suitable culture environment,
they will a ach, divide and grow.
 Physical disaggrega on: Breaking down ssue with physical force. It is
inexpensive, rapid, and simple but they damage cells.
 Enzyma c disaggrega on: Diges ng enzymes are added to the ssue
fragments to dissolve the compound holding the cells together. This creates a
suspension of single cells that are then placed into culture vessels containing
culture medium and allowed to grow and divide.
  Primary explant technique: Used for disaggrega on of small quan es of
ssues (e.g. skin biopsies). Tissue is chopped finely, rinsed, and pieces
are placed on the surface of the culture medium and incubated.
 Chela ng Agents: Chela ng agents like EDTA along with ions like Ca, Mg etc.,
are used to prepare cell suspensions by maintaining the integrity of cells.
 Density Gradient Centrifuga on: Separates cells based on their density. Used
for isola ng specific cell types or enriching for certain cell popula ons based
on density differences.
 Filtra on: Involves passing a ssue or cell suspension through a filter to
separate cells based on size. Used for obtaining single-cell suspensions from
ssues with a heterogeneous cell popula on.

2. Secondary Culture:
When cells from primary culture on
confluency are isolated and cultured in new
media, it is called as secondary culture.
Secondary culture is also known as
subculture. Subculture allows fresh nutrients
and more space for the expansion of the
cells.
The sequence of subcultures prepared from
primary culture is called “Cell lines”.
Cells from primary culture are splits by
trypsin/EDTA treatment. Trypsin digests the
extracellular protein so that cells get free. EDTA which is a chela ng agent chelates
calcium ion because calcium helps in cell adhesion.
Some cell lines can be harvested by slightly scrapping off the bo om of the cell
vessel.
Based on the culture life span, the cell lines are categorized into two types:
 Finite cell lines: Cell lines with limited number of cell division and having limited
life span.
 Con nuous cell lines: Some cells of the finite cell line undergo muta on and
acquire the ability to divide indefinitely.
Once cells have grown in the subculture, they are treated with suitable
cryoprotec ve agents like dimethylsulfoxide (DMSO) or glycerol, carefully frozen
and then stored at cryogenic temperature below –130°C un l they are needed.
Differences between primary culture and secondary culture
SN Primary Culture Secondary Culture
1 The growing and maintaining of A cell line or sub-clone sub-cultured
the selected cell type taken from a from primary cell culture
normal parental ssue
2 Contains the cells directly obtained Contains sub-cultured cells from
from a host ssue either through primary cell culture
mechanical or enzyma cal
diges on.
3 Heterogeneous: Has different Homogeneous: Has same kind of cells
types of cells in culture. in culture.
4 Have the same biological response Adapted to the culture condi ons by
as the cells in the host ssue. altering their biology
5 Have similar gene c makeup to Have altered gene c makeup
the cells of the host ssue.
6 High risk of contamina on Low risk of contamina on
7 Have a finite lifespan Have an indefinite lifespan
8 Requires a rich mixture of amino Easy to maintain
acids, micronutrients, certain
hormones, and growth factors
9 Does not contain a sufficient Can have the op mal cell density
amount of cells
10 Important in manufacturing Important for the produc on of
vaccines and therapeu c hormones, an bodies, an cancer
development agents, etc.

6. Cell coun ng & Quality control

1. Hemocytometer (Manual Cell Coun ng):
Principle: The hemocytometer is a glass slide with a grid pa ern that allows for the
manual coun ng of cells under a microscope. Cells are placed in a chamber, and the
number of cells within the grid is counted to calculate cell concentra on.
Procedure: Cells are mixed with a diluent, loaded into the hemocytometer chamber,
and observed under a microscope. The cells in the grid squares are counted, and the
concentra on is calculated based on the known volume of the chamber.
Advantages: Simple, cost-effec ve, and suitable for various cell types.
Limita ons: Time-consuming, subject to user variability, and may require staining for
accurate viability assessment.
2. Automated Cell Counter:
Principle: Automated cell counters use image analysis technology to count cells quickly
and accurately. They o en employ trypan blue staining for viability assessment.
Procedure: A cell suspension is mixed with a dye (trypan blue) that stains dead cells.
The instrument uses image recogni on so ware to dis nguish between live and dead
cells, providing total cell counts and viability percentages.
Advantages: Rapid, precise, and reduces user subjec vity. Provides both total and
viable cell counts.
Limita ons: Costlier than manual methods, and may require specific consumables and
maintenance.
3. Trypan Blue Exclusion Method:
Principle: Trypan blue is a dye that selec vely stains dead cells with compromised cell
membranes. Live cells exclude the dye, while dead cells take up the dye, becoming
blue.
Procedure: Cells are mixed with trypan blue, and both live and dead cells are counted
using a hemocytometer or an automated cell counter. Viability is determined by
calcula ng the ra o of live to total cells.
Advantages: Quick, rela vely simple, and suitable for a variety of cell types.
Limita ons: Subject to user variability, does not provide informa on about cell
morphology, and may not accurately dis nguish between apopto c and necro c cells.
4. Fluorescent Scanning (Flow Cytometry or Fluorescence Microscopy):
Principle: Fluorescent dyes are used to label cells, and a flow cytometer or
fluorescence microscope is employed to analyze the labeled cells based on
fluorescence signals.
Procedure: Cells are stained with fluorescent dyes, and the instrument detects and
quan fies fluorescence signals, allowing for the enumera on of cells. Viability can be
assessed based on the fluorescence characteris cs of the dye.
Advantages: High-throughput, provides detailed informa on about cell characteris cs,
and allows for mul parametric analysis.
 Limita ons: Equipment cost, complexity, and may require specialized training. Flow
cytometry is typically used for suspension cells, while fluorescence microscopy is
suitable for adherent cells.
7. Experimenta on & Storage
 Experiments performed on cultured cells to inves gate specific research ques ons
(tes ng the effects of drugs, studying cellular processes, or conduc ng molecular
analyses).
 Storage done using cryopreserva on where a cryoprotec ve agent (e.g., dimethyl
sulfoxide) is used and frozen in liquid nitrogen.
 Post-Thaw Quality Check: Cell viability and func onality are assessed.
 Method of harves ng from culture chosen based on the ssue type and purpose
of the experiment (enzyma c dissocia on, mechanical dissocia on, and scraping).
 Frozen cells are thawed when needed rapidly using a water bath at 37°C.
Cell Culture Techniques
Asep c Handling: To prevent contamina on and maintain a sterile environment during cell
culture procedures. (Tissue Culture Hood, Steriliza on, Personal Protec ve Equipment (PPE).)
Cell Line Establishment: To derive and establish a popula on of cells that can be cultured over
mul ple genera ons. (Cell Isola on, Primary Culture, Immortaliza on.)
Maintenance of Cell Lines: To ensure the con nuous and healthy growth of established cell
lines. (Subculturing, Regular microscopic observa on and cell coun ng to assess cell health
and density.)
Adherent Culture: Allows cells to a ach and grow on a solid substrate. Coa ng culture vessels
with extracellular matrix proteins, Detaching cells using enzymes (e.g., trypsin) for
subculturing.
Suspension Culture: Allowing cells to grow freely in a liquid medium without a aching to a
surface. Spinner Flasks or Bioreactors with gentle agita on to keep cells suspended. Cells are
harvested by centrifuga on for passaging or analysis.
Applica ons of Animal Tissue Culture
Cell Biology Studies:
 Primary Cell Culture: Isola on and culture of primary cells from animal ssues enable
the study of cell behavior, morphology, and func ons in a controlled environment.
Medical Research:
Drug Discovery and Development: Animal ssue culture is crucial in tes ng the efficacy and
safety of poten al drugs before advancing to clinical trials. It helps iden fy promising drug
candidates and understand their effects on cells and ssues.
Vaccine Produc on:
 Virus Propaga on: Animal ssue cultures are used for the propaga on of viruses that
are used in the produc on of vaccines. Cultured cells provide a controlled environment
for virus replica on.
Cancer Research:
 Cancer Cell Lines: Established cancer cell lines derived from animal ssues are used to
study the mechanisms of cancer, test poten al an -cancer drugs, and inves gate
molecular pathways involved in tumorigenesis.
Gene c Engineering and Biotechnology:
 Recombinant Protein Produc on: Animal cells are used to produce recombinant
proteins, including therapeu c proteins and enzymes, using gene c engineering
techniques.

Stem Cell Research:

 Stem Cell Culture: Animal ssue culture is fundamental for the maintenance and
differen a on of stem cells. It is crucial in understanding stem cell biology and
exploring their poten al applica ons in regenera ve medicine.
Toxicology Studies:
 Toxicity Tes ng: Animal ssue culture is used to assess the toxic effects of various
compounds and chemicals. It provides a controlled environment to study cellular
responses to poten al toxins.
Infec ous Disease Studies:
 Host-Pathogen Interac ons: Animal ssue cultures are used to study host-pathogen
interac ons in infec ous diseases. This includes inves ga ng the mechanisms of
infec on, tes ng an viral or an bacterial compounds, and understanding immune
responses.
Biomedical Engineering:
 Biocompa bility Tes ng: Animal ssue cultures are employed to assess the
biocompa bility of materials used in medical devices, implants, and ssue engineering
constructs.
Reproduc ve Biology:
 Gamete and Embryo Culture: Animal ssue culture is used in reproduc ve biology to
culture gametes (sperm and eggs) and embryos. This is essen al in assisted
reproduc ve technologies and fer lity research.
Neuroscience Research:
 Neuronal Cell Culture: Animal ssue cultures, par cularly neuronal cell cultures, are
used to study the physiology and func on of neurons. This is important for
understanding neurodegenera ve diseases and developing poten al treatments.
Environmental Monitoring:
 Biomarker Studies: Animal ssue cultures can be used to study the impact of
environmental factors on cellular biomarkers, helping to assess environmental toxicity.
PLANT TISSUE CULTURE
Plant ssue culture is the culture of all types of plant cells, ssues, and organs under asep c
condi ons. Each plant cell has the power to develop into any type of cell and a complete plant
if provided all essen al requirements and a suitable environment. Plants can be grown under
in vitro condi ons using any part of plants, such as leaves, stems, roots, bud, and meristem.
Instruments in Plant Tissue Culture
1. Laminar Flow Hood: This enclosed worksta on provides a sterile environment for
working with plant ssues. HEPA filters within the hood con nuously blow ultra-clean
air, minimizing the risk of contamina on.
2. Autoclave: This pressurized chamber uses high-temperature steam to sterilize culture
media, glassware, and other equipment, elimina ng microorganisms and spores.
3. Scalpels and Forceps: These sterilized cu ng tools are used for precise handling and
dissec on of plant ssues during explant prepara on.
4. Petri Dishes and Culture Flasks: These sterile containers hold the culture medium and
explants. Petri dishes are typically used for ini al culturing, while flasks are used for
longer-term growth and mul plica on.
5. Magne c S rrer and S r Bar: This combina on helps dissolve media components like
basal salts and sugars efficiently within the culture solu on.
6. pH Meter: This instrument measures the acidity or alkalinity (pH) of the culture
medium, which is crucial for op mal plant growth.
7. Analy cal Balance: This precise weighing scale allows for accurate measurement of
media components to ensure the correct formula on for healthy plant development.
8. Growth Chamber: This controlled environment chamber provides ideal condi ons for
plant growth, with se ngs for temperature, light intensity, and photoperiod
(light/dark cycle).
9. Light Meter: This instrument measures light intensity within the growth chamber,
ensuring plants receive the appropriate amount of light for photosynthesis.
10. Microscope: This tool allows researchers to observe plant ssues and cells at high
magnifica on, monitoring their health and development.
11. Cryopreserva on Equipment: Specialized freezers and liquid nitrogen tanks are used
for long-term storage of plant germplasm in a frozen state.
12. Flow Cytometer: This advanced instrument can analyze individual cells within a plant
ssue culture, providing informa on about cell size, viability, and DNA content.
Stages in Plant Tissue Culture

Stage 0: Stage Stage 4:

Stage 2:
Selection/ 1: Initiation/ Stage 3: Rooting Acclimatization/
Multiplication
preparation establishment hardening
 Stage 0 involves selec ng the mother plant or stock plant from which the explant (the part
of the plant used for culturing) will be obtained. The mother plant should be healthy,
disease-free, and have the desired characteris cs. The explant can be any part of the plant,
such as shoot ps, buds, leaves, stems, roots, or even anthers (pollen sacs). The explant is
then surface sterilized using disinfectants like sodium hypochlorite (bleach) or hydrogen
peroxide to remove any microorganisms present on the surface.
 In Stage 1, the sterilized explant is then placed on a nutrient culture medium contained in
a glass jar or test tube. The composi on of the culture medium can be adjusted depending
on the type of plant and the desired outcome.
 A er placing on the media, in Stage 2, the cells start growing and dividing. Culture medium
used at this stage is o en supplemented with plant hormones like growth regulators.
 In Stage 3, the regenerated shoots are induced to develop roots. This is achieved by
transferring the shoots to a new culture medium containing different plant hormones to
s mulate root development.
 In the final stage, Stage 4, plantlets are taken off from the controlled environment of the
culture vessel and introduced to the external environment. This allows the plantlets to
adapt to the outside environment and develop the necessary physiological and
morphological features to survive and grow independently.
 Once the plantlets are fully acclima zed, they can be transplanted to soil or another
growing medium.
Harves ng Plant Tissues:
Steps:
 Selec on of donor plant: Choose a healthy, disease-free plant with the desired
characteris cs.
 Steriliza on: This is crucial to prevent contamina on in the culture medium. Disinfect
workspace and all tools with 70% ethanol. Prepare a bleach solu on for surface
steriliza on of the plant ssue.
 Work asep cally: Wear sterile gloves and a lab coat.
 Use sterile instruments: Scalpels, forceps, and other instruments should be sterilized
by autoclaving or wiping with disinfectant.
 Explant selec on: The specific type of ssue harvested depends on the desired
outcome of the ssue culture experiment. (Meristem/ Embryo/ Leaf/ Stem Culture)
Techniques in harves ng ssues from Plants:
1. Mechanical Disrup on: This method is suitable for isola ng cells or organelles
from plant material. It u lizes physical methods to break down cell walls and
membranes, releasing the cellular contents. Two disrup on methods are:
 a. Grinding: You can use a mortar and pestle with liquid nitrogen or dry
ice to freeze and grind the ssue. This makes it bri le and easier to
break down.
b. Homogeniza on: For a more controlled approach, a ssue homogenizer
can be used. This electrically powered device disrupts the ssue in a
buffer solu on.
2. Punching or Coring: This method is ideal for obtaining small ssue samples for
studies like DNA extrac on or culturing specific plant parts. A sterile cork borer
or scalpel can be used to punch out small discs or cores of ssue.
Isola on of Plant Tissues:
Steps:
1. Surface Steriliza on: Immerse explants in bleach solu on for 1-5
minutes according to the plant species and ssue type.
2. Mul ple Rinses: Thoroughly rinse explants with sterile dis lled water to remove
any residual bleach solu on that could harm the cells.
3. Final Trimming: Using sterile instruments, remove any damaged or discolored
ssue from the explants to ensure a healthy star ng material for culture. This
reduces the risk of contamina on and promotes healthy growth.
4. Inocula on: The sterilized explant is then inoculated onto the culture medium in a
sterile container.
5. Incuba on: The culture is incubated under controlled condi ons of temperature,
light, and humidity.
6. Subculturing: As cells grow and mul ply, they are periodically subcultured onto
fresh medium to provide them with more nutrients and space.
7. Regenera on: Regenerate whole plant by inducing the forma on of shoots, roots,
and embryos.
8. Acclima za on: Once regenerated plantlets are formed, they need to be moved
to greenhouse or outdoor condi ons before they can be transplanted into the
field.
Techniques in isola ng ssues from Plants:
1. Enzyme-assisted extrac on: This method u lizes enzymes that break down specific
components of the cell wall, allowing for gentler isola on of cells or organelles
compared to mechanical disrup on. Different enzymes target different cell wall
components, so the choice depends on the target ssue and desired outcome.
2. Microdissec on: This is a more me culous technique using a microscope and
specialized tools for isola ng single cells or specific ssue regions. It's ideal for
studies requiring very specific cell popula ons.
 3. Pressure techniques: This method u lizes a pressure chamber to force a solu on
or buffer into the plant ssue. The pressure disrupts cell walls and allows for the
extrac on of cellular components. This can be useful for isola ng specific
metabolites or fluids within the plant.
4. Vacuum infiltra on: Similar to pressure techniques, vacuum infiltra on uses a
vacuum to draw a solu on into the plant ssue. This can be helpful for introducing
substances into the plant cells for experimenta on.
5. Laser microdissec on: This advanced technique uses a laser to precisely cut out
specific regions of interest within the plant ssue. This allows for highly targeted
extrac on and analysis of specific cell types.
Prepara on of Growth Media:
Steps:
1. Gather materials: You'll need a clean workspace, Erlenmeyer flask or beaker, magne c
s r plate, s r bar, ssue culture grade water, basal medium powder (pre-mixed
nutrients), sucrose (sugar source), gelling agent (agar is common), pH meter, sodium
hydroxide (NaOH) or hydrochloric acid (HCl) for pH adjustment, and plant growth
regulators (op onal).
2. Prepare the water: Add around 75-80% of the final desired medium volume (e.g.,
800ml for 1L medium) of ssue culture grade water to your flask or beaker.
3. Dissolve the basal medium: While s rring with the magne c s r plate, slowly add the
basal medium powder to the water. Ensure it completely dissolves. You might need
slight hea ng to aid this process.
4. Add supplements: Here's where you add the remaining ingredients one by one:
a. Sucrose: The amount varies depending on the plant and media recipe.
b. Gelling agent (like agar): Usually around 0.5-1% of the final volume.
c. Plant growth regulators (auxins, cytokinins): If required for your experiment,
add them according to the specific recipe.
5. Adjust pH: Use a pH meter to measure the solu on's pH. The ideal range for plant
ssue culture is typically between 5.5 and 5.8. Adjust the pH using small amounts of
dilute NaOH (to raise pH) or HCl (to lower pH).
6. Steriliza on: There are two op ons:
a. Autoclave before adding gelling agent: If your medium tolerates high heat, you
can autoclave the en re solu on at 121°C and 15 psi for about 15 minutes.
A erward, the medium is dispensed into sterile culture vessels.
b. Autoclave a er adding gelling agent: For heat-sensi ve media, autoclave only
the water, basal medium, and other heat-stable supplements. Then, add the
gelling agent and dispense the medium into sterile vessels while it's s ll
molten.
 7. Adding heat-labile components (op onal): A er autoclaving and the medium cools
slightly, you can add any heat-sensi ve components like vitamins or specific growth
factors.
Important components of Plant Tissue Culture Media:

Growth Condi ons:

Storage of Plant ssues:

1. Short-term Storage:
 Culture at room temperature: For short periods (days to weeks), ac vely growing
cultures can be maintained on solidified media at room temperature under
appropriate light condi ons. This is a simple and low-maintenance approach for
ongoing experiments.
 Slow growth storage: For longer storage (weeks to months), cultures can be
maintained on media with reduced nutrient concentra ons or at slightly cooler
temperatures. This slows down growth and extends the storage me by reducing
nutrient deple on and minimizing the risk of contamina on.
2. Long-term Storage: Cryopreserva on
 Cryopreserva on is the method of choice for long-term storage (months to years) of
plant ssues in plant ssue culture. It involves preserving ssues at ultra-low
 temperatures (-196°C or lower) using liquid nitrogen. Here's a breakdown of the
process:
o Prepara on: Plant ssues intended for cryopreserva on are carefully selected
and precondi oned to improve their tolerance to freezing. This might involve
exposure to specific chemicals or cold acclima on.
o Exposure to Cryoprotectants: Cells are exposed to cryoprotectants, which are
chemicals that protect cellular structures from damage caused by ice crystal
forma on during freezing.
o Controlled Freezing: A controlled-rate freezing process is employed to
gradually decrease the temperature, allowing for ice forma on outside the
cells while minimizing intracellular ice forma on. This is crucial for cell survival.
o Storage in Liquid Nitrogen: The frozen ssues are stored in cryovials immersed
in liquid nitrogen, which maintains the ultra-low temperature necessary for
long-term storage.
o Thawing and Regenera on: When needed, the cryopreserved ssues are
rapidly thawed using a specific protocol. Viable ssues can then be recultured
on appropriate media for regenera on into whole plants.
Types of Plant Cell Culture Techniques:
The ssue obtained from a plant to be cultured is called an explant. Explants can be taken
from leaves, stems, roots, bud, and meristem. Thus, based on the parts of the plant used in
the process, ssue is branched into different methods.
1. Callus Culture:
 A callus is an undifferen ated mass of cells, having the poten al to develop any part
of the plants.
 Also, callus can also be developed under in vitro condi ons using explants from any
part of the plant.
 Under the suitable condi on, the callus develops into shoot primordia and form
soma c embryos.
 The callus formed from explants of different plant species might have different
structures and growth pa erns. Also, to prevent nutrient deple on they are required
to be cultured for every 28 days interval.
 The callus growth mainly depends on two factors, which include the type of explant,
nutrient composi on, and growth condi ons.
 Applica on: Plant regenera on and produc on of secondary metabolites.
2. Protoplast Culture
 Protoplast is a plant cell without a cell wall. In this method, the cell wall of plant cells
is removed by using either mechanical or enzyma c methods. Then, they are purified
and the wall is again regenerated in ar ficial condi ons and transferred to suitable
media for development into a complete plant.
 The technique is mainly used to produce hybrid plants or gene cally transformed
plants. Generally, during the process, two protoplasts from two different species are
fused together and cultured in a suitable environment.
 The techniques generally used to grow protoplasts are:
i. Hanging-drop cultures
ii. Micro culture chambers
iii. So agars matrix
 Applica ons: Plant breeding and crop improvement
3. Embryo Culture:
 Embryo culture, as the name suggests, is isola ng and culturing either immature or
mature embryos of the plant for the whole plant development. In this method,
embryos are not separately sterilized, rather the organ, such as ovule, seed, or fruit,
from which they are isolated is sterilized and then used in the process.
 In some plants, seed dormancy due to mechanical resistance or chemical inhibitors
restricts the growth and development of plants. Thus, in such cases, embryos from
the plant are isolated and cultured in ar ficial to develop into viable seedlings. The
technique is also helpful to rescue embryos from ge ng aborted due to
incompa ble intergeneric or interspecific crosses.
 Applica ons: Rescue of embryo and germina on of difficult-to-germinate seeds.

4. Cell Suspension culture

 Cell suspensions can be obtained either directly from the explant or callus. In this
method, ssues are transferred to liquid media and con nuously agitated to obtain a
suspension of single cells.
 The obtained single cells can be cultured using many techniques, such as:
 Filter paper ra nurse ssue technique
 Microchamber technique
 Microdrop method
  Bergmann’s pla ng technique
 Ovary Culture
 In this technique, fer lized or unfer lized ovaries of the plant species are cultures in a
suitable environment to develop into a whole plant. It’s also known as gymnogenesis.
 The technique is mainly used to overcome pre-fer liza on and post-fer liza on
barriers. It has also been used to achieve interspecific hybrids.
 Applica ons: Large-scale produc on of secondary metabolites or studying plant cell
physiology

5. Organ Culture:
 Focuses on culturing isolated plant organs, such as shoots, roots, or leaves, to induce
their growth and development.
 By providing a sterile nutrient medium and ideal condi ons, scien sts can coax these
organs to develop into whole plants. This method offers advantages like rapid
propaga on of desirable varie es, disease-free plant produc on, and even the
preserva on of endangered species. Applica ons: Micropropaga on, in vitro
fer liza on, embryo rescue etc.
6. Meristem Culture:
 Plant meristem culture is a specific type of plant organ culture that focuses on growing
new plants from ny ssue samples containing meristems.
 Meristems are the rapidly dividing cells found in shoot and root ps, and because of
their ac ve cell division, they are typically free of viruses plaguing the rest of the plant.
 By isola ng these meristems and placing them on a special nutrient medium, scien sts
can cul vate disease-free plantlets that are gene c copies of the parent plant.
 This technique is valuable for mass producing desirable plant varie es and preserving
rare or endangered species.
 Applica ons: It is used to produce disease-free plants, regenerate a whole
plant, produce transgenic plants, and in crop improvement.

7. Cell Culture:
 Involves isola ng and culturing cells from plant explants to grow a complete plant.
 These cells are nurtured in a special nutrient broth containing sugars, vitamins, and
some mes hormones. This allows scien sts to study plant growth at the cellular level,
create large numbers of iden cal plants for research or agriculture, and even develop
en rely new plant varie es.
 Applica ons: Produce useful secondary metabolites that are important for the
pharmaceu cal, nutraceu cal, cosme c, and industrial sectors.
Applica ons of plant ssue culture:
1. Micropropaga on: This is perhaps the most widespread applica on. It involves using
meristem or shoot p culture to rapidly produce large numbers of gene cally iden cal
plants (clones) from a single parent plant. This is par cularly valuable for:
 Hor culture: Micropropaga on allows for the mass produc on of commercially
valuable plants like orchids, fruits, and ornamentals, ensuring consistent quality and
disease-free stock.
 Forestry: This technique helps in the rapid mul plica on of desired tree varie es for
reforesta on projects and conserva on efforts.
2. Disease elimina on: Viruses and other pathogens can be eliminated from plants using
meristem p culture. Since meristema c ssues (growing ps) are o en virus-free, this
technique allows for the regenera on of healthy plants from infected ones. This is crucial
for maintaining healthy stock in agriculture and hor culture.
3. Cryopreserva on: Plant ssue culture allows for the long-term storage of plant
germplasm (gene c material) in a frozen state. This is vital for preserving rare or
endangered plant species and maintaining valuable gene c diversity for future use.
4. In vitro selec on: This technique involves applying selec ve pressure (like exposure to
herbicides) on cultured plant cells or ssues. This allows for the selec on of cells with
desired traits like resistance to diseases or herbicides. These selected cells can then be
regenerated into whole plants with the desired characteris cs.
5. Synthe c seed produc on: Soma c embryogenesis, a type of plant ssue culture, can be
used to produce synthe c seeds. These are encapsulated embryos that can be stored and
transported easily, offering advantages over tradi onal seeds, especially for recalcitrant
species with difficult seed storage requirements.
6. Produc on of secondary metabolites: Many plants produce valuable secondary
metabolites with medicinal or industrial applica ons. Plant cell culture allows for the
controlled produc on of these compounds under asep c condi ons. This can be a more
efficient and reliable method compared to extrac ng them directly from whole plants.
7. Gene c modifica on: Plant ssue culture serves as a pla orm for plant transforma on
techniques. Isolated plant cells can be bombarded with foreign genes or manipulated to
introduce desired gene c changes. These gene cally modified (GM) plants can then be
regenerated for research purposes or agricultural applica ons like herbicide resistance or
improved nutri onal content.
8. Basic research: Plant ssue culture is a powerful tool for studying various aspects of plant
biology, including cell differen a on, developmental processes, and the response to
environmental stresses. This allows scien sts to gain a deeper understanding of plant
physiology and develop new strategies for crop improvement.
9. Interspecific hybridiza on: Plant ssue culture techniques can overcome breeding barriers
between sexually incompa ble plant species. This is achieved through protoplast fusion,
where isolated cells from different species are fused electrically or chemically. The
 resul ng hybrid cells can then be regenerated into en rely new plant varie es with
poten ally combined traits from both parents. This opens doors for crea ng crops with
improved characteris cs like disease resistance or enhanced yields.
10. Microspore embryogenesis: This is an advanced technique where immature pollen grains
(microspores) are cultured to develop into haploid embryos. Haploid plants contain only
one set of chromosomes, which makes them ideal for breeding purposes. By doubling the
chromosome number of these haploid plants, researchers can create homozygous lines in
a single genera on. This significantly reduces the me required for developing stable plant
varie es compared to tradi onal breeding methods.