Tissue Culture

Acknowledgement

My Project Work could get ended in a very even and productive way with those support and help from various areas. For, I would like to borrow all the languages of the world to express my heartfelt gratitude to my subject tutor for his support and laid hand in my need towards my approach in doing this project into a real shape of cognition.And also for his instruction to be abided in writing project work.

My sincere thanks also goes to my brother who helped me to get most of the information and coverage from the website mentioned at the end. Also for his type-written and cover design too.

I also would like to congratualate our librarian for the catered of needed text books and necessary diagrammatic information and coverage.

I won’t forget to extend my gratitude towards my friends for instructing me on this particular topic and let me to be successful in shaping my project into this very shape. Also for their support and help in searching the references for this topic.

Once again I wholeheartedly like to extend my gratitude and felicitation to my subject tutor for his generous helping hand in shaping my project work into productive and knowledgeable one particularly for me and other upcoming friends too. My gratitude for him has been beyond the horizons of earth and heaven.

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Tissue Culture

Introduction

Biology the scientific study of life includes relevant branches and various applications in human

which ease our life comfort due to the advancement of modern technology. The major field of

applications would include agriculture, biochemistry, genetics, immunology, Biotechnology and

so on.

But in the rapid multiplication and preservation of noble, extinct and endangered species of

plants, modern technology called BIOTECHNOLOGY has been applied by advanced experts.

This method can be applied vastly in the field called TISSUE CULTURE cum

MICROPROPATION. Biotechnology the applied biology dealing with the biological systems,

living organisms or derivatives thereof to make or modify products or species that is specified to

use.

TISSUE CULTURE actually to technique of growing plant cells, tissues, organs, seeds or other

plant parts in a sterile environment on a nutrient medium.MICROPROPAGATION too refers

to the practice of rapidly stock plant material to produce a large number of offspring plants,

using modern plant tissue culture methods.

Therefore, in the very project work. I did on Tissue Culture and Micropropagation. Tissue

Culutre project work basically includes: Overview of tissue culture and micropropagation, their

steps in culturing the tissues, advantages and disadvantages of tissue culture, Tissue culture

methods and its steps, safety consideration and their applications in general.

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Tissue Culture

Micropropagation and Tissue Culture

Microprogation

Tissue culture often called micropropagation is a special type of asexual propagation where a

very small piece of tissue (shoot apex, leaf section, or even an individual cell) is excised (cut-

out) and placed in sterile (aseptic) culture in a test tube, petri dish or tissue culture container

containing a special culture medium.

Micropropagation is also the practice of rapidly multiplying stock plant material to produce a

large number of offspring plants, using modern plant tissue culture methods.

Micropropagation is used to multiply novel plants, such as those that have been genetically

modified or bred through conventional plant breeding methods.

Tissue culture in animal is basically based on cellular totipotency.This is all the multicellular

organisms basically formed from single cell- zygote.by repeated multiplication and

differentiation

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Tissue Culture

Methods

Establishment of Culture Medium and Explanting

In vitro culture of plants in a controlled, sterile environment

Micropropagation begins with the selection of plant material to be propagated. Clean stock

materials that are free of viruses and fungi are important in the production of the healthiest

plants. Often plants are first virus indexed to determine if they are clean and free of viruses.

Once the plant material is chosen for culture, the collection of explants begins and is dependent

on the type of tissue to be used; including stem tips, anthers, petals, pollen and others plant

tissues. The explants material is then surface sterilized, usually in multiple courses of bleach

and alcohol washes and finally rinsed in sterilized water. This small portion of plant tissue,

sometimes only a single cell, is placed on a growth medium, typically containing sucrose as an

energy source and one or more plant growth regulators (plant hormones). Usually the medium

is thickened with agar to create a gel which supports the explants during growth. Some plants

are easily grown on simple media but others require more complicated media for successful

growth; some media include vitamins, minerals and amino acids. The medium is sterilized

during preparation to prevent and bacterial contamination, which can outgrow and smother the

growing explants. The plant tissue grows and differentiates into new tissues depending on the

medium. For example, media containing cytokinins are used to create branched shoots from

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Tissue Culture

plant buds.

Explants can be pieces of any part of the plant (leaves, stems, flowers, etc.),

or even individual isolated cells.

Multiplication

Multiplication is the taking of tissue samples produced during the first stage and increasing

their number. Following the successful introduction and growth of plant tissue, the

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Tissue Culture

establishment stage is followed by multiplication. Through repeated cycles of this process, a

single explant sample may be increased from one to hundreds or thousands of plants.

Depending on the type of tissue grown, multiplication can involve different methods and media.

If the plant material grown is callus tissue, it can be placed in a blender and cut into smaller

pieces and recultured on the same type of culture medium to grow more callus tissue. If the

tissue is grown as small plants called plantlets, hormones are often added that cause the

plantlets to produce many small offshoots that can be removed and recultured.

A mass of callus tissue is formed that is just starting to make new plantlets.

New plantlets (shoots with leaves) are forming.

New plantlets (shoots with leaves) are forming.

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Tissue Culture

If the conditions are right a small "forest" of plants

will develop in the tissue culture container

Pretransplant

This stage involves treating the plantlets/shoots produced to encourage root growth and

"hardening." It is performed in vitro, or in a sterile "test tube" environment.

Root growth does not always occur in the earlier stages in plant cell culture, and is of course a

requirement for successful plant growth after the micro propagation procedure. It is often

performed in vitro by transferring the plantlets to a growth medium containing auxin(s) which

stimulate root initiation. The pretransplant stage is not always performed; some plants are micro

propagated and grown in culture and normal cuttings are made that are then rooted ex vitro.

"Hardening" refers to the preparation of the plants for a natural growth environment. Until this

stage, the plantlets have been grown in "ideal" conditions, designed to encourage rapid growth.

Due to lack of necessity, the plants are likely to be highly susceptible to disease and often do

not have fully functional dermal coverings and will be inefficient in their use of water and

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Tissue Culture

energy.

Hardening typically involves slowly weaning the plantlets from a high-humidity; low light,

warm environment to what would be considered a normal growth environment for the species

in question. This is done by moving the plants to a location high in humidity, such as a green

house with regular mist watering.

Some of the small plantlets can be removed and transferred to new tissue culture containers.

Transfer from culture

In the final stage of plant micropropagation, the plantlets are removed from the plant media and

transferred to soil or to potting compost for continued growth by conventional methods.

This stage is often combined with the "pretransplant" stage.

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Tissue Culture

After acclimation, the young plants can be transplanted and grown in pots in a greenhouse to

produce new plants.

Advantages

Micropropagation has a number of advantages over traditional plant propagation techniques:

The main advantage of micropropagation is the production of many plants that are

clones of each other.

Micropropagation can be used to produce disease-free plants.

Micropropagation produces rooted plantlets ready for growth, saving time for the

grower when seeds or cuttings are slow to establish or grow.

It can have an extraordinarily high fecundity rate, producing thousands of propagules

while conventional techniques might only produce a fraction of this a number.

It is the only viable method of regenerating genetically modified cells or cells after

protoplast fusion.

Micropropagation often produces more robust plants, leading to accelerated growth

compared to similar plants produced by conventional methods - like seeds or cuttings.

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Tissue Culture

A greater number of plants can be produced per square meter and the propagules can be

stored longer and in a smaller area.

Disadvantages

Micropropagation is not always the perfect means of multiplying plants, conditions that limits

include:

It is very expensive, and can have a labor cost of more than 70%

As monoculture is produced after micropropagation, in case of infection whole crop can

get damaged

Not all plants can be successfully tissue cultured, often because the proper medium for

growth is not known or the plants produce secondary metabolic chemicals that stunt or

kill the explants.

Sometimes plants or cultivars do not come true to type after being tissue cultured; this is

often dependent on the type of explants material utilized during the initiation phase or

the result of the age of the cell or propagule line.

Some plants are very difficult to disinfest of fungal organisms.

Tissue Culture Methods

I. TYPES OF CELLS GROWN IN CULTURE

Tissue culture is often a generic term that refers to both organ culture and cell culture and the

terms are often used interchangeably. Cell cultures are derived from either primary tissue

explants or cell suspensions.

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Tissue Culture

II. WORK AREA AND EQUIPMENT

1. Laminar flow hoods. There are two types of laminar flow hoods, vertical and horizontal.

The vertical hood, also known as a biology safety cabinet, is best for working with hazardous

organisms since the aerosols that are generated in the hood are filtered out before they are

released into the surrounding environment. Horizontal hoods are designed such that the air

flows directly at the operator hence they are not useful for working with hazardous organisms

but are the best protection for your cultures. Both types of hoods have continuous displacement

of air that passes through a HEPA (high efficiency particle) filter that removes particulates from

the air. In a vertical hood, the filtered air blows down from the top of the cabinet; in a

horizontal hood, the filtered air blows out at the operator in a horizontal fashion. NOTE: these

are not fume hoods and should not be used for volatile or explosive chemicals. They should

also never be used for bacterial or fungal work. The hoods are equipped with a short-wave UV

light that can be turned on for a few minutes to sterilize the surfaces of the hood, but be aware

that only exposed surfaces will be accessible to the UV light.

B. CO2 Incubators. The cells are grown in an atmosphere of 5-10% CO2 because the medium

used is buffered with sodium bicarbonate/carbonic acid and the pH must be strictly maintained.

Culture flasks should have loosened caps to allow for sufficient gas exchange. Cells should be

left out of the incubator for as little time as possible and the incubator doors should not be

opened for very long. The humidity must also be maintained for those cells growing in tissue

culture dishes so a pan of water is kept filled at all times.

C. Microscopes. Inverted phase contrast microscopes are used for visualizing the cells.

Microscopes should be kept covered and the lights turned down when not in use. Before using

the microscope or whenever an objective is changed, check that the phase rings are aligned.

D. Preservation. Cells are stored in liquid nitrogen.

E. Vessels. Anchorage dependent cells require a nontoxic, biologically inert, and optically

transparent surface that will allow cells to attach and allow movement for growth. The most

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Tissue Culture

convenient vessels are specially-treated polystyrene plastic that are supplied sterile and are

disposable. These include petri dishes, multi-well plates, microtiter plates, roller bottles, and

screw cap flasks.

III. PRESERVATION AND STORAGE. Liquid N2 is used to preserve tissue culture cells,

either in the liquid phase (-196°C) or in the vapor phase (-156°C). Freezing can be lethal to

cells due to the effects of damage by ice crystals, alterations in the concentration of electrolytes,

dehydration, and changes in pH. To minimize the effects of freezing, several precautions are

taken. First, a cryoprotective agent which lowers the freezing point, such as glycerol is added.

A typical freezing medium is 90% serum, 10% glycerol. In addition, it is best to use healthy

cells that are growing in log phase and to replace the medium 24 hours before freezing. Also,

the cells are slowly cooled from room temperature to -80°C to allow the water to move out of

the cells before it freezes. The optimal rate of cooling is 1°-3°C per minute.

IV. MAINTENANCE

Cultures should be examined daily, observing the morphology, the color of the medium and the

density of the cells. A tissue culture log should be maintained that is separate from your regular

laboratory notebook. The log should contain: the name of the cell line, the medium components

and any alterations to the standard medium, the dates on which the cells were split and/or fed, a

calculation of the doubling time of the culture

A. Growth pattern. Cells will initially go through a quiescent or lag phase that depends on the

cell type, the seeding density, the media components, and previous handling. The cells will then

go into exponential growth where they have the highest metabolic activity. The cells will then

enter into stationary phase where the number of cells is constant, this is characteristic of a

confluent population.

B. Harvesting. Cells are harvested when the cells have reached a population density which

suppresses growth. Ideally, cells are harvested when they are in a semi-confluent state and are

still in log phase. Cells that are not passaged and are allowed to grow to a confluent state can

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Tissue Culture

sometime lag for a long period of time and some may never recover. It is also essential to keep

your cells as happy as possible to maximize the efficiency of transformation. Most cells are

passaged three times a week.

1. Suspension culture. Suspension cultures are fed by dilution into fresh medium.

2. Adherent cultures. Adherent cultures that do not need to be divided can simply be fed by

removing the old medium and replacing it with fresh medium.

When the cells become semi-confluent, several methods are used to remove the cells from the

growing surface so that they can be diluted:

Mechanical - A rubber spatula can be used to physically remove the cells from the

growth surface. This method is quick and easy but is also disruptive to the cells and may

result in significant cell death. This method is best when harvesting many different

samples of cells for preparing extracts.

Proteolytic enzymes - Trypsin, collagenase, or pronase, usually in combination with

EDTA, causes cells to detach from the growth surface. This method is fast and reliable

but can damage the cell surface by digesting exposed cell surface proteins. The

proteolysis reaction can be quickly terminated by the addition of complete medium

containing serum

EDTA - EDTA alone can also be used to detach cells and seems to be gentler on the

cells than trypsin. The standard procedure for detaching adherent cells is as follows:

a. usually inspect daily.

b. Release cells from monolayer surface

  c. wash once with a buffer solution

d. treat with dissociating agent

e. observe cells under the microscope. Incubate until cells become rounded and

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Tissue Culture

loosen when flask is gently tapped with the side of the hand.

f. transfer cells to a culture tube and dilute with medium containing serum.

g. Spin down cells, remove supernatant and replace with fresh medium.

h.Count the cells in a hemacytometer, and dilute as appropriate into fresh

medium.

C. Media and growth requirements.

1. Physiological parameters

A. temperature - 37C for cells from homeother

B. pH - 7.2-7.5 and osmolality of medium must be maintained

C. humidity is required

D. gas phase - bicarbonate conc. and CO2 tension in equilibrium

E. visible light - can have an adverse effect on cells; light induced production of toxic

compounds can occur in some media; cells should be cultured in the dark and exposed

to room light as little as possible.

2. Medium requirements:

A. Bulk ions - Na, K, Ca, Mg, Cl, P, bicarbonate or CO2

B. Trace elements - iron, zinc, selenium

C. sugars - glucose is the most common

D. amino acids

E. vitamins - B, etc.

F. choline, inositol

G. serum - contains a large number of growth promoting activities such as buffering toxic

nutrients by binding them, neutralizes trypsin and other proteases, has undefined effects on the

interaction between cells and substrate, and contains peptide hormones or hormone-like growth

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Tissue Culture

factors that promote healthy growth.

H. antibiotics - although not required for cell growth, antibiotics are often used to control the

growth of bacterial and fungal contaminants.

3. Feeding - 2-3 times/week.

4. Measurement of growth and viability.

The viability of cells can be observed visually using an inverted phase contrast microscope.

Live cells are phase bright; suspension cells are typically rounded and somewhat symmetrical;

adherent cells will form projections when they attach to the growth surface. Viability can also

be assessed using the vital dye, which is excluded by live cells but accumulates in dead cells.

Cell numbers are determined using a hemocytometer.

V. SAFETY CONSIDERATIONS

The following safety precautions should also be observed:

pipetting: use pipette aids to prevent ingestion and keep aerosols down to a minimum

no eating, drinking, or smoking

wash hands after handling cultures and before leaving the lab

decontaminate work surfaces with disinfectant (before and after)

autoclave all waste

use biological safety cabinet (laminar flow hood) when working with hazardous

organisms.

use aseptic technique

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Tissue Culture

VI. TISSUE CULTURE PROCEDURES

The cells to be cultured should be monitored daily for morphology and growth characteristics,

fed every 2 to 3 days. A minimum of two 25 cm2 flasks should be carried for each cell line;

these cells should be expanded as necessary for the transfection experiments. Each time the

cells are subculture, a viable cell count should be done, the subculture dilutions should be

noted, and, after several passages, a doubling time determined. As soon as you have enough

cells, several vials should be frozen away and stored in liquid N2. One vial from each freeze

down should be thawed 1-2 weeks after freezing to check for viability. These frozen stocks will

prove to be vital if any of your cultures become contaminated.

Procedures:

1. Media preparation. The concern person will be responsible for maintaining his own stock

of cell culture media; the particular type of media, the sera type and concentration, and other

supplements will depend on the cell line. Any media for culture should not be shared because if

a culture or a bottle of media gets contaminated, you have no back-up. Most of the media

components will be purchased prepared and sterile. In general, all we need to do is sterily

combine several sterile solutions. Use only media that has been sterility tested. For this reason,

you must anticipate your culture needs in advance so you can prepare the reagents necessary.

But, please try not to waste media.

All media preparation and other cell culture work must be performed in a laminar flow hood.

Before beginning your work, turn on blower for several minutes, wipe down all surfaces with

70% ethanol, and ethanol wash your clean hands. Use only sterile pipets, disposable test tubes

and autoclaved pipet tips for cell culture.

2. Growth and morphology. Frequent feeding is important for maintaining the pH balance of

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the medium and for eliminating waste products. Cells do not typically like to be too confluent

so they should be subcultured when they are in a semi-confluent state.

3. Cell feeding. Use prewarmed media and have cells out of the incubator for as little time as

possible. Use 10-15 ml for T-25's, 25-35 ml for T-75's and 50-60 ml for T-150's.

a. Suspension cultures. Feeding and subculturing suspension cultures are done simultaneously.

About every 2-3 days, dilute the cells into fresh media. Typically 1:4 to 1:20 dilutions are

appropriate for most cell lines.

b. Adherent cells. About every 2-3 days, pour off old media from culture flasks and replace

with fresh media. Subculture cells as described below before confluency is reached.

4. Subculturing adherent cells. When adherent cells become semi-confluent, subculture using

trypsin/EDTA.

Trypsin-EDTA helps to:

a. Remove medium from culture dish and wash cells in a balanced salt solution without Ca++

or Mg++. Remove the wash solution.

b. Add enough trypsin-EDTA solution to cover the bottom of the culture vessel and then pour

off the excess.

c. Place culture in the 37°C incubator for 2 minutes.

d. Monitor cells under microscope. Cells are beginning to detach when they appear rounded.

e. As soon as cells are in suspension, immediately add culture medium containing serum. Wash

cells once with serum containing medium and dilute as appropriate (generally 4-20 fold).

5. Thawing frozen cells.

a. Remove cells from frozen storage and quickly thaw in a 37°C waterbath by gently agitating

vial.

b. As soon as the ice crystals melt, pipet gently into a culture flask containing prewarmed

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growth medium.

c. Log out cells in the "Liquid Nitrogen Freezer Log" Book.

6. Freezing cells.

Resuspend cells in complete medium and determine cell count/viability.

Centrifuge and resuspend in ice-cold freezing medium: 90% calf serum/10% DMSO, at

106 - 107 cells/ml. Keep cells on ice.

Transfer 1 ml aliquots to freezer vials on ice.

Place in a Mr. Frosty container that is at room temperature and that has sufficient

isopropanol.

Place the Mr. Frosty in the -70°C freezer overnight. Note: Cells should be exposed to

freezing medium for as little time as possible prior to freezing

Next day, transfer to liquid nitrogen (DON'T FORGET) and log in the "Liquid Nitrogen

Freezer Log" Book.

7. Viable cell counts.

USING A HEMOCYTOMETER TO DETERMINE TOTAL CELL COUNTS AND

VIABLE CELL NUMBERS.

Trypan blue is one of several stains recommended for use in dye exclusion procedures for

viable cell counting. This method is based on the principle that live cells do not take up certain

dyes, whereas dead cells do.

1. Prepare a cell suspension, either directly from a cell culture or from a concentrated or

diluted suspension and combine 20 μl of cells with 20 μl of trypan blue suspension. Mix

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thoroughly and allow standing for 5-15 minutes.

2. With the cover slip in place, transfer a small amount of trypan blue-cell suspension to both

chambers of the hemocytometer by carefully touching the edge of the cover slip with the pipette

tip and allowing each chamber to fill by capillary action. Do not overfill or underfill the

chambers.3.

Total cells = cells/ml x the original volume of fluid from which the cell sample was removed;

% Cell viability = total viable cells (unstained)/total cells x 100.

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Tissue Culture

Applications

Plant tissue culture is used widely in plant science; it also has a number of commercial

applications. Applications include:

Micropropagation is widely used in forestry and in floriculture. Micropropagation can

also be used to conserve rare or endangered plant species.

A plant breeder may use tissue culture to screen cells rather than plants for advantageous

characters, e.g. herbicide resistance/tolerance.

Large-scale growth of plant cells in liquid culture inside bioreactors as a source of

secondary products, like phytochemicals (secondary metabolites) and recombinant

proteins used as biopharmaceuticals.

To cross distantly related species by protoplast fusion and regeneration of the novel

hybrid.

To cross-pollinate distantly related species and then tissue culture the resulting embryo

which would otherwise normally die (Embryo Rescue).

For production of doubled monoploid (dihaploid) plants from haploid cultures to achieve

homozygous lines more rapidly in breeding programmes, usually by treatment with

colchicine which causes doubling of the chromosome number.

As a tissue for transformation, followed by either short-term testing of genetic constructs

or regeneration of transgenic plants.

Certain techniques such as meristem tip culture can be used to produce clean plant

material from virused stock, such as potatoes and many species of soft fruit.

micropropagation using meristem and shoot culture to produce large numbers of identical

individuals.

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Tissue Culture

Conclusion

The project work: Tissue Culture, I would say is not an easy task as it needs a lot of hands and

heads to be implemented in writing of project and information findings mainly. On the evidence

that, I now have the better understanding of Tissue Culture and Micro propagation in which it

required certain critical ideas, knowledge and skills as well. This project work has catered me with

ample experiences and correct sequences that are required for our writing.

More importantly, it catered me the knowledge in the areas of tissue culture and micro

propagation. Basically, it taught me the steps and procedures of tissue culture and micro

propagation. Despite its availability and expensiveness, I would say that I am fully acquainted

with the tissue culture and its steps and techniques. Moreover, I am acquainted with its

advantageous of rapid multiplication within a short period of time with special retention of novel

characteristics.

Such a project work is an immense cognitive and knowledgeable for every one of us that

bestowed both our hands and heads to do research for this very project work. It also leads me in

gaining vocabulary and improving the ways of doing the project work. Not only do this

assignment really help us in understanding applications of Tissue Culture but also revealed the

ways of writing the project work with the norms abiding the certain rules.

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Tissue Culture

Lists of References

David, W. (n.d.) Cloning Plants: Tissue Culture-Horticulture and the Science of Plants-Youth

Adventure Program.Texas: A&M University.

Freshney,I.R.,& Liss, W.(1987). Culture of Animal cells- A manual of basic techniques.

Retrieved 8th April, 2010 from website

http://en.wikipedia.org/wiki/MicropropagationMicropropagation - Definitions from

Dictionary.com" dictionary.reference.com.

Retrieved from http://dictionary.reference.com/browse/Micropropagation.

Retrieved 8th April, 2010 from website

http://images.google.com/imgres?imgurl=http://generalhorticulture.tamu.edu/

YouthAdventureProgram/TisueCulture/P81F1.GIF&imgrefurl

Retrieved 17th June 2010 from http://en.wikipedia.org/wiki/Plant_tissue_culture#Applications.

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