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TECHNIQUES IN BIOTECHNOLOGY: TISSUE CULTURE
Department of Chemical Engineering Technology, UniMAP
PTT 104: Introduction to Biotechnology
COURSE OUTCOME
CO2: Ability to demonstrate important recent advance in methods and application of biotechnology with regards to microorganisms and plants
WHAT IS TISSUE CULTURE
Tissue culture is the growth of tissues and/or cells separate from the organism. This is typically facilitated via a liquid, semi-solid, or solid growth medium, such as broth or agar. Tissue culture commonly refers to the culture of animal cells and tissues, while the more specific term plant tissue culture is being named for the plants.
.This growth of cells and tissues is called in-vitro growth.
Contrasted to in-vivo growth which is growth within the body.
In modern usage, tissue culture generally refers to the growth of cells from a tissue from a multicellular organism in vitro. These cells may be cells isolated from a donor organism, primary cells, or an immortalised cell line. The term tissue culture is often used interchangeably with cell culture
Tissue culture is an important tool for the study of the biology of cells from multicellular organisms. It provides an in vitro model of the tissue in a well defined environment which can be easily manipulated and analyzed
WHAT IS CELL CULTURE
Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established.
PRIMARY CULTURE
Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e., passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth.
CELL LINE
After the first subculture, the primary culture becomes known as a cell line or subclone. Cell lines derived from primary cultures have a limited life span (i.e., they are finite), and as they are passaged, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population.
TISSUE CULTURE IN PLANT
Tissue culture in plants is the technique of growing plant cells, tissues and organs in an artificial prepared nutrient medium static or liquid under aseptic conditions.
HISTORY
In vitro cultures in which isolated organs, tissues, cells, protoplasts are used has made considerable advances in last two decades.
Though the technique is latest but history of this began more than 225 years back when first callus formation was done by Duhamel du Monceau in 1756.
Haberlandt 1898 successfully cultured somatic cells of higher plants in simple nutrient solutions.
Although he was able to maintain the cells in nutrient medium, the cell division was not recorded until much later.
The first real success was made by Nobecourd, Gautheret and White who successfully cultured cambium tissue and maintained them for more than a year through 5 or 6 sub segments sub cultures.
APPLICATION OF TISSUE CULTURE IN PLANT
Micropropagation: The term represents the vegetative multiplication of plants in artificial media under aseptic conditions from tissue; organs of plants e.g. root tip, shoot tip, embryo, stem and callus etc. It is used for rapid multiplication of stock plant material to produce large no of progeny plants. (plants that have been genetically modified/ gone thru selective breeding)
Production of disease free plants: By using tissue culture in plants healthy disease free plants of potato, sugarcane, sweet potato, and strawberry have been produced.
Embryo rescue for successful hybridisation: The hybrid embryos produced as a result of interspecific or intergeneric cross usually collapse due to incompatibility. Such embryos are isolated from female plants and rescued by growing them on synthetic medium.
Induction and selection of mutants: By adding chemical mutagens into the medium for growing various traits, useful viable mutants can be produced.
Somaclonal variation: These are variations produced in the plants regenerated from tissue cultures involving callus formation. The source of variation due to chromosomal rearrangements. variations appearing during tissue culture in plants are called somaclonal variation. Benefit of somaclonal variation: improvements of genetics. Plants that resist to pesticides/herbicides.
TISSUE CULTURE METHOD: CLONING
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.
Overview of the Tissue Culture Process
The culture medium contains a gel (agar) with the proper mixture of nutrients, sugars, vitamins and hormones, which causes the plant part to grow at very rapid rates to produce new plantlets.
It has been estimated that one chrysanthemum apex placed in tissue culture could produce up to 1,000,000 new plantlets in one year.
Thus, tissue culture is used for rapid multiplication of plants. A very specialized laboratory is required for tissue culture.
All the procedures are done in a laboratory and special ventilated cabinet that is as sterile as an operating room
STEP IN TISSUE CULTURE
Explant: Cut-out Plant Tissue and Place in Tissue Culture Container
Multiplication: Tissue Grows and Produces Small Plants
Rapid Multiplication by Transfer of Cultures
Transplanting
EXPLANT
The first step is to obtain what is called and explant.
This means to simply cut-out a very small piece of leaf or stem tissue, or even isolate individual cells, and place them in a tissue culture container.
The tissue has to be sterilized so it will not have any contaminating bacteria or fungus.
It is then placed inside the tissue culture contain on a gel called agar.
In the agar are dissolved sugar, nutrients and hormones the plant needs.
Explants can be pieces of any part of the plant (leaves, stems, flowers, etc.), or even individual isolated cells.
MULTIPLICATION
The tissue will begin to grow. It may make a big blob of tissue called callus, or it may make new shoots directly from the explant tissue that was inserted in the container.
A mass of callus tissue is formed that is just starting to make new plantlets.
New plantlets (shoots with leaves) are forming.
If the conditions are right a small "forest" of plants will develop in the tissue culture container.
RAPID MULTIPLICATION
Once the plantlets start developing, some can be removed and placed in new tissue culture containers.
Thus, another "forest"' of plants is produced. This results in a rapid multiplication of the cultures and many thousand of plants can be produced in a few months.
TRANSPLANTING
When the plantlets are large enough, they can be removed from the tissue culture container and transferred into pots with potting soil.
The young plants are growth in a greenhouse just like you would any young seedling or cutting.
When the small plant clones are removed from the culture containers, they must be transplanted into some type of acclimation container or kept under a mist system until the acclimate to the ambient environment.
After acclimation, the young plants can be transplantedand grown in pots in a greenhouse to produce new plants.
THE NEXT REVOLUTION
Plant Transgenesis – transferring genes to plants directly Development of plant vaccines, plants that produce
their own pesticides and are resistant to herbicides 17 countries are growing more than 200 million
acres of crops improved through biotechnology Plants have a natural pathogen, Agrobacterium
tumefeciens, that causes tumours. A. tumefeciens inserts its own DNA into the plant, making it a natural vector.
Genes can be put into A.tumefeciens surrounded by the recognition splice sequences, and the bacteria will introduce them into the host.
METHODS USED IN PLANT TRANSGENESIS
Conventional Selective Breeding and Hybridization
Cloning Protoplast fusion Leaf fragment technique Gene guns Chloroplast engineering Antisense technology
METHODS USED IN PLANT TRANSGENESIS
Conventional Selective Breeding and Hybridization Sexual cross between two lines and repeated
backcrossing between hybrid offspring and parent For example, a mildew-resistant pea may be crossed
with a high-yielding but susceptible pea, the goal of the cross being to introduce mildew resistance without losing the high-yield characteristics.
Can take years or unsuccessful Polyploid plants (multiple chromosome sets greater
than normal, usually more than 2n) Increases desirable traits, especially size. For example
many seedless fruit varieties are seedless as a result of polyploidy..
Whole chromosomes can be transferred rather than single genes esults in production of enlarged fruits.
METHODS USED IN PLANT TRANSGENESIS
Cloning – growing plants from a single cell Protoplast fusion is the fusion of two protoplast
cells from different species/different varieties from same species Protoplast cell is a callus cell whose cell wall has been
dissolved by the enzyme cellulase Fusion of the two protoplast cells creates a cell that can
form a hybrid plant Hybrids have been produced either between the different
varieties of the same species (e.g. between non-flowering potato plants and flowering potato plants) or between two different species (e.g. between wheat triticum and rye secale to produce Triticale).
* callus- when plant injured, callus may grow over the side of the wound. Callus can redifferentiate into shoots and roots.
PROTOPLAST FUSION METHOD
The somatic/protoplast fusion process occurs in four steps:
The removal of the cell wall of one cell of each type of plant using cellulase enzyme to produce a somatic cell called a protoplast
The cells are then fused using electric shock (electrofusion) or chemical treatment to join the cells and fuse together the nuclei. The resulting fused nucleus is called heterokaryon.
The somatic hybrid cell then has its cell wall induced to form using hormones
The cells are then grown into calluses which then are further grown to plantlets and finally to a full plant, known as a somatic hybrid.
METHODS USED IN PLANT TRANSGENESIS
METHODS USED IN PLANT TRANSGENESIS
Cloning Leaf fragment technique Small discs are cut from leaf Discs are cultured in the to start a new plant Early in the regeneration process, the bacterium
Agrobacterium tumefaciens carrying a Ti plasmid is introduced into the culture
A soil bacterium that infects plants Bacterium contains a plasmid, the Ti plasmid, that can be
genetically modified DNA from the Ti plasmid integrates with DNA of the host
cell
The plasmid DNA combines with the plant chromosome Discs are treated with hormones to encourage shoot
and root development and then the new plant is planted in the soil
AGROBACTERIUM TUMEFACIENS AS A VECTOR FOR TRANSFERRING FOREIGN GENES INTO PLANT CHROMOSOME
This bacterium naturally infects plant cells causing cancerous growths - crown gall disease
Infection (vir) genes carried on Ti plasmid
INFECTION PROCESS
Vir genes copy T-DNA Open channel in bacterial
cell membrane for T-DNA to pass through
T-DNA enters plant through wound, integrates itself into plant chromosome
http://www.bio.davidson.edu/people/kabernd/seminar/2002/method/dsmeth/ds.htm
METHODS USED IN PLANT TRANSGENESIS
METHODS USED IN PLANT TRANSGENESIS
Cloning Gene Guns
Used to blast tiny metal beads coated with DNA into an embryonic plant cell
Aimed at the nucleus or the chloroplast Use marker genes to distinguish genetically
transformed cells Antibiotic resistance
Technique is useful in plants that are resistant to Agrobacter
GEN
E G
UN
SGene gun
stopped
METHODS USED IN PLANT TRANSGENESIS
Cloning Chloroplast engineering
DNA in chloroplast can accept several new genes at once
High percentage of genes will remain active Advantage: DNA in chloroplast is completely separate
from DNA released in pollen – no chance that transformed genes will be carried on wind to distant crops
METHODS USED IN PLANT TRANSGENESIS
Cloning Antisense technology
Process of inserting a complementary copy of a gene into a cell
Gene encodes an mRNA molecule called an antisense molecule
Antisense molecule binds to normal mRNA (sense molecule) and inactivates it
Example is Flavr Savr tomato Enzyme polygalacturonase breaks down structural
polysaccharide pectin in wall of a plant. This is part of the natural decay process in a plant Monsanto identified the gene than encodes the enzyme
and made another gene that blocked the production of the enzyme.
METHODS USED IN PLANT TRANSGENESIS
FIRST COMMERCIAL TRANGENIC PLANT PRODUCT: THE FLAVR SAVR TOMATO
The Flavr Savr tomato is a genetically altered tomato developed by Calgene.
It contains an antisense RNA which inhibits the expression of a gene that normally causes fruit to soften, therefore, the fruit stays firm longer.
This allows producers a greater period of time for transportation and the opportunity for mechanical harvesting with little bruising.
TISSUE CULTURE IN ANIMAL
Animal culture was first successfully undertaken by Ross Harrison in 1907.
This was followed by a series of developments that made cell culture widely available tool for scientists including development of antibiotics, use of trypsin to remove cells from culture vessels, development of standardized, chemically defined culture media that made far easier to grow cells.
The term tissue culture refers to the culture of whole organs, tissue fragments as well as dispersed cells on a suitable nutrient medium. It can be divided into
(1) organ culture and
(2) cell culture mainly on the basis of whether the tissue organisation is retained or not.
In organ cultures, whole embryonic organs or small tissue fragments are cultured in vitro in such a manner that they retain their tissue architecture.
intent to study their continued function or development.
In contrast, cell cultures is when cells are removed from the organ fragments prior to, or during cultivation, thus disrupting their normal relationships with neighboring cells
Freshly isolated cell cultures are called primary cultures; they are usually heterogeneous and slow growing, but are more representative of the tissue of their origin both in cell type and properties.
Once a primary culture is subcultured, it gives rise to cell lines, which may either die after several subcultures (such cell lines are known as finite cell lines) or may continue to grow indefinitely (these are called continuous cell lines).
HISTORY OF ANIMAL TISSUE CULURE
HISTORY1880 Roux maintained Embryonic chicken in saline
1900 Harrison
• Frog neuroblast in lymph medium •Anchorage dependent• Nutrients• Relative slow growth rate
• Doubling 1 day vs 20 minutes bacteria• Contamination
frog embryo
characteristics for in vitro cell growth:
1. Cells require an anchor like the lymph clots (the cover slip)
2. Cells require nutrients provided by the lymph.3. Cells grow very slow; 20 hours doubling time
compared to 20 minutes for bacteria This means cell cultures are vulnerable to contamination
1900 Harrison
Carrel (surgeon, 1923)
Aseptic techniques
Carrel Flask
1912-1946 Culture Chicken Embryo Fibroblast
Plasma+tissue homogenate
Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology.
Growing viruses in cell cultures allowed preparation of purified viruses for the manufacture of vaccines.
The injectable polio vaccine developed by Jonas Salk was one of the first products mass-produced using cell culture techniques.
This vaccine was made possible by the cell culture research of John Franklin Enders, Thomas Huckle Weller, and Frederick Chapman Robbins, who were awarded a Nobel Prize for their discovery of a method of growing the virus in monkey kidney cell cultures.
Cell Cultures -Cell cultures may contain the following three types of cells:
(1) stem cells,
(2) precursor cells and
(3) differentiated cells.
STEM CELL
Stem cells are undifferentiated cells, which have unlimited capacity for poliferation, and they can differentiate under correct inducing conditions into one of several kinds of cells; different kinds of stem cells differ markedly in terms of the kinds of cells they will differentiate into.
PRECURSOR CELL
Precursor cells are derived from stem cells, are committed to differentiation, but are not yet differentiated; these cells retain the capacity for proliferation.
DIFFERENTIATE CELL
differentiated cells, usually, do not have the capacity to divide. Some cell cultures, e.g., epidermal keratinocyte cultures, contain all the three types of cells.
Cell cultures can be grown as
(1) monolayers or as
(2) suspension cultures.
INITIATION OF CELL CULTURES
The initiation of cell cultures may be conveniently dealt with under the following heads:
(1) preparation and sterilization of the substrate (culture vessels),
(2) preparation and sterilization of the medium,
(3) isolation of explant,
(4) disaggregation of the explant, and
(5) subculture and cloning.
CELLS ARE EITHER….
Anchorage – dependant
Anchorage - independant
ANCHORAGE – INDEPENDANT CELLS
Cells associated with body fluid-blood cells
Grown in suspension
Will eventually need subculturing
ANCHORAGE – DEPENDANT CELLS Most animal derived cells
Adhere to bottom of a flask and form a monolayer
Eventually cover entire surface of substratum (confluence)
Proliferation then stops
Need to subculture cells at this point (remove to fresh medium)
Proliferation can begin again
In 1997, cloning was revolutionized when Ian Wilmut and his colleagues at the Roslin Institute in Edinburgh, Scotland, successfully cloned a sheep named Dolly. Dolly was the first cloned mammal. Wilmut and his colleagues transplanted a nucleus from a mammary gland cell of a Finn Dorsett sheep into the enucleated egg of a Scottish blackface ewe. The nucleus-egg combination was stimulated with electricity to fuse the two and to stimulate cell division. The new cell divided and was placed in the uterus of a blackface ewe to develop. Dolly was born months later.
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