1. TISSUE CULTURE TECHNIQUE Miss Noorulnajwa Diyana Yaacob
PTT 104
TISSUE CULTURE TECHNIQUE
Miss Noorulnajwa Diyana Yaacob

2. Course Outcome
CO2: Ability to demonstrate important recent advance in methods and application of biotechnology with regards to microorganisms and plants

3. What is Tissue Culture
Tissue culture is the growth of tissues and/or cells separate from the organism. This is typically facilitated via use of 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.

4. .This growth of cells and tissues is called in- vitro growth.
Contrasted to in-vivo growth which is growth within the body.

5. 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

6. 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.

7. 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.

8. 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.

9. 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.

10. 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 one 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.

11. Application of Tissue Culture in Plant
Micro propagation: 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.
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.
Androgenic haploids and their use in breeding: With the help of tissue culture in plants haploid embryos or haploid plants are raised by another culture technique.
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. Vitiations appearing during tissue culture in plants are called somaclonal variation

12. 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. 

13. Overview of the Tissue Culture Process

14. 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

15. 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

16. 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 is dissolved all the sugar, nutrients and hormones the plant needs.

17. Explants can be pieces of  any part of the plant (leaves, stems, flowers, etc.), or even individual isolated cells.

18. 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.

19. 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.

20. 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.

21. 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 transplanted and grown in pots in a greenhouse to produce new plants.

22. 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

23. Methods Used in Plant Transgenesis
Conventional Selective Breeding and Hybridization
Cloning
Protoplast fusion
Leaf fragment technique
Gene guns
Chloroplast engineering
Antisense technology

24. Methods Used in Plant Transgenesis
Conventional Selective Breeding and Hybridization
Sexual cross between two lines and repeated backcrossing between hybrid offspring and parent
Can take years
Polyploid plants (multiple chromosome sets greater than normal)
Increases desirable traits, especially size
Whole chromosomes can be transferred rather than single genes

25. Methods Used in Plant Transgenesis
Cloning – growing plants from a single cell
Protoplast fusion is the fusion of two protoplast cells from different 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 grow into a hybrid plant
Examples include broccoflower

26. Methods Used in Plant Transgenesis

27. Methods Used in Plant Transgenesis
Cloning
Leaf fragment technique
Small discs are cut from leaf
Cultured in a medium containing genetically modified Agrobacter (Agrobacterium tumefaciens)
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
Leaf discs are treated with plant hormones to stimulate shoot and root development

28. Methods Used in Plant Transgenesis

29. 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

30. 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
DNA in chloroplast is completely separate from DNA released in pollen – no chance that transformed genes will be carried on wind to distant crops

31. 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

32. Methods Used in Plant Transgenesis

33. 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.

35. 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.

36. 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.

37. In organ cultures, whole embryonic organs or small tissue fragments are cultured in vitro in such a manner that they retain their tissue architecture.
In contrast, cell cultures are obtained either by enzymatic or mechanical dispersal of tissues into individual cells or by spontaneous migration of cells from explants; they are maintained as attached monolayers or as cell suspensions.

38. 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).

39. History of Animal Tissue Culure

40. History 1880 Roux Embryonic chicken in saline 1900 Harrison
frog embryo
1900 Harrison
Anchorage dependent
Nutrients
Relative slow growth rate
Doubling 1 day vs 20 minutes bacteria
Contamination
Roux was the first to maintain embryonic chicken cells alive in a saline solution. Around 1900 Harrison was the first to grow cells in vitro by means of the hanging drop technique. He entrapped small pieces of frog embryo tissue in clotted lymph fluid and observed elongation of nerve fibers. From this he deduced the following characteristics for in vitro cell growth:
Cells require an anchor like the lymph clots and the cover slip.
Cells require nutrients provided by the lymph.
Cells grow very slow; 20 hours doubling time compared to 20 minutes for bacteria This means cell cultures are vulnerable to contamination

41. characteristics for in vitro cell growth:
Cells require an anchor like the lymph clots (the cover slip)
Cells require nutrients provided by the lymph.
Cells grow very slow; 20 hours doubling time compared to 20 minutes for bacteria This means cell cultures are vulnerable to contamination
1900 Harrison

42. 1912-1946 Culture Chicken Embryo Fibroblast
Carrel (surgeon, 1923)
Aseptic techniques
Carrel Flask
Culture Chicken Embryo Fibroblast
Plasma+tissue homogenate
Contamination was a main bottleneck in cell culture. Thus, it is not surprising that a surgeon Alexis Carrel was able to really routinely culture cells without contamination by applying surgical procedures for aseptic handling of cell cultures. He developed the Carrel flask which is the ancestor of our present T-flask. The techniques were however difficult to adapt by others making routine cell culture still difficult.
Carrel cultured chicken embryo fibroblast during a long term experiment form 1912 to He concluded from this that cells were immortal in culture. However, in the 1960s Hayflick and Moorhead showed that cells had a finite life-span. Carrel used as a growth medium plasma and a homogenate of tissue and very likely reinocculated his culture on every medium change.

43. Cell Cultures - Cell cultures may contain the following three types of cells: (1) stem cells, (2) precursor cells and (3) differentiated cells.

44. 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.

45. 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.

46. 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.

47. Cell cultures can be grown as (1) monolayers or as (2) suspension cultures.

48. 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.

49. Cells are either….
Anchorage – dependant
Anchorage - independant

50. Anchorage – independant cells
Cells associated with body fluid
-blood cells
Grown in suspension
Will eventually need subculturing

51. 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

52. 2 main categories of animal cell cultures….
Primary culture
Continuous cell line

53. Primary Cultures Taken from fresh tissue Limited life span in culture
Treated by proteolytic enzyme (Trypsin)
Separate into single cells
-epithelial cells
-fibroblasts

54. Continuous Cell Line Derived from humans Been transformed
-lose sensitivity to factors associated with growth control
Produce immortalised cell lines
Often lose their anchorage-dependence
More easily cultured

55. 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.

56. Thank You