TECHNIQUES IN BIOTECHNOLOGY: TISSUE CULTURE

1. TECHNIQUES IN BIOTECHNOLOGY: TISSUE CULTURE Department of Chemical Engineering Technology, UniMAP PTT 104: Introduction to Biotechnology khadijahhanim@unimap.edu.my

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 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 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 • 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.

12. 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.

13. 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. 

14. Overview of the Tissue Culture Process

15. 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

16. 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

17. 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.

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

19. 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.

20. 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.

21. 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.

22. Transplanting 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. • 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.

23. 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, Agrobacteriumtumefeciens, that causes tumours. A. tumefeciensinserts 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.

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

25. 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.

26. 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.

27. 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.

28. Methods Used in Plant Transgenesis

29. 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 Agrobacteriumtumefaciens 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

30. This bacterium naturally infects plant cells causing cancerous growths - crown gall disease Infection (vir) genes carried on Ti plasmid Agrobacteriumtumefaciensas a vector for transferring foreign genes into plant chromosome

31. 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 Infection Process http://www.bio.davidson.edu/people/kabernd/seminar/2002/method/dsmeth/ds.htm

32. Methods Used in Plant Transgenesis

33. 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

34. stopped Gene gun Gene guns

35. 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

36. 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 FlavrSavrtomato • 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.

37. Methods Used in Plant Transgenesis

38. First Commercial Trangenic Plant Product: The FlavrSavr Tomato • The FlavrSavr 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.

40. 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.

41. 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.

42. 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

43. 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).

44. History of Animal Tissue Culure

45. 1900 Harrison History 1880 Roux maintained Embryonic chicken in saline frog embryo • Frog neuroblast in lymph medium • Anchorage dependent • Nutrients • Relative slow growth rate • Doubling 1 day vs 20 minutes bacteria • Contamination

46. 1900 Harrison • 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

47. Carrel (surgeon, 1923) Aseptic techniques Carrel Flask 1912-1946 Culture Chicken Embryo Fibroblast Plasma+tissue homogenate

48. 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 injectablepolio 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.

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

50. 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.