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DEFINATION : DEFINATION It is an aseptic technique wherein by proper manupulation of the nutrients,culture conditions, temperature and hormonal level, one may be able to produce desired quality and quantity of plants as well as metabolites. Tissue culture involves both plant and animal cells. Requirement for Plant tissue culture : Requirement for Plant tissue culture Appropriate tissue (some tissues culture better than others) A suitable growth medium containing energy sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can over run a culture Slide 4: Growth regulators - in plants, both auxins & cytokinins. In animals, this is not as well defined and the growth substances are provided in serum from the cell types of interest Frequent subculturing to ensure adequate nutrition and to avoid the build up of waste metabolites Defination : Defination 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 surface-sterilized so it will not have any contaminating bacteria or fungus. It is then placed inside the tissue culture vessel (dish,jar, etc.)containing a gel called agar. In the agar is dissolved all the sugar, nutrients and plant growthregulators the explant needs Explant: Slide 6: Totipotency or Totipotent: The capacity of a cell (or a group of cells) to give rise to an entire organism. Differentiation (De-): The physiological and morphological changes that occur in a cell, tissue, or organ during development. Organogenesis: The development of tissues and/or organs from individual cells not from pre-existing meristems. Culturing Animal Tissue-Step-1 : Culturing Animal Tissue-Step-1 Animal tissue is obtained either from a particular specimen, or from a ‘tissue bank’ of cryo-preserved (cryo = frozen at very low temperatures in a special medium) Establishment of the tissue is accomplished in the required medium under aseptic conditions Culture vessels and medium for animal cell culture Step-2 : Step-2 Growing the cells / tissue requires an optimum temperature, and subculturing when required Human cells, for example are grown at 37degrees and 5% CO2 Incubator Animal tissue/cell culture - differences from plant tissue culture : Animal tissue/cell culture - differences from plant tissue culture Animal cell lines have limited numbers of cell cycles before they begin to degrade Animal cells need frequent subculturing to remain viable Tissue culture media is not as fully defined as that of plants - in addition to inorganic salts, energy sources, amino acids, vitamins, etc., they require the addition of serum (bovine serum is very common, but others are used) Slide 10: Animal tissue cultures can pose biohazard concerns, and cultures require special inactivation with hypochlorite (e.g. Janola,Chlorox, etc.) and then incineration The pipettes are disposable Gloves and labcoat are always worn Uses of Animal Tissue Culture : Uses of Animal Tissue Culture Growing viruses - these require living host cells Making monoclonal antibodies, used for diagnosis and research Studying basic cell processes Genetic modification & analysis Photo courtesy of Sigma Aldrich Slide 12: ‘Knockout’ technology - inactivating certain genes and tracing their effects Providing DNA for the Human Genome Project (and other species’ genome projects) Biotransformation : Biotransformation The chemical conversion of exogenously supply substance by living cell culture, permeabilized cells and immobilized enzymes derived from cell culture is termed as biotransformation. The substrate use for biotransformation may be natural or synthetic. The reaction may be single or multistep reaction.That may include Esterification, Hydrolysis,Isomerization, Methylation,Demethylation,Dehydrogenation. Slide 14: Examples of biotransformation Cardiac glycosides produced by Digitalis species, has been used in treatment of heart diseases. The biotransformation of digitoxin to digoxin using D-lanata cell which are unable to produce cardiac glycosides De-novo is the most famous examples of biotransformation.This is a commercial interest, since, digoxin is a better drug for heart disease than digitoxin. APPLICATION OF PTC : APPLICATION OF PTC 1)Agricultural application For production of weather and disease free, high yielding, salt resistant and insect resistant plant material in a uniform physiological state. Such material can be used to generate undiscovered novel compounds in addition to potentially known compounds. 2)Agronomical application For rapid multiplication of selected plants by multiple production of plants identical to original plants Slide 16: 3)Industrial Application The technique is employed for production of known molecule, using biosynthesis capacities of plant cells bred in a bio-reactor. The technique is also useful for the production of economically valuable chemicals. 4)For production of secondary metabolites Plant tissue culture technique is widely useful for production and to enhance the production of secondary metabolites in plants. Slide 17: 5)Biotechnological application -For production of natural or secondary product, pharmaceutical, colors, dyes, flavours. -For biotransformation -Production of human protein via genetic engineering. -Useful for micropropagation -Useful in the enzyme production. 6)To study biogenesis pathway of secondary metabolites. References : References Textbook of Industrial Pharmacognosy By A.N.Kalia First edition Reprint 2009 Page no. 97-130 Pharmacognosy By W.C.Evans, Fifteenth edition Page no.67-79 Pharmaceutical biotechnology By S.P.Vyas , V.K.Dixit First edition, Reprint 2007 Page no. 225-272 Pharmacognosy By C.K.Kokate Page no. 71-80 Essential of pharmacognosy By Dr.S.H.Ansari Third edition 2008-2009 Page no. 551-574. www.une.edu.au/agronomy/AgSrHortTCinfo.html http://aggie-horticulture.tamu.edu /tisscult/pltissue/pltissue.html http://www.liv.ac.uk/~sd21/tisscult/what.htm You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.