Tissue culture and their applicat by Dr.U.Srinivasa
PLANTTISSUE CULTURE & APPLICATIONS BY U.SRINIVASA
WHAT IS IT ?Tissue culture is the term used for “ the process of growing cells artificially in the laboratory ”Tissue culture involves both plant and animal cellsTissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture) OR
Tissue culture is the culture and maintenance of plant cells, tissues or organs (explants) in sterile, nutritionally (synthetic media) and environmentally, (controlled) supportive conditions (in vitro) What conditions do plant cells need to multiply in vitro?Freedom from competitionNutrients and removal of waste productsA controlled environment
Explant - Definition 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 growth regulators the explant needs.
WHAT IS NEEDED? TISSUE CULTURE, BOTH PLANT AND ANIMAL HAS SEVERAL CRITICAL REQUIREMENTS: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 semisolidAseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can over run a culture
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 interestFrequent subculturing to ensure adequate nutrition and to avoid the build up of waste metabolites
APPLICATIONS OF PLANT TISSUE CULTUREA single explant can be multiplied into several thousand plants in less than a year - this allows fast commercial propagation of new cultivarsTaking an explant does not usually destroy the mother plant, so rare and endangered plants can be cloned safelyOnce established, a plant tissue culture line can give a continuous supply of young plants throughout the year
In plants prone to virus diseases, virus free explants (new meristem tissue is usually virus free) can be cultivated to provide virus free plantsPlant ‘tissue banks’ can be frozen, then regenerated through tissue culturePlant cultures in approved media are easier to export than are soil-grown plants, as they are pathogen free and take up little space (most current plant export is now done in this manner)
Tissue culture allows fast selection for crop improvement - explants are chosen from superior plants, then clonedTissue culture clones are ‘true to type’ as compared with seedlings, which show greater variability
TYPES OF CULTURE1.Cell culture2.Organ culture3.Embryo culture4.Protoplast culture
CELL CULTURECultivation of cells on a solid, semisolid or in a liquidmedium is called cell culture Based on the type of medium used they areclassified into1. Callus culture2. Suspention culture
SUSPENSION CULTUREHere cells are cultivated on liquid medium Liquid suspension culture consists of mixtures ofcell aggregates, cell clusters and single cells
CALLUS CULTURE TECHNIQUEEstablish the callus culture from the seeds of T.Foenum-Graecum seedsProcedure:1.Perform all the operations under asepticconditions.2. Immerse the seeds in 70% ethanol for 2 minutes and rinse thrice with sterile distilled water
3.Carry the surface sterilization of seeds by submergingfor 5 minutes in 2% v/v bromine solution or 2% aqueoussolution of sodium hypochlorite. Wash the seeds threetimes sterile water to totally remove the sterilizing agent4.Germinate the seeds in dark for 2 to 3 days on sterilefilter paper or cotton wool, previously moistened withsterile distilled water in Petri dishes at 26 + 2C
5. Remove the cotyledon portion by cutting with sterilescalpel and transfer the explant portion onto solid sterilemedium(25ml) in culture flasks6. Incubate the culture at 26+ 1C in darkness for 3weeks .Transfer the cultures aseptically on sterile freshmedium at an interval of 4 weeks.
7. Calculate the growth rate in terms of GrowthIndex (G.I) as followsG.I= Final weight of callus/Initial weight of callus8. Use these static cultures for detection of plantmetabolites and precursor studies inbioproduction of secondary products
IMPORTANCE OF CALLUS CULTURE1. Production of plantlets through somatic embryogenesis or organogenesis.2. For obtaining virus-free plants.3. As a source of protoplasts and suspension cultures.4. Production of useful secondary metabolites5. For biotransformation studies.6.Selection of cell lines with valuable properties such as resistance to disease, herbicides, overproduction of secondary metabolites etc.7. For mutagenetic studies.
SUSPENSION CULTURE When cells or cell aggregates are cultured in liquidmedium, it is known as suspension cultureTypes of suspension culture 1. Batch culture 2. Continuous culture
Batch culture:This is the type of suspension culture in which cells growin a definite volume of nutrient medium is called BatchcultureContinuous culture: This is a type of culture where cells are separatedmechanically from outflowing medium and againbalanced by inflowing the fresh medium is calledContinuous culture
SUSPENSION CULTURE TECHNIQUE1.The suspension medium is taken in the conicalflask ,autoclaved and used for this technique2. A Pre-established callus culture is taken and it isintroduced inside the conical flask keeping all stepsaseptic culture.
5.The filtrate ( i.e. free cells) is centrifuged and the supernatant are poured off. The residue is the free cells and cell aggregates6.These cells are again cultured in a fresh liquid medium and the flasks again agitated by a shaker so that the cells are suspended equally in the flask
3.The conical flask is closed with cotton plug and placed with in the clamps of rotary shaker moving at the 8-120 rpm4.After considerable time (3-7 days depending on the growth of the callus),the entire contents of the flask taken out, filtered through sterilized sieve and collected the filtrate in a presterilized container.
7. From this the culture used as inoculum and a subcultred in an another presterlized liquid medium containing flask dispensing equally the cells or cell aggregates8.Cell aggregates are taken and kept in culture room for the study of regeneration of plant
IMPORTANCE1.The metabolic events of individual cells may be studied2.It forms important tools for the development of organs such as embryo3.Induction of polyploidy