Tissue culture is the process of growing cells artifically in the laboratory. Tissue culture produces clones, in which all product cells have the same genotypes. Plant Tissue Culture Is used to create exact copies of plants that produces particularly good flowers or fruits.
Tissue culture is the process of growing cells artifically in the laboratory. Tissue culture produces clones, in which all product cells have the same genotypes. Plant Tissue Culture Is used to create exact copies of plants that produces particularly good flowers or fruits.
Tissue culture is the process of growing cells artifically in the laboratory. Tissue culture produces clones, in which all product cells have the same genotypes. Plant Tissue Culture Is used to create exact copies of plants that produces particularly good flowers or fruits.
The term tissue culture is commonly used in a very
wide sense to include in vitro aseptic culture of plant cells, tissue and organs. Is the term for the process of growing cells artifically in the laboratory. Involves both plant and animal cells Tissue culture produces clones, in which all product cells have the same genotypes (unless affected by mutation during culture).
Haberlandt
early 1900S proposed concept of totipotency cells cultured under right conditions Callus cultured from tree cambium
Gautheret, Nobecourt, Whire
In the 1930s. cells kept alive but did not develop
Plant Tissue Culture Is a practice used to propagate clones of a plant There are various reasons this may be done: 1)To create exact copies of plants that produces particularly good flowers or fruits. 2)To quickly produce mature plants. 3)To produce multiple of plants in the absence of seeds or necessary pollination to produce seeds. 4)Used to regenerate the whole plants from plant cells that have been genetically modified.
What is needed? Tissue culture, both plant and animal has several critical requirements: 1)Appropriate tissue -Some tissue culture better than others
2)A suitable growth medium -Containing energy sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid.
Aseptic conditions - Microorganisms grow much more quickly than plant and animal tissue and can over run a culture
4)Growth regulators -In plants, both auxins and cytokins. -In animal, this is not as well defined and the growth substances are provided in serum from the cell types of interest 5)Frequent subculturing - To ensure adequate nutrition and to avoid the build up of waste conditions.
Aseptic Technique Is the exclusion of invading microorganisms during experimental procedures Using sterile instruments and culture media Media and apparatus are sterile by autoclaving (121C for 15 minutes) Aseptic transfer performed in a transfer chamber such as laminar flow hood which also preferably equipped with a bunsen burner. Common sterilants are ethyl alcohol an clorox with an added surfactants.
Culturing (micropropagating) plant Tissue the steps. 1)Selection of the plant tissue -Plant tissue (explant) from a healthy vigorous mother plant -Often the apical bud, but can be other tissue
2)Sterilization -This tissue must be sterilized to remove microbial contamination
3)Establishment of the explant -Establishment in a culture medium. The medium sustain the plant cells and encourage cell division. It can be solid or liquid. -Each plant species has particular medium requirements that must be established by trial and error.
4)Multiplication -The explant gives rise to a callus ( a mass of loosely arranged cells) which is manipulated by varying sugar concentrations and the auxin (low): cytokinin (high) ratios to form multiple shoots. -The callus may be subdivided a number of times
5)Root formation -The shoots are transfered to a growth medium with relatively higher auxin: cytokinin ratios.
Benefits of plant tissue culture In plants prone to virus diseases, virus free explants (new meristem tissue is usually virus free) can be cultivated to provide virus free plants. Plant tissue banks can be frozen, the regenerated through tissue culture. Plant culture 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 selections for crop improvement- explants are chosen from superior plants, then cloned.
Laboratory Requirements for Tissue Culture General Organization Localize each portion of the tissue culture procedure in a specified place in the laboratory. An assembly-line arrangement of work areas (such as, media preparation, glassware washing, sterilization, microscopy, and aseptic transfers) facilitates all operations and enhances cleanliness. Media (tissue culture and nutrient agar) are available from Carolina Biological Supply Co., Burlington, NC. Laminar flow hoods are available from several suppliers. Any laboratory in which tissue culture techniques are performed, regardless of the specifi c purpose, must contain a number of basic facilities. These usually include the following: A general washing area A media preparation, sterilization, and storage area An aseptic transfer area Environmentally controlled incubators or culture rooms An observation/data collection area. Washing Area The washing area should contain large sinks, draining boards, and racks, and have access to deionized/distilled water. Space for drying ovens or racks, automated dishwashers, acid baths, pipet washers and driers, and storage cabinets may be necessary in the washing area, depending on the work being performed Media Preparation Area The media preparation area should have ample storage space for the chemicals, culture vessels and closures, and glassware required for media preparation and dispensing. Bench space for hot plates/stirrers, pH meters, balances, water baths, and mediadispensing equipment should be available. Other necessary equipment may include air and vacuum sources, Bunsen burners with a gas source, refrigerators and freezers for storing stock solutions and chemicals, a microwave or convection oven, and an autoclave or domestic pressure cooker for sterilizing media, glassware, and instruments. In preparing culture media, analytical grade chemicals should be used and good weighing habits practiced. To insure accuracy, an exact, step-by-step routine should be developed for media preparation. This routine should be contained in a complete media preparation checklist required to be completed by all media preparers, even for the simplest media. The water used in preparing media should be highly purifi ed though deionization and/or distillation. Tap water is not recommended because it may contain undesirable salts and dissolved gases, microorganisms (algae, fungi, bacteria), and particulate matter (silt, oils, organic matter, etc.). Water used for plant tissue culture should meet, at a minimum, the standards for type II reagent grade water, i.e., be free of pyrogens, gases, and organic matter and have an electrical conductivity less than 1.0 mho/cm. The most common and preferred method of purifying water to type II standards is a deionization treatment followed by one or two glass distillations. The deionization treatment removes most ionic impurities, and the distillation process removes large organic molecules, microorganisms, and pyrogens. Three other methods that will produce type II purity water are absorption fi ltration, which uses activated carbon to remove organic contaminants and free chlorine; membrane fi ltration, which removes particulate matter and most bacterial contamination; and reverse osmosis, which removes approximately 90% of the bacterial, organic, and particulate matter as well as about 90% of the ionized impurities Transfer Area Under very clean and dry conditions, tissue culture techniques can be successfully performed on an open laboratory bench. However, it is advisable that a laminar fl ow hood or sterile transfer room be utilized for making transfers. Within the transfer area there should be a source of electricity, gas, compressed air, and vacuum. The most desirable arrangement is a small dust-free room equipped with an overhead ultraviolet light and a positivepressure ventilation unit. The ventilation should be equipped with a high-effi ciency particulate air (HEPA) fi lter. A 0.3-m HEPA fi lter of 99.97-99.99% effi ciency works well. All surfaces in the room should be designed and constructed in such a manner that dust and microorganisms do not accumulate and the surfaces can be thoroughly cleaned and disinfected. A room of such design is particularly useful if large numbers of cultures are being manipulated or large pieces of equipment are being utilized. Another type of transfer area is a laminar fl ow hood. Air is forced into the unit through a dust fi lter then passed through a HEPA fi lter. The air is then either directed downward (vertical fl ow unit) or outward (horizontal fl ow unit) over the working surface. The constant fl ow of microbe-free fi ltered air prevents non-fi ltered air and particulate matter in the room from settling on the working surface. The simplest type of transfer area suitable for tissue culture work is an enclosed plastic box commonly called a glove box. This type of culture hood is sterilized by an ultraviolet light and wiped down periodically with 70% alcohol when in use. This type of unit is used when relatively few transfers are performed. Culture Room All types of tissue cultures should be incubated under conditions of well-controlled temperature, humidity, air circulation, and light quality and duration. These environmental factors may infl uence the growth and differentiation process directly during culture or indirectly by affecting their response in subsequent generations. Protoplast cultures, low-density cell suspension cultures, and anther cultures are particularly sensitive to environmental condition. Typically, the culture room for growth of plant tissue cultures should have a temperature between 15 and 30 C, with a temperature fl uctuation of less than 0.5 C; however, a wider range in temperature may be required for specifi c experiments. It is also recommended that the room have an alarm system to indicate when the temperature has reached preset high or low temperature limits, as well as a continuous temperature recorder to monitor temperature fl uctuations. The temperature should be constant throughout the entire culture room (i.e., no hot or cold spots). The culture room should have enough fl uorescent lighting to reach the 10,000 lux; the lighting should be adjustable in terms of quantity and photoperiod duration. Both light and temperature should be programmable for a 24-hr period. The culture room should have fairly uniform forced-air ventilation, and a humidity range of 20-98% controllable to 3%. Many incubators, large growth chambers, and walk-in environmental chambers meet these specifi cations. Water Purifi cation System; water should have a resistivity of at least 200,000 ohms/ cm and a conductivity 5.0 micromhos/cm NA Purifi cation of water for media preparation 1 Electronic Balance (0.01 g readability; 200 g minimum capacity) B933 295.00 Measuring out biochemicals and media 1 pH meter (range 0-14 +/- 0.01; automatic temperature compensation 0-600 C; one or two point calibration) P976 79.90 Measurement and adjustment of media pH 1 Hot Plate/Stirrer (7 x 7 ceramic top; variable heating range from ambient to 4000 C; variable stirring speed from 50-150 rpm; chemically resistant) H926 295.00 Mixing & heating media and stock 1 Refrigerator/freezer; capable of maintaining a refrigerator temperature of 2-60 C with a freezer temperature of approximately 200 C NA Storage of stock solutions, media, hormones 1 Laminar Flow Transfer Hood; incoming air should be HEPA fi ltered to remove 99.99% of particles larger that 0.3m; should meet or exceed the Class 100 Clean Standard 209D; should maintain a fl ow of 90 fpm +/- 20% at static pressures of 0.6-1.2 NA Provide a sterile atmosphere to transfer cultures 1 4 liter 70% Isopropyl alcohol (or several pint bottles purchased from a local pharmacy) NA Used to sterilize instruments and work areas 1 roll Aluminum foil, heavy duty; (18 x 100 ft roll Culture tubes, 25 x 150 mm, borosilicate glass; 500 tubes/ case C930 146.50 Starting cultures in Stage I 1 case Culture tube racks; holds 40, 25 mm culture tubes; withstands temperatures up to 1210 C C908 90.50 Holding culture tubes 500 Closures, for 25 mm culture tubes, 500 each C945 139.25 Sealing culture tubes 1 case Culture vessel, baby food jar; glass culture vessel; autoclavable; uses Magenta B Cap (C903) as closure; 6 oz; 100/ case autoclavable; uses Magenta B Cap (C903) as closure; 4 oz height; 100/ case C900 38.50 Culture vessel for maintaining plant cultures 1 case Magenta B Caps; autoclavable closure for baby food jars; fi ts both C904 and C900; clear polypropylene closure; 100/ case C903 215.50 Closure for baby food culture vessel 2 cases Culture vessels; autoclavable culture vessel and lid made from clear polypropylene; round vessel measures; 250/ case C913 173.00 Culture vessel for maintaining plant cultures 1 gal Detergent NA Cleaning glassware 1 case Culture dishes, disposable, sterile, 100 x 15 mm D940 75.50 Sterile surface for cutting explants (for Stage I cultures) 1 gal Chlorine bleach (sodium hypochlorite) PREPARATION OF CULTURE MEDIA:
Take 50 ml of stock solution 1 + 5ml of stock solution 2 & 4 in a beaker. The stock solution 3 prepared separately in a other 450ml flask by adding double distilled water and heat with constant stirring. Mix two solutions and adjust PH to 5.5.
STERILISATION OF MEDIA
The prepared media should be sterilized by ISI mark Autoclave( for large amounts) at 121 Domestic pressure cookers( for small amounts)
For the sterilization of glassware and metallic equipments Hot air oven with adjustable tray is required. ESTABLISHMENT OF PLANT TISSUE CULTURE In vitro culturing of plant tissue culture involves the following steps. Collecting & sterilization of glassware tools/vessels. Preparation of explant. Surface sterilization of Explant. Production of callus from explant. Proliferation of culture. Sub culturing of callus. Suspension culture EXPLANT PREPARATION
EXPLANT : It is defined as a portion of plant body, which has been taken from the plant to establish a culture Explant may be taken from any part of the plant like root,stem,leaf,or meristematic tissue like cambium, floral parts like anthers, stamens etc.. Age of the explant. Homozygous plants are preferred.
SURFACE STERILISATION OF EXPLANT For surface sterilization chromic acid, Hgcl(0.11%),calcium hypochlorite, sodium hypochlorite(1-2%),alcohal(70%) are used. Process depends on the type of explant. SEED : absolute ethyl alcohol calcium hypochlorite bromine water sterile water FRUIT : ethyl alcohol sodium hypochlorite sterile water STEM : running water sodium hypochlorite sterile water LEAF : surface clean Hgcl2 sterile water dried
PRODUCTION OF CALLUS FROM EXPLANT
Sterilized explant is transferred aseptically onto defined medium. Transfer to BOD incubator. Temperature (25 2 ) and light is necessary for callus production. Callus produced with in 3-8 days.
PROLIFERATION OF CULTURE
if callus is well developed, it should cut into small pieces & transferred to another fresh medium containing hormones, which supports growth. The medium used for production of more amount of callus is called proliferation medium.
SUBCULTURING OF CALLUS
After sufficient growth of callus it should be periodically transferred to fresh medium to maintain viability of cells.
This subculture will be done at the interval of 4-6 weeks.
After a maximum growth transfer into a pottling soil under required condition.
SUSPENSION CULTURE
It contains a uniform suspension of separate cells in a liquid medium callus
liquid medium
agitated continuously
finally cells separated
sub-culture the cells
This can be achieved by rotary shaker attached within the incubator at a rate of 50- 150 rpm. TYPES OF CULTURE Callus culture
Suspension culture
Root tip culture
Leaf or leaf primordial culture
Shoot tip culture
Complete flower culture
Anther & pollen culture
Ovule & embryo culture
Protoplast culture APPLI CATI ONS alkaloids virus-free plants forest trees
saponins apical meristem culture fruit crops
secondary metabolite PTC in industry micro propagation
chemicals III. Sterilization and Use of Supplies and Equipment: A. Sterilizing tools, media, vessels etc. 1. Autoclaving Autoclaving is the method most often used for sterilizing heat-resistant items and our usual method for sterilizing items. In order to be sterilized, the item must be held at 121C, 15 psi, for at least 15 minutes. It is important that items reach this temperature before timing begins. Therefore time in the autoclave will vary, depending on volume in individual vessels and number of vessels in the autoclave. Most autoclaves automatically adjust time when temperature and psi 4 are set, and include time in the cycle for a slow decrease in pressure. There are tape indicators that can be affixed to vessels, but they may not reflect the temperature of liquid within them. There are also test kits of microorganisms that can be run through the autoclave cycle and then cultured. Empty vessels, beakers, graduated cylinders, etc., should be closed with a cap or aluminum foil. Tools should also be wrapped in foil or paper or put in a covered sterilization tray. It is critical that the steam penetrate the items in order for sterilization to be successful. 2. Autoclaving and Fiter-sterilizing Media and Other Liquids Two methods (autoclaving and membrane filtration under positive pressure) are commonly used to sterilize culture media. Culture media, distilled water, and other heat stable mixtures can be autoclaved in glass containers that are sealed with cotton plugs, aluminum foil, or plastic closures. However, solutions that contain heat-labile components must be filter-sterilized. For small volumes of liquids (100 ml or less), the time required for autoclaving is 15-20 min, but for larger quantities (2-4 liter), 30-40 min is required to complete the cycle. The pressure should not exceed 20 psi, as higher pressures may lead to the decomposition of carbohydrates and other components of a medium. Too high temperatures or too long cycles can also result in changes in properties of the medium. Organic compounds such as some growth regulators, amino acids, and vitamins may be degraded during autoclaving. These compounds require filter sterilization through a 0.22 m membrane. Several manufacturers make nitrocellulose membranes that can be sterilized by autoclaving. They are placed between sections of a filter unit and sterilized as one piece. Other filters (the kind we use) come pre-sterilized. Larger ones can be set over a sterile flask and a vacuum is applied to pull the compound dissolved in liquid through the membrane and into the sterile flask. Smaller membranes fit on the end of a sterile syringe and liquid is pushed through by depressing the top of the syringe. The size of the filter selected depends on the volume of the solution to be sterilized and the components of the solution. Nutrient media that contain thermo labile components are typically prepared in several steps. A solution of the heat-stable components is sterilized in the usual way by autoclaving and then cooled to 35-50 C under sterile conditions. Solutions of the thermo labile components are filter-sterilized. The sterilized solutions are then combined under aseptic conditions to give the complete medium. In spite of possible degradation, however, some compounds that are thought to be heat labile are generally autoclaved if results are found to be reliable and reproducible. These compounds include ABA, IAA, IBA, kinetin, pyridoxine, 2-ip and thiamine are usually autoclaved. 3. Ethylene Oxide Gas Plastic containers that cannot be heated are sterilized commercially by ethylene oxide gas. These items are sold already sterile and cannot be resterilized. Examples of such items are plastic petri dishes, plastic centrifuge tubes etc. 4. UV Radiation 5 6 It is possible to use germicidal lamps to sterilize items in the transfer hood when no one is working there. We do not do this. UV lamps should not be used when people are present because the light is damaging to eyes and skin. Plants left under UV lamps will die. 5. Microwave It is also possible to sterilize items in the microwave; we do not do this. 6. More Comments Know which of your implements, flasks, etc. are sterile and which are not. Sterile things will have been autoclaved and should be wrapped with some kind of protective covering, e.g. foil, for transport from the autoclave to the hood. Our usual autoclave time of 20 minutes is intended for relatively small volumes. Large flasks of media, water, etc. may require longer autoclaving periods. It is preferable to put no more than one liter of liquid in a container to be autoclaved. Also, be sure to leave enough room in the container so that the liquid does not boil over. Items that come packaged sterile, e.g. plastic petri plates, should be examined carefully for damage before use. If part of a package is used, seal up the remainder and date and label. Use up these items unless there is some question about their sterility; they are expensive
Two methods (autoclaving and membrane filtration under positive pressure) are commonly used to sterilize culture media. Culture media, distilled water, and other stable mixtures can be autoclaved in glass containers that are sealed with cotton plugs, aluminum foil, or plastic closures. However, solutions that contain heat-labile components must be filter-sterilized. Generally, nutrient or plant tissue culture media are autoclaved at 1.05 kg/cm 2 (15 psi) and 121C. The time required for sterilization depends upon the volume of medium in the vessel. For small volumes of liquids (100 ml or less), the time required for autoclaving is 15-20 min, but for larger quantities (2-4 liter), 30-40 min is required. The pressure should not exceed 20 psi, as higher pressures may lead to the decomposition of carbohydrates and other thermolabile components of a medium. There is evidence that medium exposed to temperatures in excess of 121C may not properly gel or may result in poor cell growth. The minimum times required for sterilization of different volumes of medium are listed below. Since many proteins, vitamins, amino acids, plant extracts, hormones, and carbohydrates are thermolabile and may decompose during autoclaving, filter sterilization may be required. The porosity of the filter membrane should be no larger than 0.2 microns (m). Empty glassware that is to hold media must be sterilized in an autoclave before filter sterilization. Nutrient media that contain thermolabile components can be prepared in several steps. That is, a solution of the heat-stable components is sterilized in the usual way by autoclaving, then cooled to 35-50 C under sterile conditions; in a separate operation, solutions of the thermolabile components are filter-sterilized. The sterilized solutions are then combined under aseptic conditions to give the complete media. indexing It is testarious plant parts ie cuttings for theing of vfor the presence of plant pathogen. If the plant is pathogenic then it is subjected to various procedures for elimanation of pathogens. Indexing programms are being used in many commericial labs, universities and government labs. Normally micropropagated plants are indexed and common crops are bannana, vannial, spices etc.. Careful evaluation of an artificial media is required prior to initation of elimination procedures Indexing techniques Indexing strategies recolve around the host crop studied as well as specific pathogens affeciting the crop. It need to be developed that 10exploit the biology of the pathogen within the host2)utilize dettection /eradiction tech that is feconomically easible & sensitive.3)repeated over months to reduce probability of infection4)performed under strict sanitation eradication One methdod for eliminaton of systemic fungal and bacterial plant pathogens from geraniums is accomplised by using culture indexing It is the simplest,very effective method