accomplished through tissue culture of small tissue pieces from the plant of Tissue culture involves the use of small pieces of plant tissue (explants) which. PDF | On Nov 18, , Gaurav Kumar Sharma and others published General Techniques of Plant Tissue Culture. Plant cell and tissue culture in a simple fashion refers to techniques which utilize either single plant cells, groups of unorganized cells (callus) or organized tissues or organs put in culture, under controlled sterile conditions. Their theory was in fact the foundation of plant.
|Language:||English, Spanish, Portuguese|
|Distribution:||Free* [*Registration needed]|
Advantages of tissue culture over intact plants. 1. The biochemical engineer can grow plant cells in liquid culture on a large scale--Bioreactor. h d. f d h l d l. A micropropagation unit includes a tissue culture laboratory and a propagation The laboratory for plant tissue culture requires a basic organization that. PDF Drive is your search engine for PDF files. As of today we have 78,, eBooks for you to download for free. No annoying ads, no download limits, enjoy .
Then the required amount is carefully weighed using an appropriate instrument. Excess material that has to be removed from the balance should be discarded rather than returned to the chemical container. The balance and weighing area around it must be kept clean. This involves cleaning the balance after each use.
Often a brush is used for this. Balances are accurate for specific weight ranges and should never be overloaded. Measuring Liquids Calibrated glassware e. Tarek Kapiel for many measuring operations; pipettes are used for smaller volumes. Volumetric flasks are much less commonly used than they used to be.
When measuring solutions with pipettes or graduated cylinders, the bottom of the curved air- liquid interface should be aligned with the measuring mark. Again, do not return excess to the original container. Pipettes should be filled with a hand-operated pipettor, never by mouth! There are several types of pipettors; individuals usually find that they are most comfortable with a particular type.
Water The quality of the water used can make a significant difference in the final medium. Type I water is most desirable. Type I water is produced by distillation, ion exchange, reverse osmosis, or a combination of these methods. We distill deionized lab water. The water can be tested for purity. The pH of most culture media is adjusted to 5. The pH can influence the solubility of ions in nutrient media, the ability of agar to gel, and the subsequent growth of cells. For example, if the pH is lower than about 4.
Therefore accurate determination and control of the pH of tissue culture media are necessary. Generally, pH is determined with a pH meter, which can be digital or analog.
The pH meter is calibrated before use by adjusting it using standards of known pH. Although pH drops somewhat during autoclaving, the pH of the medium is not typically readjusted after autoclaving. Tarek Kapiel IX. Pouring and Storing Media Once media is made and pHed, it may be heated to dissolve the gelling agent and dispensed into culture vessels, like tubes, baby food jars, or Magenta boxes, and then autoclaved, if no filter-sterilized components need to be added.
Alternatively, the media may be placed in a flask, which is covered, e. The medium should be stirred or shaken frequently while cooling. Any filter-sterilized reagents are then added and mixed in well. The media is then poured into vessels e. The vessels in either case are cooled in the transfer hood; lack of movement at this point promotes even gelling. All media should be labeled before it is removed from the hood.
I t should then be sealed up we use ziplock bags to prevent contamination. I t should be stored in the refrigerator or at least in the dark light causes some reagents to break down. Under good conditions, most media can be stored for at least a month. Cleaning Glassware The traditional method of washing glassware involved soaking it in a chromic acid-sulfuric acid bath followed by tap water rinses, distilled water rinses, and finally double- distilled water rinses.
Due to the corrosive nature of chromic acid, this is almost never done anymore. The glassware can be air dried or dried in a drying oven. Care should be taken after washing to keep the glassware clean, e. I f the glassware contains contaminant or recombinant material, it is autoclaved before washing.
The in vitro environment in which the plant material is grown is also ideal for the proliferation of microorganisms. I n most cases the microorganisms outgrow the plant tissues, resulting in their death. Contamination can also spread from culture to culture.
The purpose of aseptic technique is minimizing the possibility that microorganisms remain in or enter the cultures. The environmental control of air is also of concern because room air may be highly contaminated. Sneezing produces , - , aerosol droplets which can then attach to dust particles. These contaminated particles may be present in the air for weeks. Air may also contain bacterial and fungal spores, as do we. The essence of aseptic technique is the exclusion of invading microorganisms during experimental procedures.
I f sterile tissues are available, then the exclusion of microorganisms is accomplished by using sterile instruments and culture media concurrently with standard bacteriological transfer procedures to avoid extraneous contamination.
The use of dis posable sterile plasticware reduces the need for some autoclaving. Alternative sterilization techniques such as filter sterilization must be employed for heat-labile substances like cytokinins.
Aseptic transfers can be made on the laboratory bench top by using standard bacteriological techniques i. Tarek Kapiel Aseptic transfers are more easily performed in a transfer chamber such as a laminar flow hood, which is also preferably equipped with a bunsen burner.
I f experimental tissues are not aseptic, then surface sterilization procedures specific to the tissues are employed. Concentration of sterilants and exposure time are determined empirically. Bacteria, fungi, and insects 1. Bacteria Bacteria are the most frequent contaminants.
They are usually introduced with the explant and may survive surface sterilization of the explant because they are in interior tissues. So, bacterial contamination can first become apparent long after a culture has been initiated see below. Some bacterial spores can also survive the sterilization procedure even if they are on the tissue surface.
Many kinds of bacteria have been found in plant tissue cultures including Agrobacterium, Bacillus, Corynebacterium, Enterobacter, Lactobacillus, Pseudomanas, Staphylococcus, and Xanthomonas. Bacteria can be recognized by a characteristic "ooze"; the ooze can be many colors including white, cream, pink, and yellow. There is also often a distinctive odor. Fungi Fungi may enter cultures on explants or spores may be airborne. Fungi are frequently present as plant pathogens and in soil.
They may be recognized by their "fuzzy" appearance, and occur in a multitude of colors. Tarek Kapiel 3. Yeast Yeast is a common contaminant of plant cultures. Yeasts live on the external surfaces of plants and are often present in the air. Viruses, etc.
Viruses, mycoplasma-like organisms, spiroplasmas, and rickettsias are extremely small organisms that are not easily detected. Thus, plant culture is not necessarily pathogen-free even if microorganisms are not detected, and this can influence culture success. Special measures such as meristem culture are often necessary to eradicate such contaminants.
Insects The insects that are most troublesome in plant cultures include ants, thrips, and mites. Thrips often enter cultures as eggs present on the explants. Ants and mites, however, usually infest already established cultures. Mites feed on fungus and mite infestations are often first detected by observing lines of fungal infection that lead from the edge of the culture vessel to the plant tissue, having been introduced by the insect.
I t is very difficult to eradicate insect infestations. Careful lab practices and cleanliness should prevent most infestations. Initial Contaminants Most contamination is introduced with the explant because of inadequate sterilization or just very dirty material. I t can be fungal or bacterial. This kind of contamination can be a very difficult problem when the plant explant material is harvested from the field or greenhouse. I nitial contamination is obvious within a few days after cultures are initiated.
Tarek Kapiel liquid cultures. Latent Contamination This kind of contamination is usually bacterial and is often observed long after cultures are initiated. Apparently the bacteria are present endogenously in the initial plant material and are not obviously pathogenic in situ. Once in vitro, however, they increase in titer and overrun the cultures.
Latent contamination is particularly dangerous because it can easily be transferred among cultures. Introduced Contamination Contamination can also occur as a result of poor sterile technique or dirty lab conditions. This kind of contamination is largely preventable with proper care. Detection of Contaminants Contamination is usually detected by the "eyeball" method in research labs.
However, indexing is possible, and is frequently done in commercial settings. This involves taking a part of the plant tissue and culturing it in media that are specific for bacteria and fungi. Media that have been used for this purpose include PDA potato dextrose agar and NB broth with salts, yeast extract and glucose.
Tarek Kapiel II. A horizontal laminar flow unit is designed to remove particles from the air. The air is filtered by a HEPA high efficiency particulate air filter so nothing larger than 0.
This renders the air sterile. The positive pressure of the air flow from the unit also discourages any fungal spores or bacteria from entering. Depending on the design of the hood, the filters are located at the back or in the top of the box. Figure 2. Laminar airflow hoods used in commercial and research tissue culture settings. Tarek Kapiel III. Sterilizing tools, media, vessels etc. Autoclaving Autoclaving is the method most often used for sterilizing heat-resistant items and our usual method for sterilizing items.
I t 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 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.
Empty vessels, beakers, graduated cylinders, etc. Tools should also be wrapped in foil or paper or put in a covered sterilization tray. I t is critical that the steam penetrate the items in order for sterilization to be successful. Autoclaving and Filter-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.
Tarek Kapiel However, solutions that contain heat-labile components must be filter-sterilized. For small volumes of liquids ml or less , the time required for autoclaving is min, but for larger quantities liter , 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. 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. Solutions of the thermo labile components are filter-sterilized. The sterilized solutions are then combined under aseptic conditions to give the complete medium. I n 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.
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. UV Radiation I t 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. Microwave I t is also possible to sterilize items in the microwave; we do not do this. Sterile things will have been autoclaved and should be wrapped with some kind of protective covering, e. Large flasks of media, water, etc. I t 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.
Tarek Kapiel 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. I f for some reason it has been turned off, turn it on and let it run for at least 15 minutes before using.
As few things as possible should be stored in the hood. Wipe the work area and let the spray dry. I t is not necessary to flame them This is a joke. Especially keep all flasks as far back to the back of the hood as possible. Movements in the hood should be contained to small areas. A line drawn across the distance behind which one should work is a useful reminder. Use forceps, etc.
This can be dangerous if the vessel holding the alcohol tips over and an alcohol fire results. A fairly deep container, like a coplin-staining jar, should be used to hold the ethanol. Use enough ethanol to submerge the business ends of the instruments but not so much that you burn your hands. Some people wear gloves in the hood for certain procedures.
I f you do this, be very careful not to get them near the flame. Other methods of sterilization that do not require alcohol are with a bacticinerator or glass bead sterilizer.
There is not as much risk from fire with these, but the instruments can still get extremely hot, causing burns.
For a right-handed person, it is best that the flame, alcohol for flaming, and tools be placed on the right. The plant material should be placed to the left. All other items in the hood should be arranged so that your work area is directly in front of you, and between 8 and 10 inches in from the front edge. No materials should be placed between the actual work area and the filter. Keep as little in the hood as possible. Sterile petri dishes expensive , sterile paper towels, or sterile paper plates work fine.
Pre- sterilized plastic dishes have two sterile surfaces-the inside top and inside bottom. The tools should be placed on a holder in the hood to cool or should be cooled by dipping in sterile water or medium before handling plant tissues. Clean hood surface periodically while working. Glass pipettes are put into containers or wrapped and then autoclaved.
Tarek Kapiel are downloadd presterilized in individual wrappers. To use a pipette, remove it from its wrapper or container by the end opposite the tip.
Do not touch the lower two-thirds of the pipette. Do not allow the pipette to touch any laboratory surface. I nsert only the untouched lower portion of the pipette into a sterile container.
When you open a sterile tube, touch only the outside of the cap, and do not set the cap on any laboratory surface. I nstead, hold the cap with one or two fingers while you complete the operation, and then replace it on the tube. This technique usually requires some practice, especially if you are simultaneously opening tubes and operating a sterile pipette.
After you remove the cap from the test tube, pass the mouth of the tube through a flame. I f possible, hold the open tube at an angle. Put only sterile objects into the tube. Complete the operation as quickly as you reasonably can, and then flame the mouth of the tube again. Replace the lid. We use plastic ones that come sterile. I f you are moving organisms from an agar plate, touch an isolated colony with the transfer loop.
Replace the plate lid. Open and flame the culture tube, and inoculate the medium in it by stirring the end of the transfer tool in the medium. I f you are removing cells from a liquid culture, insert the loop into the culture.
Even if you cannot see any liquid in the loop, there will be enough cells there to inoculate a plate or a new liquid culture. For example, we do not measure a specific volume of medium when we pour culture plates, although after you have done it for a while, you become pretty consistent. Remove the cap or lid from the solution to be transferred. Thoroughly flame the mouth of the container, holding it at an angle as you do so.
Remove the lid from the target container. Tarek Kapiel at an angle. Quickly and neatly pour the contents from the first container into the second.
When using a sterile graduated cylinder, complete the transfer as quickly as you reasonably can to minimize the time the sterile liquid is exposed to the air. All cultures must be sealed before leaving the hood.
Situate the cultures so that the contaminated part is closest to the front of the hood. Empty e. All needles go in the sharps box, needles used with bacteria get autoclaved. Small bags used in the hood for waste go in the big bag to be autoclaved; do not overfill the small bags or leave full bags in or on the hood for someone else to dispose of.
Glassware that comes in contact with bacteria is placed in a separate pan to be autoclaved. Special problems are contaminated cultures, dirty dishes and solutions where microorganisms can grow. We will discuss this area later. Check cultures every days for contamination. Tarek Kapiel V. Preparation of Stock Plants Prior good care of stock plants may lessen the amount of contamination that is present on explants. Overhead watering increases contamination of initial explants. Likewise, splashing soil on the plant during watering will increase initial contamination.
I t is sometimes possible to harvest shoots and force buds from them in clean conditions. The forced shoots may then be free of contaminants when surface- sterilized in a normal manner. Seeds may be sterilized and germinated in vitro to provide clean material. Covering growing shoots for several days or weeks prior to harvesting tissue for culture may supply cleaner material.
Explants or material from which material will be cut can be washed in soapy water and then placed under running water for 1 to 2 hours. Ethanol or Isopropyl Alcohol Ethanol is a powerful sterilizing agent but also extremely phytotoxic. Therefore, plant material is typically exposed to it for only seconds or minutes.
The more tender the tissue, the more it will be damaged by alcohol. Tissues such as dormant buds, seeds, or unopened flower buds can be treated for longer periods of time since the tissue that will be explanted or that will develop is actually within the structure that is being surface-sterilized.
Sodium Hypochlorite Sodium hypochlorite, usually downloadd as laundry bleach, is the most frequent choice for surface sterilization. I t is readily available and can be diluted to proper concentrations. Commercial laundry bleach is 5. Plant material is usually immersed in this solution for 10 - 20 minutes. A balance between concentration and time must be determined empirically for each type of explant, because of phytotoxicity. Calcium Hypochlorite Calcium hypochlorite is used more in Europe than in the U.
I t is obtained as a powder and must be dissolved in water. The concentration that is generally used is 3. The solution must be filtered prior to use since not all of the compound goes into solution. Calcium hypochlorite may be less injurious to plant tissues than sodium hypochlorite. Mercuric Chloride Mercuric chloride is used only as a last resort in the U. I t is extremely toxic to both plants and humans and must be disposed of with care. Since mercury is so phytotoxic, it is critical that many rinses be used to remove all traces of the mineral from the plant material.
Tarek Kapiel that hydrogen peroxide is useful for surface-sterilizing material while in the field. Tween 20 is frequently added to the sodium hypochlorite.
Rinsing After plant material is sterilized with one of the above compounds, it must be rinsed thoroughly with sterile water.
Typically three to four separate rinses are done. Use of Antibiotics and Fungicides in Vitro We have found that the use of antibiotics and fungicides in vitro is not very effective in eliminating microorganisms and these compounds are often quite phytotoxic.
The recommended dose is 0. Higher doses are required to treat endogenous contamination and for Agrobacterium. Tarek Kapiel fungicides. Many antibiotics adversely affect plant materials. This is useful when routine surface sterilization is insufficient. Sterilizing Glassware and Instruments Metal I nstruments are best sterilized using a glass bead sterilizer.
The instruments simply need to be inserted into the heated glass beads for a period of 10 to 60 sec. The instruments should then be placed on a rack under the hood to cool until needed. Metal instruments, glassware, aluminum foil, etc. Autoclaving is not advisable for metal instruments because they may rust and become blunt under these conditions. After an instrument has been used, it can again be dipped in ethyl alcohol, re-flamed, and then reused. This technique is called flame sterilization.
Safety is a major concern when using ethyl alcohol. Alcohol is flammable and if spilled near a flame will cause an instant flash fire. Tarek Kapiel hoods due to the strong air currents blown towards the worker. Fires commonly start when a flamed instrument is thrown back into the alcohol beaker. I n case of fire do not panic. Limiting the supply of oxygen can easily put out fires. Autoclaving is a method of sterilizing with water vapor under pressure.
Cotton plugs, gauze, labware, plastic caps, glassware, filters, pipettes, water, and nutrient media can all be sterilized by autoclaving. Nearly all microbes are killed by exposure to the super-heated steam of an autoclave for minutes. Sterilizing Nutrient Media 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. The time required for sterilization depends upon the volume of medium in the vessel.
For small volumes of liquids ml or less , the time required for autoclaving is min, but for larger quantities liter , 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. Tarek Kapiel 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. 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. The sterilized solutions are then combined under aseptic conditions to give the complete media. Table 3. Tarek Kapiel Remember: Place or move materials out of the way accordingly. At the end of the day, rinse all tools to remove the rubbing alcohol. Shoes are to be worn at all times.
It is best not to wear open toed shoes or sandals, since they offer no protection from spills. We will be using substances that can ruin your clothes, e.
We will not be using many dangerous chemicals, but safety glasses will be provided when we are using acids, etc. No eating, drinking or smoking in the laboratory at any time.
If you spill a chemical on yourself, wash immediately with copious amounts of water and notify the TA. In the event of a spill on the floor or a bench involving hazardous materials such as strong acid or base or a volatile organic compound notify us immediately and receive instructions regarding cleanup before attempting to clean it up. Be careful! Pay attention to what you are doing at all times.
If you injure yourself in the laboratory in any way however minor you may think the injury is , report it to us immediately. Broken Glass: Everybody breaks glass occasionally. If you break something, don't rush to clean it up with your hands.
Find a broom or dust brush, sweep up the glass and place it in the appropriate broken glassware container. Do not ever put any glass in the regular trash can. Tarek Kapiel 6. Other Waste: Do not put any waste chemicals down the sink. We will instruct you as to disposal. We will also be using sharps, e. Mouth pipetting is forbidden. Use pipettors at all times. Volatile Chemicals: Use the fume hood when working with volatile chemicals. Check to make sure the hood is working before opening the volatile chemical.
Dirty Labware: Follow the TAs instructions on how to deal with dirty labware. Make sure that all of your cultures, etc. Transgenic procedures: Genetic manipulation experiments must be carried out in accordance with guidelines laid down by local Environmental Health and Safety on our campus. We will instruct you in this. Tarek Kapiel Experiment 1: Fast tissue culture with rapid- cycling Brassica rapa Introduction: This is an introductory experiment to cell and tissue culture practical work.
Plant tissue culture refers to the growth of individual cells, tissues or, as in this case, organs on an artificial medium. The aseptic techniques which are usually associated with tissue culture experiments are not necessary here. This is because the explants will continue to grow on agar alone.
Serious bacterial or fungal contamination is unlikely because the medium contains no sugar and the time scale is so short. Seedlings of Sinapis alba and rapid-cycling Brassica rapa grow very quickly. New leaves should appear on explants within days and even new roots or flower buds within days. Procedure Read the following instructions carefully before you start. What, if anything, would you expect the seedling explants to obtain from the agar? Short-necked test tubes or McCartney bottles are ideal for this experiment.
If you use long test tubes you should pour more agar into each tube than is suggested in step 3. Why is this? Why should you cover the tubes with a transparent lid e.
Why should you not open the tubes again once you have set them up? It is always best to try and obtain quantitative data from experimental work. Suggest what measurements can be made as you watch the explants grow. What further information would you gain by doing this?
Vary the growth conditions e.
Compare material from different species. Remove one or both cotyledons and compare growth with that of complete explants. Try growing isolated cotyledons only.
Explant response is enhanced by the addition of nutrients to the agar. Tarek Kapiel Experiment 2: The aim of this experiment is to examine the growth of root explants over a 7-day period. The addition of sugar to the medium increases the risk of fungal and bacterial contamination unless you take precautions to keep your explants and apparatus sterile.
With banana being a perennial crop and can be harvested at least once a year coupled with its vegetative-propagation nature, the crop is popular among small holder farmers in East Africa as a staple food crop MoA ; ISAAA The growing urban markets are making bananas a more important source of cash for smallholders Kalyebara et al.
East Africa has a dynamic cross-border trade in cooking and beer bananas Jagwe et al. Burundi is a net importer, bringing in cooking bananas from Rwanda and Tanzania and beer bananas from Tanzania and DR Congo. Kenya imports cooking and dessert banana from Uganda and Tanzania.
Kenya also exports some bananas to the Middle East and the European Union. Among local communities, bananas have other various functions. The leaves and stems are chopped and used as cattle feed. Some species of banana yield fibre, which is used for making ropes Naduvinamani, The tip of inflorescence is cooked as a vegetable in some places. The plant is also used for decoration purpose in wedding, festivals and fairs.
It is also used as raw material in industries for preparation of banana powder, chips, juices and beer. The juice of banana stem is used in making paper bond, tissue paper among others Naduvinamani, Despite such vitality, banana production in East Africa has been on a decline for the past two decades Carter et al.
In north western parts of Tanzania in Kagera region, production decreased from 10 tons to 4 tons per hectare due to declining soil fertility Galez et al.
This is very low production compared with banana production in other parts of the world like India where the production was on an average of The emerging of Banana bacteria wilt in East Africa in has caused tremendous loss to the small scale farmers whereby some farmers lost their whole crop if their varieties were very susceptible to Banana Bacterial Wilt such as beer bananas Tushemereirwe, In addition, a complex of various pest imperil banana in the region poses a big threat to the banana industry.
The banana weevil and a complex of nematodes are considered the most important pests on East African highland bananas Niere et al. One of the major factors behind all the above bottle necks to banana production is that most banana growers plant new gardens with suckers from old plants, exacerbating pests and diseases.
Moreover, suckers are bulky, making it slow to disseminate improved varieties that would be resistant to the prevailing diseases. Tissue culture TC technology offers mass propagation and clean planting material. In vitro banana production technology is a superior technology over traditional method Sucker-propagated of banana production with respect to optimal yield, uniformity, disease-free planting material and true to type plants Sadik et al.
Mass multiplication of tissue culture plants could be done in a short time.
They are cheaper to transport than conventional suckers and the coupling with virus indexing allows for safe movement, and exchange and conservation of germplasm Muchui et al. In addition, bananas produced using the TC technology are reported to be more vigorous, higher yielding and produce better quality fruits than those produced by conventional means Dubois et al.
For commercialization, it is necessary that consistent supplies of good quality bananas are produced to meet the increasing demand. The high rate of multiplication of genetically uniform, pest and disease free planting material can be achieved through tissue culture. Tissue culture approaches would also allow for off-season production of fast growing plants compared with conventionally propagated ones Ngomuo et al.
The in vitro propagation technique for producing banana using shoot tip cultures is a necessary approach in dealing with the problems encountered in tissue culture. These techniques will ensure sustainable production of banana planting materials. Consequently, enormous research efforts have targeted enhancing the existing banana micro propagation protocols and adapting them to different EAAAA banana cultivars.
However, tissue culture is determined by various contaminating factors for one to obtain explants. Therefore, assessment of these factors will be a big boost in understanding how to carry out the tissue culture procedures, the right way. Also, knowledge about and the propagation potential of bananas in vitro in particular is still not wide spread in agricultural scientists especially in the breeding field where it is of paramount importance.
Knowledge about and experience on in vitro proliferation of EA-AAA banana cultivars would enhance further development, adoption and use of in-vitro micro propagation banana multiplication as well as other crops whose physiology rhymes well with in vitro propagation. Increased awareness of in vitro procedures will be an additional boost to the banana plantlet production industry and overall banana production in Uganda.
Commercial rooting hormone solutions and powders are also available from hardware stores. A MS mixture was dissolved in ml of distilled water. Some cultured tissues are slow in their growth. For them there would be two options: i Optimizing the culture medium; ii Culturing highly responsive tissues or varieties. Generally, plant varieties differ in susceptibility to tissue culture necrosis. Thus, by culturing highly responsive varieties or tissues it can be managed. However, they are more easily removed from the explant by gentle rinsing, and the remainder usually can be killed by surface sterilization.
Most of the surface microflora do not form tight associations with the plant tissue. Such associations can usually be found by visual inspection as a mosaic, de-colorization or localized necrosis on the surface of the explant.
An alternative for obtaining uncontaminated explants is to take explants from seedlings which are aseptically grown from surface-sterilized seeds. The hard surface of the seed is less permeable to penetration of harsh surface sterilizing agents, such as hypochlorite , so the acceptable conditions of sterilization used for seeds can be much more stringent than for vegetative tissues.
Tissue cultured plants are clones. If the original mother plant used to produce the first explants is susceptible to a pathogen or environmental condition, the entire crop would be susceptible to the same problem. Conversely, any positive traits would remain within the line also. Applications[ edit ] Plant tissue culture is used widely in the plant sciences, forestry, and in horticulture. Applications include: The commercial production of plants used as potting, landscape, and florist subjects, which uses meristem and shoot culture to produce large numbers of identical individuals.
To conserve rare or endangered plant species. Large-scale growth of plant cells in liquid culture in bioreactors for production of valuable compounds, like plant-derived secondary metabolites and recombinant proteins used as biopharmaceuticals. To rapidly study the molecular basis for physiological, biochemical, and reproductive mechanisms in plants, for example in vitro selection for stress tolerant plants.
For chromosome doubling and induction of polyploidy ,  for example doubled haploids, tetraploids , and other forms of polyploids. This is usually achieved by application of antimitotic agents such as colchicine or oryzalin.
As a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants.