Organizations in the tissue culture lab
In each laboratory where tissue culture techniques used must have a number of facilities that include among others:
• Outdoor washing
• Outdoor media preparation, sterilization and storage
• aseptic transfer room
• Space culture or the environment-controlled incubator
• Space observation and data collection
Washing room should have a sink, a desk made of materials resistant to acids and alkalis, drying rack and a channel for demineralisasi or distilled water, space for the oven drying, equipment / washing machines and dryers, and storage rack or equipment cabinet .
Media Preparation Room
In the medium preparation room should be available space for storage of chemicals, glass culture and the lid, and glass equipment needed to manufacture the media. Sturdy table or "bench" for storage of "hot plate magnetic stirrer", pH meters, scales, and dispensers must be available. Other equipment that normally is in the preparation of media including vacuum devices, distiling units, Bunsen, refrigerator (fridge) and freezers for storage of stock solution and chemicals, microwave, gas stove, oven and autoclave for sterilization mdia, glassware and other equipment . In the manufacture of culture media, chemicals that are used to the standard analytic and penimbangannya be good and true. To be more accurate, in the manufacture of the media must be done step by step and the materials used must be in "checklist".
Water used in the manufacture of medium to high quality with a high purity level. Tap or well water is not used for the manufacture of the media because it contains cations (ammonium, calcium, iron, magnesium, sodium, etc..), Anions (bicarbonate, chloride, flourida, phosphate, etc..), Microorganisms (algae, fungi, bacteria), the gases (oxygen, CO2, nitrogen) and other materials (oil, organic materials, etc..). Water used in tissue culture should have a standard type II (minimum) free of pyrogen, gas, and organic materials and has an electrical conductivity less than 1.0 μmho / cm.
The most common method for water purification is standard with type II deionosasi followed by one or two glasses of distilled. Deionisasi remove from materials that are ionic and distillation processes remove organic molecules, microorganisms and pyrogens. Other methods that can be used to obtain pure water type II is (1) screening by means of absorption, by using activated carbon to remove organic contaminants and free klorine; (2) with membrane filtration, which removes particulate material and contamination by bacteria; and (3) reverse osmosis, which removes about 99% of bacteria, organic materials and particulate materials.
Tissue culture techniques can take place successfully if done under laboratory conditions are very clean. Therefore, removal or transfer of cultures done in a sterile transfer room or laminar flow of water. Laminar flow of water used in plant tissue culture is the horizontal type and are designed to have a room free of fine dust particles and equipped with ultraviolet (UV) and the air filter unit. Air filter air filter should have a high efficiency or "high-efficiency particulate air (HEPA filters). HEPA filter must have pores of about 0.3 μm with efficiencies ranging from 99.97 - 99.99%. All surfaces in a laminar study should be designed and has a construction such that the dust and microorganisms are not able to accumulate and work surfaces can be easily cleaned and disinfected.
All kinds of culture should be kept in place well controlled temperature, air circulation, humidity and the quality and duration of light. Environmental factors will affect the process of growth and differentiation of cultured either directly or indirectly. Protoplas culture, suspension cells and anther culture is the most sensitive to environmental conditions. Room temperature for the growth of culture generally range between 15o - 30oC, with fluctuations less than ± 0.5oC; but the range of temperatures greater may be required for the purpose of the experiment. Culture should have a space up to 10,000 lux lighting. Temperature and light must be programmed for 24 hours. Should be well ventilated with 20-98% humidity range.
Figure 1. Diagram of plant tissue culture laboratory
Equipment and basic materials in the tissue culture lab
Equipment needed from a general laboratory is as follows:
1) Hot plate / magnetic stirrer or stove
2) glassware (measuring cups, erlenmeyer) or stainless steel to heat up and dissolve the media
3) Equipment with pressure steam sterilization (autoclave)
4) pH meter
5) Scales (analitical and bench top loading)
6) glass measuring gradual
7) culture bottle with lid
9) Tools dissection (spatula, scalpel (tweezers), forceps, scissors)
11) Distiling units or water deionizer
15) Pipette measuring
17) Laminar air flow
19) Chemicals needed to manufacture the media (Appendix)
Glassware used in lab tissue culture generally made of pyrex. Erlenmeyer of various sizes (50, 125, 250, 500, 1000 or 2000 ml) is used for container culture and media production. Glass tube, petri dish, a bottle of jam jam or used too often used as a culture bottle. Tesebut glassware must withstand the heat during the sterilization process with an oven or autoclave. Other glassware often used is the glass cup, measuring cups, measuring pipettes and pumpkins.
Basic laboratory procedures
Generally operational use in the lab plant propagation by tissue culture can be studied with ease. The last thing to note is the accuracy, cleanliness and safety when working with tissue culture techniques.
At the time of making the media, all materials must be weighed carefully for it even to the media in a commercial scale. Any use of scales or other tools should pay attention to instructions from the manufacturer. Types of scales frequently used in the lab include top-loading analytical balance and balance that enables the accuracy of weighing up to milligram scale. Some of the requirements that must be considered in order to obtain an accurate weighing is (i) the scale should be placed in a hard place, stable, flat surface that is free of vibration and leakage, (ii) the area around the weighing should be wake hygiene, (iii) the most important thing weighing not more to never overload, (iv) weighing the container or suggested using a lightweight pads or paper rather than placing the material weighed directly on the plate scales.
(2) Measurement of fluid / solution
Glassware that has a size such as goblets, erlenmeyer and pipettes required to manufacture the media. Measuring cup capacity 10, 25, 100 and 1000 ml can be used to measure the volume, but a more accurate measurement is required measure and pipette pumpkin. Measurement solution by using a pipette and measured gourds will only accurate if the base of the basin between the water and the air is right at the sign measurements.
The use of pipettes should be assisted by vacuum solution (pipetor). Never use mouth to memipet. Pipetor types of commonly used include (i) type of suction ball equipped with a valve controller, (ii) suction pipette operated using a small wheel at the top of the vacuum, (iii) vacuum with the aid of an electric air pump. Diiisap fluid into the pipette by pressing the top button and release the liquid by pressing the bottom button, (iv) micro pipette, used for making solutions with very small volumes (micro liter).
(3) Cleaning glassware
Conventional methods of cleaning glassware made by soaking the glass in Chromic acid solution followed by rinsing with tap water and distilled water. Because chromate acid can cause corrosive, so this means a lot left except for contaminated glassware high. Washing a more secure is by hot water (> 70oC) + soap, followed by rinsing with hot water and distilled water. Glassware that had been washed, dried in an oven at 150oC temperature wrapped in aluminum foil, then stored in closed cabinets.
A very important part in the in vitro technique is sterilization plant material and the media, maintaining aseptic conditions that have been achieved. Bacteria and fungi are two contaminants most often found in the culture. Fungal spores is very light and there around the neighborhood. If contact with the spores of the fungus culture medium and optimal conditions for fungal germination, then the contamination will occur.
• Sterilization of the culture and the living room transfer.
Sterilization of culture space is best done with the use of ultraviolet (UV). Sterilization time varies depending on the size of the transfer itself and should be done if there is no activity in the space. UV radiation is harmful to eyes and skin. Transfer space can also be sterilized by washing / swab 1-2 times each month with anti-fungal (fungicide) commercial. Study in laminar flow is usually already equipped with UV light, so sterilisasinya done by UV and followed by washing / wiping the surface of the work in the laminar with 95% alcohol before starting work. Culture room should be cleaned with soap and then wipe with Na-hypoklorit 2% (commercial brands like Sunclin, Bayclin or other floor cleaners that contain disinfectants) or 95% alcohol. The floor and walls of the room should be cleaned once a week with the same material.
• Sterilization of glassware and other equipment.
Equipment made of metal, glass, aluminum foil, etc.., Can disterilsasi by drying in an oven at a temperature of 130o-170oC for 2-4 hours. All equipment should be wrapped up before the oven, but do not use paper because it will be decomposed at a temperature of 170oC. Using autoclave sterilization is not recommended for materials made of metal because it will cause rust.
For dissection equipment to be used in the transfer or laminar, after sterilized in the oven should be soaked first in 96% alcohol and then burned in the Bunsen lamp. This technique is called sterilization combustion (flame sterilization). This technique should be done with extra caution because alcohol is very flammable.
Autoclave sterilization is the method by using water vapor pressure. The materials or tools that can be sterilized by autoclave include cotton cover tube, nylon sieves, lab clothing, plastic lid, glassware, pipettes, water, and the culture medium. Almost all of the microbes may die if diautoclave temperature 121oC at 15 psi pressure for 15-20 minutes.
• Sterilization of Media
There are two methods for sterilization of media commonly used, namely the autoclave and the filter membrane. Culture media, distilled water and a stable mixture can be sterilized in the autoclave by using a closed container with cotton, aluminum foil or plastic. However, the solution of the materials are not stable (heat-labile) must use a filter.
Diautoclave media generally at a pressure of 15 psi with a temperature of 121oC. For the volume of solution per container that small (<100 ml), the time required is 15-20 minutes, but for large quantities (2-4 liters) for 30-40 minutes. Melebhi not pressure from 20 psi may cause decomposition of carbohydrates and other ingredients in a medium that is thermolabile.
Several compounds belonging to the group of proteins, vitamins, amino acids, extracts tanama, hormonal and carbohydrates that are thermolabile there may be cause decomposition when sterilized by autoclave, so that should be sterilized with a filter. Millipore filter with a porosity ± 0.2 microns (μm) is one of the filters that are widely used for sterilization of materials that are thermolabile. Glassware that will accommodate the media should be sterilized with the filter was first sterilized by autoclave.
Media is part containing thermolabile components, can be made by: (i) a solution containing heat-stable components sterilized by autoclave, then cooled until the temperature of 50o-60oC in sterile conditions (usually in a laminar), (ii) in other parts of the state a sterile solution containing besifat thermolabile components sterilized with a filter, (iii) the two solutions that have been sterilized by different methods are combined in aseptic conditions.
• Sterilization of Plant Material
Obtain a sterile plant material is difficult. Although many precautions have been conducted, 95% will experience a culture eksplan contamination if not disinfected. Plant organ or tissue should be sterilized with disinfectant solution, because as a biological material can not be done by the extreme heat. The solution most widely used for sterilization of plant material can be seen in Table B-2.1. There is no standard method for sterilization eksplan, so the time soaking in a disinfectant solution is a range because it depends on the type of materials and plants that will be sterilized. The solution used must be safe for the network / eksplan but are able to kill contaminants, both bacteria and fungi.
For woody plants, bulbs etc.. usually before sterilized with disinfectant solution must be cleaned first with soap and rinsed with water, but not for herbaceous plant species. All surfaces should be eksplan which disteriliasi submerged in sterilan, and thereafter be rinsed with sterile akuades at least three times.
5) Determining pH
pH is measured by the concentration of hydrogen ions in solution. The pH scale ranging from 0 (very acidic) to 14 (very alkaline) and the scale of 7 is neutral point. pH of culture medium is generally regulated 0.1 ± 5.7 before diautoclave. pH can affect the solubility of ions in the media, so the ability to be a gel and further influence cell growth. Therefore, the accuracy of the pH of the media becomes an important factor Untk note. Generally, the media pH measurements using a pH meter.
1. Bhojwani, 1990. Plant Tissue Culture: Application & Limitations.
2. Jalil, M., N. Khalid & RY. Othman, 2003. Plant regeneration from embryogenic suspension cultures of Musa acuminata cv. Mas (AA). Plant Cell, Tissue and Organ Culture 75: 209-214, 2003.
3. Lee, S.W. 2003. Micropropagation of Cavendish banana in Taiwan. FFTC, Pingtung.
4. Morris, Scragg, Stafford & Fowler, 1986. Secondary Metabolism in Plant Cell Cultures
5. Pierik, 1987. In vitro Culture of Higher Plants.
6. Reinert & Yeoman, 1983. Plant Cell & Tissue Culture: A Laboratory Manual.
7. Rodrigues, I., 2007. Low-cos tissue culture technology. Center of Nuclear Energy for Agriculture (CENA). San Paulo.
8. Stafford & Warren, 1990. Plant Cell & Tissue Culture.
9. Taji, A., P. Kumar and P. Lakshamanan, 2002. In vitro plant breeding. The Haworth Press, Inc.. New York.
10. Trigiano, R.N. and D.J. Gray, 2000. Plant tissue culture concepts and laboratory exercises. 2nd EDT. CRC Press. London.
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Definitions and tissue culture applications
Plant tissue culture is a method or technique to isolate parts of plants (protoplasm, cells, tissues, and organs) and grow them on artificial media in aseptic conditions in a controlled space so that parts of these plants can grow and develop into complete plants. The use of tissue culture techniques in the beginning just to prove the theory of "totipotensi" ( "total genetic potential") is expressed by Schleiden and Schwann (1838) which states that the plant cell as the smallest unit can grow and thrive if kept in appropriate conditions. We have used tissue culture techniques not only as a means for studying aspects of plant physiology and biochemistry, but has developed into methods for various purposes such as:
• Mikropropagasi (micro propagation of plants)
Tissue culture techniques have been used to help produce crops in large scale through mikropropagasi or klonal propagation of various plants. Plant tissue in very small amounts can produce hundreds or thousands of plants continuously. This technique has been used in industrial scale in various countries to commercially produce various types of plants such as ornamental plants (orchids, cut flowers, etc..), Fruit crops (like bananas), crops and forestry industries (coffee, teak, etc.) . By using tissue culture methods, millions of plants with the same genetic characteristics can be obtained only from one eye buds. Therefore, this method becomes an alternative in the vegetative propagation of plants.
• Improved crop
In crop improvement efforts through the glorification of the conventional methods, to obtain pure strains can take six to seven generations of self-pollination or crosses. Through tissue culture techniques, can be obtained homosigot plants in a short time by producing haploid plants through pollen culture, anther or ovaries followed by chromosome doubling. Homosigot plants can be used as plant breeding material in order to improve the nature of the plant.
• Production of disease-free plants (virus)
Tissue culture technology has contributed in a plant that is free from viruses. In plants that have been infected with the virus, the cells in the bud tip (meristem) is an area that is not infected with the virus. In this way the meristem will mengkulturkan obtained virus-free plants.
• Genetic transformation
Tissue culture techniques have become an important part in helping the success of plant genetic engineering (gene transfer). For example, bacterial gene transfer (such as cry genes from Bacillus thuringiensis) into the plant cells will be expressed after transgeniknya achieved plant regeneration.
• The production of secondary metabolites, compounds
Plant cell culture can also be used to produce biochemical compounds (secondary metabolites) such as alkaloids, terpenoids, phenyl etc. propanoid. This technology is now available in industrial scale. For example, the commercial production of compounds "shikonin" from Lithospermum erythrorhizon cell culture.
History of tissue culture
The use of tissue culture techniques initiated by Gottlieb Haberlandt in 1902 in an attempt mengkulturkan hair cells of the leaf mesophyll tissue monocot plant. But the effort failed because the cells do not have cleavage, it was alleged failures because they do not use growth regulator substances needed for cell division, proliferation and induction of the embryo. In the year 1904, Hannig planting embryos isolated from several plant crucifers. In 1922, Knudson and separately each Robbin conduct investment business and culture of orchid seedlings root tip. After the 1920s, the discovery and development of tissue culture techniques continues. The following table shows the historical development of the field of plant tissue culture which was adapted from various sources
Important discoveries in the history of plant tissue culture
YEAR important findings
Schleiden & Schwann 1838 theorized Totipotensi
1902 Haberlandt:: The first person who tried to isolate and mengkulturkan monocot plant tissue, but failed
1922 Knudson: mengecambahkan orchid seeds
Blumenthal & Meyer 1924: The formation of callus from carrot roots eksplan
1929 Laibach & Hered: Culture of embryos to overcome the incompatibility in plants Linum spp.
Gautheret 1934: Culture in vitro of woody plant tissue kambium and shrubs, but failed.
White: Successful culture of tomato roots in a long time
Kogl et.al. : Identification of the first plant hormone, IAA, for the elongation of cells.
1936 LaRue: Culture of embryos in some plants Gymnospermae
1939 Gautheret: Successfully growing culture kambium carrot and tobacco plants
1941 Overbeek: The use of coconut water for the young embryo culture menumbuhkam Datura plants
1944 The first in vitro culture of tobacco plants to study the formation of buds adventif
Skoog and Tsui 1948: The formation of shoots and roots of tobacco adventif
1949 Nitsch: in vitro culture of fruit plants
1952 Morel & Martin:
meristem culture to obtain a free Dahlia plant viruses. The first success of micro-called grafting.
1953 Tulecke: Kalus haploid pollen plants of Ginkgo biloba
Miller 1955: The discovery of the structure and synthesis of kinetin
Skoog & Miller 1957: Menemuan that the formation of roots and shoots in comparison tergatung culture auksin: cytokines
1958 Maheswari & Rangaswamy: Regeneration of somatic embryos Citrus nuselus ovul
Reinert & Steward: Growth and development of carrot suspension cultures
Cocking 1960: enzymatic degradation of cell walls to get protoplas
Morel: Vegetative Propagation of orchids by meristem culture
1962 Murashige & Skoog: Development of MS medium
1964 Guha & Maheswari: The discovery of the first haploid plants by androgenesis Datura plant
Erickson & Jonassen 1969: Isolation protoplas of cell suspension Hapopappus
1970 Power: Figaro protoplas
Chilton 1977: The success of T-DNA integration in plants
Noguchi et al.: Planting of tobacco cells in Bioreaktor capacity of 20 000 L.
1978 Melchers et al.: Somatic hybridization between tomato and potato plants
Tabata et al.: Production of shikonin on the industrial scale cell culture
Zimmermann 1982: Figaro protoplas electrically (Electrofusion)
Mitsui Petrochemicals 1983: The first secondary metabolite production in industrial scale through suspension culture at the plant Lithospermum spp.
1985-1990 Development of gene transfer in rapidly growing crops, such as the use of Agrobacterium, particle bombardment (gene gun), electroporasi, mikroinjeksi.
1990 - Development of genetic and metabolic engineering challenges. growing rapidly
Marketing products of genetic engineering
1. Bhojwani, 1990. Plant Tissue Culture: Application & Limitations.
2. Gleba & Sytnik, 1984. Protoplast Fusion : Genetic Engineering in Higher Plants.
3. Green, Somers, Hacket & Biesboer, 1987. Plant Tissue and Organ Culture.
4. Pierik, 1987. In vitro Culture of Higher Plants.
5. Stafford & Warren, 1990. Plant Cell & Tissue Culture.
6. Taji, A., P. Kumar and P. Lakshamanan, 2002. In vitro plant breeding. The Haworth Press, Inc. New York.
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