When selecting the basic medium, in addition to the genotype of the plant to be cultured, the type of the medium, the total ion concentration, the total nitrogen level, and the nitrogen source type (nitrate nitrogen and ammonium nitrogen) and ratio, calcium and chlorine should generally be considered. The content of the compound, etc. Herbaceous tissue is cultured in widely used MS medium, while woody plant tissue culture is usually carried out using a low salt basic medium such as 1/2 or 1/4 MS or WPM (Woody Plant Medium). In addition to the total ion concentration, the total nitrogen level, the nitrogen source type (nitrate nitrogen and ammonium nitrogen) and the proportion also have a great influence on the tissue culture. Therefore, changing the nitrogen source type and concentration of the basic medium according to the specific conditions can improve the culture effect. [15~17].
Different genotypes may require different basic media, but the same media will be required for the same plant at different stages of culture or for different culture purposes. Usually, according to the culture stage and purpose, it can be divided into start-up or induction medium, proliferation medium, rooting medium, etc. This division may be only the adjustment of hormones, or the change of certain components of the same basic medium, or even basic culture. The difference in the base type. For example, most plants use 1/2~1/4 MS or other low-salt medium such as White medium in rooting culture. Somatic embryogenesis often changes the type and proportion of nitrogen source at different stages. The type of basic medium may also affect the effect of growth regulating substances, so it is necessary to consider the interaction between different basic medium and different concentrations of growth regulating substances.
Usually, before carrying out tissue culture on a plant, firstly, the relevant data of plant tissue culture of the same genus, the same species or the same species should be searched, and the research results of the predecessors should be referred to. Unable to find the relevant information, you can start some experiments with some famous basic media such as MS, WPM or B5. If the screening effect of the existing basic medium is not satisfactory, it can be improved based on basic medium such as MS, WPM or B5. The improvement methods generally include the following:
(1) Halving the bulk or inorganic salt components of the basic medium to three-quarters (1/2, 1/3, 2/3, 1/4, or 3/4), or different minimal medium a combination of components such as combining the inorganic salt component of B5 with the organic component of MS;
(2) increase or decrease the level of nitrogen source and the type and proportion of ammonium and nitrate nitrogen (ammonium salt and nitrate). The nitrogen source usually has a great influence on the quality and development route of the culture;
(3) increasing calcium salts to improve calcium deficiency in woody plant culture;
(4) Adding a variety of vitamins, amino acids and organic additives, which is more effective for plants with complex nutritional requirements or difficult to cultivate;
(5) Measuring the content of nutrients in the soil and the body of the plant, and providing reference for the improvement of the basic medium;
(6) The inorganic salt, organic components, auxin and cytokinin are divided into three levels of high, medium and low, and 81 (34) combinations are tested, which is more accurate. In summary, the basic culture medium appropriate to the specific requirements of a given plant material can be optimized and selected by media quality (ingredients) and quantity (concentration) adjustments.
Frequently altered factors in plant tissue culture media are growth regulators, especially auxin and cytokinin. The type, amount and proportion of auxin and cytokinin are usually determined by experience or the nature and function of the hormone. But the more scientific approach is to use a one-factor test and a multi-factor test (orthogonal design) so that the results can be analyzed using biometric methods. For example, based on a minimal medium, auxin (such as NAA) and cytokinin (such as BA) are at 9 levels (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mg). /l) Combine into 81 treatments to obtain the best combination of auxin and cytokinin concentration levels. Although the orthogonal design is scientific and reasonable, considering the interaction between the two types of hormones, the workload is large.
In practice, fixed auxin concentrations can also be used to change the cytokinin species or concentration, and the optimal cytokinin species and concentration can be selected; and auxin can be selected by the same method. Or sometimes to reduce the workload, can be representatively formulated in a small number of combinations (such as a relatively high concentration of auxin, a high concentration of cytokinin, a balance of auxin and cytokinin phase ), first determine which type is more suitable, and then use the single factor or multi-factor test method to further determine the optimal hormone combination according to a suitable category. The same is true for the method of determining the optimum factors such as light and temperature.
Preparation, sterilization and disinfection:
First, the purpose of the experiment
Understand the preparation principle of the medium; master the general methods and procedures for preparing the medium; understand the basic principles and methods of common sterilization and cleaning; master the operation methods of dry heat, high-pressure steam sterilization and filtration sterilization.
Second, the experimental principle
The medium is a mixture of nutrients required for the growth and reproduction of microorganisms and the synthesis of metabolites, artificially formulated in a certain ratio. The raw materials of the medium can be classified into a carbon source, a nitrogen source, an inorganic salt, a growth factor, and water. Depending on the type of microorganism and the purpose of the experiment, the medium also has different types and preparation methods.
Beef extract peptone medium is one of the most widely used and most common bacterial basal media, sometimes referred to as normal media. Since this medium contains the most basic nutrients required for general cell growth and reproduction, it can be used for the growth and reproduction of microorganisms.
The method of killing microorganisms is mainly to denature the protein by heating to achieve the effect of killing microorganisms.
Third, reagents and equipment
Equipment
Test tubes, flasks, beakers, measuring cylinders, glass rods, media, dispensers, balances, horn spoons, autoclaves, pH paper, cotton, kraft paper, markers, twine, gauze, straws, petri dishes , electric oven, syringe, microporous membrane filter, tweezers, etc.
2. Reagents beef extract, peptone, NaCl, agar
Fourth, the experimental content
1. Weighing → Dissolving → Adjusting pH → Filtration → Dispensing → Plugging → Bandaging → Sterilization → Sterility Inspection
2. Dry heat sterilization: loading the items to be sterilized → warming → constant temperature → cooling → unpacking
3. High-pressure steam sterilization: adding water → loading items → capping → heating → discharging cold air → pressurizing → constant pressure → reducing pressure back to zero → exhausting steam → taking material → sterilizing inspection
4. Filtration and sterilization: assembly sterilization → connection → pressure filtration → sterility inspection → cleaning sterilization
Five, key steps and precautions
1. It must be prepared in strict accordance with the formula.
2. Do not overdo the pH.
3. Dry heat sterilization should pay attention to the items should not be piled too tight, pay attention to the time control of temperature, discharge and take objects below 70oC.
4. High-pressure sterilization should pay attention to not too much items, remove the cold air after heating, and then reduce the pressure to zero.
5. Filtration and sterilization should pay attention to the sterilization of various components. When pressure filtration, the pressure should be appropriate, not too fast and too fast, and the filter should be cleaned and stored.
Different genotypes may require different basic media, but the same media will be required for the same plant at different stages of culture or for different culture purposes. Usually, according to the culture stage and purpose, it can be divided into start-up or induction medium, proliferation medium, rooting medium, etc. This division may be only the adjustment of hormones, or the change of certain components of the same basic medium, or even basic culture. The difference in the base type. For example, most plants use 1/2~1/4 MS or other low-salt medium such as White medium in rooting culture. Somatic embryogenesis often changes the type and proportion of nitrogen source at different stages. The type of basic medium may also affect the effect of growth regulating substances, so it is necessary to consider the interaction between different basic medium and different concentrations of growth regulating substances.
Usually, before carrying out tissue culture on a plant, firstly, the relevant data of plant tissue culture of the same genus, the same species or the same species should be searched, and the research results of the predecessors should be referred to. Unable to find the relevant information, you can start some experiments with some famous basic media such as MS, WPM or B5. If the screening effect of the existing basic medium is not satisfactory, it can be improved based on basic medium such as MS, WPM or B5. The improvement methods generally include the following:
(1) Halving the bulk or inorganic salt components of the basic medium to three-quarters (1/2, 1/3, 2/3, 1/4, or 3/4), or different minimal medium a combination of components such as combining the inorganic salt component of B5 with the organic component of MS;
(2) increase or decrease the level of nitrogen source and the type and proportion of ammonium and nitrate nitrogen (ammonium salt and nitrate). The nitrogen source usually has a great influence on the quality and development route of the culture;
(3) increasing calcium salts to improve calcium deficiency in woody plant culture;
(4) Adding a variety of vitamins, amino acids and organic additives, which is more effective for plants with complex nutritional requirements or difficult to cultivate;
(5) Measuring the content of nutrients in the soil and the body of the plant, and providing reference for the improvement of the basic medium;
(6) The inorganic salt, organic components, auxin and cytokinin are divided into three levels of high, medium and low, and 81 (34) combinations are tested, which is more accurate. In summary, the basic culture medium appropriate to the specific requirements of a given plant material can be optimized and selected by media quality (ingredients) and quantity (concentration) adjustments.
Frequently altered factors in plant tissue culture media are growth regulators, especially auxin and cytokinin. The type, amount and proportion of auxin and cytokinin are usually determined by experience or the nature and function of the hormone. But the more scientific approach is to use a one-factor test and a multi-factor test (orthogonal design) so that the results can be analyzed using biometric methods. For example, based on a minimal medium, auxin (such as NAA) and cytokinin (such as BA) are at 9 levels (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mg). /l) Combine into 81 treatments to obtain the best combination of auxin and cytokinin concentration levels. Although the orthogonal design is scientific and reasonable, considering the interaction between the two types of hormones, the workload is large.
In practice, fixed auxin concentrations can also be used to change the cytokinin species or concentration, and the optimal cytokinin species and concentration can be selected; and auxin can be selected by the same method. Or sometimes to reduce the workload, can be representatively formulated in a small number of combinations (such as a relatively high concentration of auxin, a high concentration of cytokinin, a balance of auxin and cytokinin phase ), first determine which type is more suitable, and then use the single factor or multi-factor test method to further determine the optimal hormone combination according to a suitable category. The same is true for the method of determining the optimum factors such as light and temperature.
Preparation, sterilization and disinfection:
First, the purpose of the experiment
Understand the preparation principle of the medium; master the general methods and procedures for preparing the medium; understand the basic principles and methods of common sterilization and cleaning; master the operation methods of dry heat, high-pressure steam sterilization and filtration sterilization.
Second, the experimental principle
The medium is a mixture of nutrients required for the growth and reproduction of microorganisms and the synthesis of metabolites, artificially formulated in a certain ratio. The raw materials of the medium can be classified into a carbon source, a nitrogen source, an inorganic salt, a growth factor, and water. Depending on the type of microorganism and the purpose of the experiment, the medium also has different types and preparation methods.
Beef extract peptone medium is one of the most widely used and most common bacterial basal media, sometimes referred to as normal media. Since this medium contains the most basic nutrients required for general cell growth and reproduction, it can be used for the growth and reproduction of microorganisms.
The method of killing microorganisms is mainly to denature the protein by heating to achieve the effect of killing microorganisms.
Third, reagents and equipment
Equipment
Test tubes, flasks, beakers, measuring cylinders, glass rods, media, dispensers, balances, horn spoons, autoclaves, pH paper, cotton, kraft paper, markers, twine, gauze, straws, petri dishes , electric oven, syringe, microporous membrane filter, tweezers, etc.
2. Reagents beef extract, peptone, NaCl, agar
Fourth, the experimental content
1. Weighing → Dissolving → Adjusting pH → Filtration → Dispensing → Plugging → Bandaging → Sterilization → Sterility Inspection
2. Dry heat sterilization: loading the items to be sterilized → warming → constant temperature → cooling → unpacking
3. High-pressure steam sterilization: adding water → loading items → capping → heating → discharging cold air → pressurizing → constant pressure → reducing pressure back to zero → exhausting steam → taking material → sterilizing inspection
4. Filtration and sterilization: assembly sterilization → connection → pressure filtration → sterility inspection → cleaning sterilization
Five, key steps and precautions
1. It must be prepared in strict accordance with the formula.
2. Do not overdo the pH.
3. Dry heat sterilization should pay attention to the items should not be piled too tight, pay attention to the time control of temperature, discharge and take objects below 70oC.
4. High-pressure sterilization should pay attention to not too much items, remove the cold air after heating, and then reduce the pressure to zero.
5. Filtration and sterilization should pay attention to the sterilization of various components. When pressure filtration, the pressure should be appropriate, not too fast and too fast, and the filter should be cleaned and stored.
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