First, the five components that make up the culture medium
At present, most culture media are composed of inorganic nutrients, carbon sources, vitamins, growth regulators and organic additives.
1. Inorganic nutrients
Inorganic nutrients are mainly composed of macroelements and microelements. Among a large number of elements, nitrate nitrogen or ammonium nitrogen is usually used as nitrogen source, but nitrate nitrogen is mostly used in culture medium, and some of it is mixed with ammonium nitrogen. Phosphorus and sulfur are usually provided by phosphates and sulfates. Potassium is the main cation in culture medium, and its quantity in modern culture medium tends to increase gradually. However, the requirements of calcium, sodium and magnesium are less. The sodium and chlorine required by the culture medium are provided by calcium salts, phosphates or micronutrients. Trace elements include iodine, manganese, zinc, molybdenum, copper, cobalt and iron. Most of the iron ions in the culture medium exist in the form of chelated iron, that is, the mixture of FeSO4 and Na2—EDTA (chelating agent).
2. Carbon source
The photosynthesis of cultured plant tissues or cells is weak. Therefore, it is necessary to add some carbohydrates to the culture medium to meet the demand. The carbohydrate in the culture medium is usually sucrose. Sucrose is not only used as carbon source and energy source in culture medium, but also plays an important role in maintaining osmotic pressure of culture medium.
vitamin
Adding vitamins to culture medium is often beneficial to the development of explants. Vitamins in the culture medium belong to B vitamins, among which vitamin B 1, vitamin B6, biotin, calcium pantothenate and inositol are the best.
4. Organic additives
Include synthetic or natural organic additives. The most commonly used are casein hydrolysate, yeast extract, coconut milk and various amino acids. In addition, agar is also the most commonly used organic additive, which is mainly used as the support of culture medium to make the culture medium in a solid state, which is beneficial to the culture of various explants.
5. Growth regulators
Commonly used growth regulators generally include the following three categories:
(1) auxin. Such as indoleacetic acid (IAA), naphthylacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d).
(2) Cytokinin. Such as luteolin (Zt), 6- benzylpurine (6-BA or BAP) and kinetin (Kt).
(3) gibberellin. There is only one gibberellin used in tissue culture, namely gibberellic acid (GA3).
Second, the commonly used culture medium formula
1. Common medium formula
The success of tissue culture depends largely on the choice of medium. Different media have different characteristics and are suitable for different plant species and inoculation materials. When carrying out tissue culture activities, it is necessary to understand and analyze various media in order to select and use them. The formulas of several media commonly used in tissue culture are shown in the following table 9- 1.
The types and quantities of hormones in the culture medium vary with different culture stages and materials, so they are not included in each formula.
2. Characteristics of several common culture media
(1) MS is medium. At present, MS medium is widely used. It contains high concentration of inorganic salts, which is very beneficial to ensure the mineral nutrition needed for tissue growth and accelerate the growth of callus. Because of the high ion concentration in the formula, even if some components are slightly different in the process of preparation, storage and disinfection, it will not affect the balance between ions. MS solid medium can be used for callus induction, or for embryo, stem segment, stem tip and anther culture, and its liquid medium has achieved remarkable success in cell suspension culture. The amount and proportion of inorganic nutrients in the culture medium are sufficient to meet the nutritional and physiological needs of plant cells. Therefore, in general, there is no need to add organic additives such as amino acids, casein hydrolysate, yeast extract and coconut juice. Compared with the basic components of other media, the contents of nitrate, potassium and ammonium in MS media are higher, which is its obvious characteristic.
(2)B5 medium. The main feature of B5 medium is low ammonium content, because ammonium may inhibit the growth of many cultures. It was found that some plant calli and suspension cultures grew better on MS medium than on B5 medium, while others were more suitable for growth on B5 medium.
(3)N6 medium. N6 medium is especially suitable for anther and pollen culture of cereal plants and has been widely used at home and abroad. In tissue culture, While(While, 1963) medium and Nisch (Nitsch, 195 1) medium are often used. Their basic components are similar. White medium has low inorganic salt content, which is more suitable for tissue culture of woody plants.
There are many other media, which can be determined according to the cultivated plants.