培养基中的矿质元素

1、刘彤石河子大学生命科学学院,1,Nutrient Media for Plant Tissue Cultures,刘彤 石河子大学生命科学学院,刘彤石河子大学生命科学学院,2,One of the first decisions that must be made when developing a tissue culture system is what medium to use. Nutrient media for plant tissue culture are designed to allow plant tissues to be maintained in a totall

2、y artificial environment.,刘彤石河子大学生命科学学院,3,Functions of medium,Provide water Provide mineral nutritional needs Provide vitamins Provide growth regulators Access to atmosphere for gas exchange Removal of plant metabolite waste,4,A. Macroelements consist of N, K, P, Ca, Mg, and S.,1. Nitrogen (N) - Nit

3、rogen is essential for plant growth. Most inorganic nitrogen is converted to amino acids and then to proteins. Nitrogen is typically added to plant nutrient media as the nitrate ion (NO3-, oxidized) and/or the ammonium ion (NH4+, reduced), which are added as inorganic salts. Inorganic nitrogen gener

4、ally ranges from 25-60 mM in nutrient media. In devising media, both the total amount of nitrogen as well as the relative amounts of NO3- and NH4+ are important. There are usually lower levels of NH4+ than NO3- in medium; nitrate is usually added at concentrations between 25 and 20 mM and ammonium a

5、t concentrations between 2 and 20 mM.,刘彤石河子大学生命科学学院,5,2. Potassium (K) - Potassium is the major ion in plants with a positive charge, balancing negative ions. Although the amount of potassium required varies widely among different species, in media potassium concentration is generally correlated wit

6、h that of nitrate and ranges between 20-30 mM.,刘彤石河子大学生命科学学院,6,3. Phosphorous (P) - Phosphorus is an integral part of nucleic acids and other structural compounds. It is added to culture medium as phosphate (PO4-) in sodium or potassium hydrogen phosphates in concentrations ranging from 1-3 mM. 4. C

7、alcium (Ca) - Calcium is a co-factor of many enzymes and is particularly important in cell wall synthesis. It is supplied mostly as calcium chloride or calcium nitrate, concentrations ranging between 1 and 3 mM. In plant cultures, calcium deficiency may result in shoot tip necrosis.,刘彤石河子大学生命科学学院,7,

8、5. Magnesium (Mg) - Magnesium is critical for the functioning of enzymes, is an integral component of the chlorophyll molecule, and is a cation that balances negative ions. It is usually added as magnesium sulfate in concentrations similar to that of calcium. 6. Sulfur (S) - Sulfur is a part of seve

9、ral amino acids and has an important function in protein structure. It is supplied as the SO4- ion, generally with magnesium as the cation, in concentrations ranging from 1-3 mM.,刘彤石河子大学生命科学学院,8,B. MicronutrientsFe, Mn, Zn, B, Cu and Mo, Co, and I.,1. Iron (Fe) - Iron is necessary for chlorophyll sy

10、nthesis and functions in many oxidation/reduction reactions. It is generally present in media at approximately 1 M. The major problem in supplying iron in vitro is that it forms insoluble compounds in alkaline pH, a problem that is particularly evident in liquid culture, where it may be seen as a pr

11、ecipitate. The use of chelating agents, which bind metal ions, makes iron more stable and available to plant tissues over wider pH ranges. Although there are several of these, the sodium or potassium form of ethylenediaminetetraacetic acid (EDTA) is most often used because it is not as toxic as othe

12、r chelating agents and it enables iron to be available to cultures over a wider pH range than other agents. Fe-EDTA may be purchased as a salt or prepared from ferric sulfate and EDTA.,刘彤石河子大学生命科学学院,9,2. Manganese (Mn) - Manganese is required for enzyme reactions, particularly in respiratory and pho

13、tosynthetic processes and is usually added as manganese sulfate in concentrations of 5-30 M. 3. Zinc (Zn) - Zinc is also required in many enzyme activities and is added to medium in concentrations similar to that of manganese. The most common form in which zinc is added is as the sulfate salt. 4. Bo

14、ron (B) - Boron is an essential element involved in lignin biosynthesis and metabolism of phenolic acids and is supplied as boric acid in culture medium (25-100 M). Boron deficiency results in the death of shoot tip meristems.,刘彤石河子大学生命科学学院,10,5. Copper (Cu) - Copper is critical in many enzyme react

15、ions, including the cytochrome oxidase system. It is added to culture medium (as cupric sulfate) in very low concentrations (0.1 M), because high amounts can be toxic. 6. Molybdenum (Mo) - Molybdenum functions in the transformation of nitrate to ammonium. It is added as sodium molybdate in low conce

16、ntrations (1 M) in culture medium. 7. Cobalt (Co) - Cobalt is not considered to be an essential mineral by plant physiologists, but is included in many of the most widely used media formulations. Cobalt is supplied in concentrations similar to that of copper, again because it may be toxic at higher

17、concentrations. 8. Iodine (I) - Iodine is not considered to be an essential element, but it is often added to plant culture media (5 M) because it has been found to improve growth of roots and callus in vitro.,刘彤石河子大学生命科学学院,11,II. Organic Compounds,Organic compounds are also added to plant culture m

18、edium. Some of these compounds, such as sugars, are absolutely needed for growth, while others, such as vitamins, undefined compounds, and organic acids, may not be essential but may enhance growth.,刘彤石河子大学生命科学学院,12,A. Sugars,Most plant tissue cultures are not highly autotrophic, that is, capable of

19、 fixing carbon through photosynthesis, due to limitations in culture of CO2 availability, among other factors. Therefore, sugar is added to the medium as an energy source. Sucrose is the most common sugar added, although glucose, fructose, and sorbitol are also used in certain instances. Sucrose is

20、the sugar form most commonly transported in plants; it is broken down into glucose and fructose during metabolism. It is also partially hydrolyzed into glucose and fructose during autoclaving. The concentration of sugars in nutrient media generally ranges from 20 to 40 g/l. Sugars also act as an osm

21、oticum in the medium. Osmotic potential can have an important effect on in vitro response. Nutrient salts contribute from 20% to 50% of the osmotic potential of media, with sucrose making up the rest. When sucrose is hydrolyzed, as during autoclaving, its contribution to the osmotic potential is fur

22、ther increased.,刘彤石河子大学生命科学学院,13,B. Vitamins,Only thiamine (vitamin B1), which is required for carbohydrate metabolism and the biosynthesis of some amino acids, has been shown to be essential for most plant cultures. Nicotinic acid (niacin) and pyridoxine (B6) are also commonly added to Murashige an

23、d Skoog medium and some other media. Other vitamins such as biotin, folic acid, ascorbic acid (vitamin C), and vitamin E (tocopherol) are sometimes added to media formulations. Vitamin concentrations are generally very low.,刘彤石河子大学生命科学学院,14,C. Myo-inositol Myo-inositol, a sugar alcohol, is added to

24、most plant culture media. Although not essential for culture viability, it can significantly improve in vitro response, especially in monocots. Although myo-inositol is not essential for growth of many plant species, its effect on growth is significant.,刘彤石河子大学生命科学学院,15,D. Complex organics,These are

25、 undefined supplements such as coconut milk, coconut water, yeast extract, fruit juices and fruit pulps. They may supply amino acids, vitamins, plant growth regulators, and/or secondary plant metabolites. Complex organics were frequently used early in the history of plant tissue culture, when growth

26、 requirements were less defined. Now they are used only when no combination of defined components supports growth. Their disadvantages are that the important compounds in them are not known, and may vary greatly from batch to batch. Only protein hydrolysates and coconut milk (at 5-20% v/v) are used

27、much today.,刘彤石河子大学生命科学学院,16,III. Activated Charcoal,Activated charcoal is sometimes added to media in order to adsorb toxic compounds released by plant tissues, particularly oxidized phenolics. It may be especially useful in rooting medium. However, activated charcoal adsorbs not only toxic compoun

28、ds, but also growth regulators and other compounds that are added to the medium. Activated charcoal is usually acid-washed prior to addition to the culture medium at a concentration of 0.5-3.0 %.,刘彤石河子大学生命科学学院,17,IV. Solidifying Agents,Solidifying agents are used to create semi-solid or solid media

29、wherein plant cultures are not submerged in the medium. Liquid medium can be used for many plants but it must usually be agitated to provide sufficient oxygen to the tissue.,刘彤石河子大学生命科学学院,18,A. Agar Agar is the most commonly used gelling agent. Marine red algae contain the structural polysaccharide

30、agar, which consists of 2 components, agarose and agaropectin. Agarose is composed of alternating D-galactose and 3,6-anhydro-L-galactose with side chains of 6-methyl-D-galactose residues. Agaropectin is like agarose but additionally contains sulfate ester side chains and D-glucuronic acid. The tert

31、iary structure of agarose is a double helix with a so-called threefold screw axis.,刘彤石河子大学生命科学学院,19,B. Agarose When greater purity is needed, agarose may be used. Agarose is extracted from agar leaving behind agaropectin and its sulfate groups. Because of the additional purification, agarose is cons

32、iderably more expensive than agar. Agarose also has higher gel strength than agar and thus less is required for solidification of media. Agarose is used in situations where the impurities of agar are a major Disadvantage, such as in protoplast culture.,刘彤石河子大学生命科学学院,20,C. Gelrite 脱乙酰吉兰糖胶 Gelrite con

33、sists of a polysaccharide produced by the bacterium Pseudomonas elodea. Medium solidified with Gelrite has the advantage of being clear, which agar-solidified medium is not. Consequently contamination is more easily detected at an early stage. Impurities in Gelrite contain inorganic ions, but no org

34、anic compounds. Gelrite requires more stirring than agar when being added to media. Unlike agar, Gelrite cannot be reheated and gelled successfully. One limitation of Gelrite is that the concentration of divalent cations such as calcium and magnesium ions must be within the range of 4-8 mM/liter. Co

35、ncentrations of these two ions either less than or greater than this range result in the media not gelling. Gelrite may also produce hyperhydric plants when used at low concentrations.,刘彤石河子大学生命科学学院,21,D. Phytagel Phytagel is an agar substitute produced from a bacterial substrate composed of glucuro

36、nic acid葡糖醛酸, rhamnose and glucose. It produces a clear, colorless, high-strength gel, which aids in detection of microbial contamination. Phytagel provides an economical alternative to agar as a gelling agent. It is used at a concentration of 1.5-2.5 g/L. To prevent clumping, Phytagel should be added to rapidly stirring culture medium which is at room temperature. Hyperhydricity may also be a pr