How to choose the best culture scheme
When the theoretical content of plant tissue culture is applied to specific plants and experimental conditions faced by everyone, there will be some problems, such as how to coordinate all links, how to choose some optimal conditions, how to judge whether the work situation is normal, how to overcome adverse effects, how to induce the culture to develop in the direction we hope, and so on. The following will introduce some methods to solve the above problems, that is, asking questions, designing experiments, and finding answers through the analysis of experimental results. In most cases, many experimental methods should be used flexibly in combination with their own work experience.
Before introducing the selection of each single factor, briefly explain the commonly used testing methods.
(A), the commonly used test methods in plant tissue culture
1, preliminary test
If you are interested in a certain plant, or have doubts about a certain factor, or think of similar problems in your own experiment after reading a paper, you can simply draw up a plan to roughly judge whether this factor has an impact. ○2 If yes, what is its approximate degree and quantity? The above two problems can be judged by roughly testing, and basically can achieve the expected purpose. Then, we can follow the path of preliminary test, design a relatively complete experimental scheme, obtain accurate and reliable experimental data and deepen our understanding of the problem.
The requirements for the initial test are relatively low, and it is not necessary to think carefully. The requirements are strict and often a brainwave. Once the conditions of plant tissue culture are met, you can do a lot of work, just put it there and observe the reaction of the culture, and then do it when there is a reaction. Usually, the more experienced people are, the more accurate their judgments are. It is because the hit rate of preliminary experiments is higher, and the breakthrough of scientific research can be found as soon as possible, thus expanding the results.
The preliminary test is small in scale and the conditions do not need to be comprehensive. Doing more preparatory experiments when energy permits can speed up the progress. The negative results of the preliminary test are also very useful information, which may indicate that the problem is not in this respect, or that the problem is not simple, and will also deepen the research ideas. In short, the preparation of the experiment consumes less time and energy, and the problem is straightforward, which can make the next stage of work more confident. Many formal studies often need to do some preparatory experiments to find out the influencing factors and their levels.
2. Single factor test
In the single factor experiment, all the conditions and factors are maintained at the general level, only one factor is changed, and the influence and degree of this factor on the experiment are found out. For example, in MS medium, other components and dosage are unchanged, and only the dosage of NAA varies between 0, 0. 1, 0.5,1.0 mg1,so as to find out the effect of NAA dosage on rooting of a culture. This is a single factor experiment. Single factor experiments should be the same or as close as possible except one variable to be studied, and the experiment should be arranged very simply. Change one factor at a time and record the whole situation in detail. Usually, after the initial test, a more comprehensive single factor test can be arranged to determine the quantitative relationship between the factor and the test results.
There are many differences between biological experiments and physical or chemical experiments. The most important and significant difference is that biological experiments need to set up a control group and an experimental group. The experimental group can have one or several groups, and the control group can have multiple groups with the increase of the complexity of the experiment. It is required that the experimental individuals in the control group and the experimental group, that is, plant tissue pieces or other cultures, must be as identical as possible in heredity, physiological state and pre-culture conditions. This is easier to do in tissue culture. This is because there are too many variables in biological research objects, and many things are difficult to be recorded intuitively and converted into numbers. The source of test materials is impure and irregular. If it changes in the future, it will be impossible to determine whether it is due to the influence of test factors or the result of differences between materials. The control group usually did not use test factors or maintained the original culture conditions.
In each treatment project, a certain number of experimental materials are usually used, so there must be a certain number of repetitions. According to the scale and requirements of the experiment, most projects should have 4- 10 bottles, and each bottle should have at least 3 cultures or 3 seedlings. The quantity and quality of these materials should be as consistent as possible. The more repetitions, the more careful the experiment and the more reliable the results. The test results obtained should be properly treated mathematically, so as to objectively judge the accuracy and credibility of the test results.
3. Multi-factor experiment and orthogonal design
In recent years, with the help of modern statistics and other mathematical methods, it has become possible to design complex experiments to test several factors at the same time. This has shown obvious advantages, that is, saving time and energy, while providing the effect of several variables in interaction. For example, with orthogonal design and L9 table, four factors, three levels of experiments and one * * * experiment with nine different combinations can be arranged, and the result is equivalent to 27 experiments with different combinations. Although orthogonal experiment is a combination of many factors, in the analysis of experimental results, the role of each factor can be clearly shown. Therefore, the test results of a system can clearly analyze the problems and gain multiple gains in a limited time. In the study of plant rapid propagation, it can be used to explore the appropriate dosage of several components in the culture medium, such as cytokinin, auxin, sugar and so on. When the culture has practical and reliable indicators that are easy to observe and calculate, even when quantitative treatment is carried out, multi-factor experiments can play a role in promoting the test process and get twice the result with half the effort. Such as seedling number, seedling height, root number, root length, transplanting survival rate and so on.
In the absence of the above-mentioned clear countable indicators, such as the growth state, growth amount and differentiation potential of callus, we can intuitively observe according to experience, use the method of evaluation and scoring, convert the state and quality indicators into numbers, and then make statistical processing. Strictly speaking, the above indicators can be quantified after careful determination and analysis. For example, the growth of callus can be weighed as an empty bottle (with culture medium) and then weighed after inoculation. The difference between the two weights is the initial weight of the initial inoculum. After a period of culture, take out the culture (because it is inconvenient to weigh aseptically, if the culture is not preserved, you can weigh it directly), transfer it to fresh culture medium with a weighing bottle, and then weigh it in weighing bottle to find out the final weight of the culture. The difference in initial weight is the growth of the culture. You can also set up a control and weigh the dry weight. It can be seen that the procedure is very troublesome. When it comes to differentiation status and potential, practical quantitative indicators can also be determined by preparing slices and observing them under a microscope, but the above process can only be used for valuable theoretical research. In most application projects, it is too complicated and has no practical value. Judging the scoring method by experience, the error coefficient is too large. Therefore, in this experiment without specific numerical indicators, it is best to use single factor experiments to illustrate the problem. Otherwise, the effects of various factors are intertwined, which often makes the results unexplained.
4, gradually increase and gradually cancel the test method.
In the study of plant tissue differentiation and regeneration, various organic nutrients are often added before reliable differentiation and regeneration, and after stable regeneration, these components can be gradually reduced. The purpose of gradual addition is to make the experiment successful, and the purpose of gradual reduction is to narrow the scope to find the most influential factors, or to simplify the culture medium in order to reduce the cost and facilitate popularization. This simple method of addition, subtraction, multiplication and division is often used in seeking the best hormone ratio.
(2) Selection of main culture conditions and influencing factors
1, selection of basic medium
MS is the most basic medium among the successful plant species in vitro propagation. So in general culture, you can try it first. If it is found that it has adverse effects or is not ideal, if the basic medium is to be improved, it is best to reduce the concentration of MS medium first. Secondly, other media with significantly different composition from MS media can be selected for trial comparison, such as B5, White and Nitsch media. Usually, trace elements and iron salts do not need to be made into many kinds, just according to the formula of MS trace elements, which can be used in other culture medium components. The concentration of mixed trace elements is generally per liter of culture medium 1 ml or 5 ml. Trace elements can be reduced or even omitted when stable differentiation and proliferation are achieved. The dosage of trace elements in the formula is also quite different. For example, the dosage in ER medium is only110 in MS formula. The organic components in the culture medium change the most, so it is not necessary to stick to the requirements of a certain formula. When encountering indistinguishable materials, it is often to increase the complexity of the culture medium, constantly add nutrients or physiologically active substances that may be promising, and then gradually subtract them after successful culture. Therefore, it may be found that some components are actually irrelevant factors to promote the test results. But some of them helped. When the experiment is unsuccessful, the total addition is better than no addition, and the amount of most organic components should not be too high, such as biotin, which is generally 0.01-0.1mg/1. Some organic substances are essential substances for the growth and differentiation of plant tissues and are electron transporters in metabolism. It can be synthesized by itself in the whole plant, but in vitro, due to the sharp increase of wound area, metabolic consumption is enhanced, synthesis is reduced or unable to be synthesized, or supply is insufficient. So even if it is rarely added, it is different whether it is added or not.
Examples of selecting basic media: The clustered seedlings of Chinese rose were inoculated on MS, B5 and White media respectively, and the components of these three media were the same except the basic inorganic salt formula. After 1 month of culture, a little change can be seen. Continue to cultivate once in the second month and once again in the third month. In this way, at the end of the third month or the beginning of the fourth month, we can see the effects of these three substrates on the growth and appreciation of Chinese rose seedlings. Among them, MS medium is the best, with fast growth and high proliferation of seedlings. B5 is average; White medium is the worst, with slow growth and low proliferation rate. The test results show that MS is 100%, B5 is about 86%, and White is about 73%. In this way, MS medium can be selected as the basic condition for in vitro propagation of Chinese rose. In this example, the three media are the control group, or the MS-treated media is the control group.
2. Selection of plant hormone ratio
According to the data accumulated in plant tissue culture literature, it is the most important link for plant tissue differentiation and rapid seedling growth to select the types of plant hormones and adjust the dosage of cytokinin and auxin. Figuratively speaking, this is the most sensitive knob.
After selecting or temporarily determining a basic medium, or at the same time, the proportion of plant hormones is usually the first consideration. For example, MS medium is used, BA is used for cytokinin and NAA is used for auxin. Dosage: BA is tentatively set at 2mg/ 1, NAA is tentatively set at 0. 1 or 0.2mg/ 1. Now, such a medium is usually written as MS+Ba2+NAA 0. 1-0.2. The culture temperature is 24-26℃ and the sunlight 10 hour. This culture medium and conditions have successfully differentiated and added hundreds of plants that are easy to regenerate, so we might as well give it a try first.
After a period of culture, the amount of phytohormones in the next medium can be modified according to the performance of most cultures and a few tissue blocks. This process is to collect the "opinions" of the culture on the culture medium and culture conditions. What problems the various manifestations of culture explain and how to adjust them will be introduced in detail later.
There may be more than one initial test, such as: MS+BA 1+NAA0. 1, MS+BA3+NAA0. 1, etc. If the culture reaction is not ideal, we can consult and refer to the culture conditions of other species in similar families, genera and genera.
The selection of different hormone dosage can also be carried out through two groups of single factor experiments: the first group finds out the appropriate dosage of BA, and the second group finds out the appropriate dosage of NAA.
The first group is1-1ms+ba1+NAA 0.1.
1-2ms +BA2+NAA0. 1
1-3ms +BA4+NAA0. 1
In this group of experiments, the dosage of NAA is unchanged, but the influence of BA on culture should be observed.
The second group is 2-1ms+ba2+naa0.05.
2-2 MS+BA2+NAA0. 1
2-3 MS+BA2+NAA0.5
In this group of experiments, the dosage of BA remained unchanged, and the effect of NAA on the culture was observed.
A simple experiment like this doesn't require both British Airways and NAA to change at the same time. Judging from the test results, we can choose one or two better treatments, or we can predict the combination that did not appear in the last two groups of tests, and the test needs to be adjusted before continuing. For example, in the above experiment, 1-2 is better, and 2-3 is better. When the experiment is repeated, the hormone ratio of MS+BA2+NAA0.5 can be selected.
You can also pre-select tests with large changes in measurement level, which means that the screened "sieve hole" is thicker and the detection range is wider. For example, BA 1, 3,5 mg/1; NAA was 0.05, 0.5 and 5 mg/1; Or 0.0 1, 0. 1, 1, 5.0mg/ 1 and so on. After the experimental results are clear, the best combination is to design a new combination and reduce the order of magnitude. After two or three rounds of experiments, the best hormone combination will be found.
For many plants, such an experiment is enough. Under special circumstances, if the problem cannot be solved, other cytokinins such as KT, ZT, 2-ip and other auxins such as IAA, IBA and 2,4-D can be further selected, and multi-factor experiments can be arranged by orthogonal test and optimization method. After careful and patient experiments, the encirclement was gradually narrowed, and finally the appropriate types and dosage of cytokinin and auxin were found, so that the plants could be propagated smoothly and at high speed.
It should be remembered that all experiments should be repeated 2-3 times on the same culture medium to confirm their effects. If a small amount of test materials are circulated on completely different media, the test results cannot be analyzed and real data cannot be provided. The best way is to increase the workload, start over, disinfect more samples, and carefully test and observe.
3. Choice of sugar concentration
The optimum sugar concentration was determined by single factor test or orthogonal test combined with the selection of plant hormones. Usually the range of choices is very small. For most plants, the suitable concentration is 20 or 30g/ 1, and in some cases it is 40g/ 1. In anther culture, the difference is large, sometimes 70- 150g/ 1.
4. Selection of 4.pH value
General plants are not strict about PH value, such as camellia, rhododendron, bougainvillea, etc., and the PH value can be reduced to 5.4 or 5.0. When trying new cultivated species, we can refer to similar plants published in the past. By using the single factor test that only changes the PH value, the suitable PH value can be quickly known. For the first time, try PH5.0, 5.4, 5.8 and 6.2. After the first result comes out, fine-tune it again to determine the best PH value.
In the process of culture, the PH value of culture medium will change with the consumption of nutrients, so when the order of magnitude difference is too small, the difference of experimental results is not obvious. As long as the growth and appreciation of the culture can meet the requirements of rapid propagation, it is not necessary to be too careful.
5. Temperature and lighting
In the absence of special equipment, the selection of temperature and lighting requirements does not need to be too detailed. Using the top, middle and bottom of the culture rack, culture bottles and thermometers are placed respectively. After a period of culture, the temperature range needed by plants can be compared. The demand for light intensity can be compared by the distance between plants and light sources. There is no special need to study the photoperiod, and most experts think that 10- 14 hour illumination is necessary.
Conditional units can buy light incubators. This device can accurately control the daily illumination time, that is, the periodic changes of illumination and darkness can be arbitrarily controlled, and the illumination intensity can be adjusted by the number of lamps. Equipped with heating and cooling devices, it can be adjusted to any range of+1℃. Light incubator is an ideal equipment to study the effects of vernalization treatment and photoperiod on plant development. Generally, the humidity in the tank can only be adjusted, but this relationship is not very important, because the humidity in the culture bottle is generally 100%. If used properly, under such experimental conditions, several most important factors that can affect the growth and development of plants have been artificially controlled. This is an ideal device that can be accurately controlled at the micro-whole level, the tissue or organ level and the cell level. With this light incubator, you can easily and quickly find out the best temperature and light conditions for rapid propagation of a certain plant.
6. Finalization of comprehensive factors
After finding the best conditions of each single factor, these best conditions are combined to carry out a comprehensive experiment. According to the experimental results, the experimental work can be ended and the research results can be popularized and applied to large-scale rapid propagation.