(1) Improve environmental conditions. The inoculation room and culture room should be disinfected and purified regularly, and the workbench or inoculation box should be opened for more than 30min before inoculation. The relative humidity of the culture room should be controlled at about 70%. When the relative humidity is too high, dehumidifier can be used to dehumidify.
(2) The training of vaccinators is very important, and aseptic operation should be strictly carried out. Tools needed for inoculation must be strictly disinfected before use. Don't put too much material in the operation area of the clean workbench to avoid airflow obstruction. Also regularly check the working quality of clean workbench [8].
(3) Strictly check the inoculation materials. Eliminate inoculation materials contaminated by fungi and bacteria.
(4) Check the sterilization quality of the sterilizer frequently, and repair it immediately when problems are found. The pressure gauge of the sterilizer can't be taken out immediately after it drops to zero. Due to the negative pressure effect caused by the action of cold and hot air, the cold air in the external environment is sucked back into the sterilized culture bottle, causing fungal pollution. In order to avoid this phenomenon, the sterilized culture bottle should be taken out of the pot after the pot is slightly cooled [9].
(5) Check whether there is any problem with the culture container. Culture containers are usually sealed with plastic covers, rubber plugs, cotton plugs, films, etc. Plastic covers are prone to aging and poor sealing when used for a long time, which will also cause pollution. Lin Sheng et al. formulated a "No.4 disinfectant" to disinfect the bottle mouth, which can control the contamination rate of the culture medium below 0.3% [10].
Prevention and control of pollution in subculture
The sterile materials obtained in the initial stage are sterile in theory, but they will also be contaminated in the later stage or subculture. Besides careless operation, they may also be contaminated by fungi spread by mites in the culture room. This kind of pollution can be prevented from two aspects: (1) There should be reasonable procedures for expanding reproduction. After obtaining the original sterile culture products, some of them should be preserved as "original seeds", and then mass reproduction and re-inspection should be carried out before mass production can be provided. (2) Antibacterial agents can be added to the culture medium to prevent [3]. Xu Wanfang and others used carbendazim to inhibit bacteria and promote growth in tissue culture of Anoectochilus roxburghii, and the effect was obviously better than metalaxyl, thiophanate-methyl and their mixtures. Anoectochilus roxburghii grown in the medium containing carbendazim was not contaminated by microorganisms, and the sterilization rate was 100%, with no albino seedlings and strong seedlings [1 1].
Endophytic bacteria in plants have deep potential and cannot be eliminated by surface disinfection. Sometimes, in the initial culture of explants, including the subculture of previous generations, it is not easy to be detected by naked eyes on the culture medium. With the increase of subculture times, the amount of bacteria gradually accumulated and developed, and then appeared on the culture medium [12]. Huang Xiaorong and others think that the cause of bacterial contamination in plant tissue culture is the result of dormant bacterial spore germination [13]. The pollution of endophytic bacteria such as bacteria can be prevented and reduced by adding antibiotics to the culture medium, cultivating stem tips and lowering PH value.
Faye et al. added 200mg/L penicillin GK during the rapid propagation of colored taro to the 4th-5th generation, which can effectively inhibit the growth of endophytes without affecting the propagation coefficient [14]. In the tissue culture of ivy, Zhai Jianzhong and others used streptomycin to inhibit the endophytic bacterium Xanthomonas. Its effect is better than gentamicin and cefazolin sodium. The simultaneous use of these two antibiotics in culture medium is beneficial to prevent bacteria from producing drug resistance and control the development of bacterial pollution for a long time. However, the increase of streptomycin dose will cause toxicity to plants [15].
It is very important to choose the appropriate concentration when adding antibacterial agents to the culture medium. Low concentration will have poor effect, while high concentration will easily poison plants, affect proliferation or blacken cultured materials, resulting in dead seedlings and albino seedlings. The combination of two or more antibiotics can prevent and control the drug resistance of bacteria. Antibiotics and carbendazim are generally not resistant to high temperature and need to be filtered and sterilized, so it is not convenient to add them in production. Sodium benzoate, potassium sorbate and sodium propionate are commonly used preservatives in food, which are resistant to high temperature and high pressure and convenient to use in tissue culture production.
Endophytes in plant materials can also be removed by repeated shoot tip culture. Because the distribution of pathogenic bacteria in plants is uneven, the meristem of shoot tip is sterile through vascular bundle transmission. Through repeated shoot tip culture, both endophytic bacteria and viruses can be removed.
Except Erwinia, most bacteria can't grow when the PH of the medium is less than 4.5. In the tissue culture of Zi Yuan, Iris and Rosa, adjusting the PH value of the culture medium from 5.8 to 3.9 ~ 4.3 can prevent a lot of bacterial pollution [7]. Hu Qing and others were cultured in water with low PH value (3. 10), and the ventilation condition of the container was improved, which could make the Hulk block with serious bacterial pollution proliferate normally and was harmless to the growth of a single plant. The proportion of rooting seedlings is higher than that of conventional solid culture, which basically solves the problem of bacterial production after serious bacterial pollution [16].
For contaminated cultures, sometimes it takes a lot of time because of the scarcity or repeated appearance of plant materials, and larger plants can also be cut and disinfected and inoculated again. Li et al. soaked the bacterial contaminated seedlings in tissue culture of Populus alba with 4 million or 6 million units /L penicillin sterile aqueous solution for 60min or 40min respectively, which could effectively prevent bacterial contamination in tissue culture. When the treated seedlings are subcultured, there is no repeated bacterial pollution and the seedlings have strong differentiation ability. When rooting, the root system is developed and the survival rate of transplanting is high [17]. Mars et al. proved that if there is only fungal pollution in subculture, it can be eliminated by soaking in 3% carbendazim sterile solution for more than 0.5h, then inoculating with sterile water or directly inoculating without washing with sterile water. If bacteria, bacteria and fungi are contaminated at the same time, the sterile seedling system can be re-established by HgCl2 disinfection method. The contaminated seedlings are cut into long stems as explants, washed with tap water for 0.5h, and then treated with ethanol and HgCl2 for a short time on the ultra-clean bench [18].
4. Reduce the organic components in the culture medium
The existence of organic matter in culture medium is an important cause of pollution, and removing organic matter is also a way to reduce pollution. In the tissue culture of pistachio, Song Fenghui and others removed organic components (such as VB 1, VB6, nicotinic acid, etc. ) In the culture medium, after 2 ~ 3 generations of culture, it can inhibit the growth of bacteria and has no effect on the growth and proliferation of clustered buds. The reason is that these are all necessary substances for the growth of some bacteria. After removing these organic substances, bacteria can't grow and develop, and the death gradually decreases [19]. Sugar-free tissue culture and rapid propagation of plants initiated by Professor Toyuki Kozai in Japan completely removes the organic components in the culture medium, inputs a controllable amount of CO2 gas as a carbon source, and promotes the photosynthesis rate of plants by controlling environmental factors, so that plants can be transformed from heterotrophic to autotrophic, and the plants can grow well and the pollution rate is obviously reduced [20]. Because of the removal of sugar, the culture of tissue culture seedlings has changed from glass bottles to box containers, and the whole culture room can also be used as a culture container, even without strict aseptic operation, with little pollution. This technique is very successful at least in the rooting promotion stage of many plant tissue culture seedlings.
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