Antibiotics in Plant Tissue Culture Technique
What is plant tissue culture?
Plant tissue culture refers to a technology in which certain tissues, cells or organs are removed from plants under sterile conditions and cultured in artificially formulated nutrient media to develop into complete plants or specific tissues. As an important part of modern biotechnology, plant tissue culture has shown great potential in plant breeding, genetic improvement, rapid propagation and bioreactor application. However, in the process of tissue culture, the pollution of seedlings is the most troublesome problem.
Contamination of plant tissue culture
In tissue culture experiments, the pathogens of pollution are divided into two categories: bacteria and fungi. The main characteristics of bacterial contamination are the appearance of slime or water marks on the medium or explants during culture, sometimes requiring careful identification. This symptom indicates that the contamination may be bacterial and can be detected within 1 to 2 days of inoculation. The identification method of fungal contamination: After inoculation, white, black, green and other colors of massive fungal spores appeared on the surface of the medium or explants, which spread quickly and could be found after 3 to 10 days of inoculation. Adding antibiotics to plant tissue culture media can effectively control this kind of pollution.
Antibiotics in plant tissue culture
The use of antibiotics is particularly critical. Antibiotics can effectively prevent the pollution of microorganisms such as bacteria and ensure the healthy growth of cells. At the same time, antibiotics can also be used to screen and retain specific genetically modified cell populations, playing an important role in transfection, gene editing and other experiments. The rational use of antibiotics not only helps to maintain the stability of cell culture, but also ensures the repeatability and accuracy of experimental results, providing a reliable experimental guarantee for researchers.
The antibacterial spectrum of each antibiotic is different, and different plants show different sensitivity to antibiotics. An antibiotic may be very toxic to some plants, while it may be very toxic to other species. Therefore, before using antibiotics, the sensitivity of the sample relative to antibiotics should be pre-tested to determine the optimal concentration of drug use and the optimal time of action, and then the formal experiment should be carried out.
In general, antibiotics need to be stored in the refrigerator. Aminoglycoside antibiotics (such as kanamycin) are hygroscopic and should be stored in a dryer. All antibiotics should be kept out of direct sunlight. Rifampicin B is very sensitive to light and should be stored in the dark. Most antibiotics are sensitive to heat and need to be filtered and sterilized, it is best to use them on the spot to minimize the number of repeated freezing and thawing of the solution to prevent the failure of antibiotics.
Antibiotics for plant tissue culture at BOC Sciences
Antibiotics for microbial infection
In plant tissue culture, antibiotics are used to remove or inhibit the growth of microorganisms. For example, the antibiotics penicillin, chloramphenicol and rifamequal are used for this purpose. These antibiotics are able to inhibit or interfere with the synthesis of certain important proteins, thereby inhibiting the growth of microorganisms.
Carbenicillin is a white to off-white, hygroscopic powder that is soluble in water. Carbenicillin is most effective against gram-negative bacteria, but it also has some effect against Gram-positive bacteria. It has been reported that carbenicillin solution is stable at 35 ℃ and pH 5.5 for 40 hours. The solution can be stored at -20 °C for 24 months. Carbenicillin is relatively low toxic to a wide variety of plants, and it has been reported that concentrations of up to 1000 mg/L can be used in plant tissue cultures.
Cefotaxime sodium is a white to off-white powder that is soluble in water. A change in the color of a newly prepared solution does not necessarily indicate a change in potency. Store in a light-proof, airtight container. The aqueous solution of cefotaxime sodium is most stable in the pH range of 4.3-6.2. The solution can be stored at -20 °C for 48 months. Cefotaxime sodium is most effective against gram-negative bacteria.
Hygromycin B is an aminoglycoside antibiotic that is effective against both prokaryotic and eukaryotic microorganisms and cells. The recommended concentration range is 10-400 µg/mL. The stock solution can be stored at 2-6 ℃ for at least 1 year; The solution should not be frozen, as this will reduce its potency.
Temetine has a wide antibacterial range against gram-positive bacteria, gram-negative bacteria, aerobic bacteria and anaerobic bacteria. Suitable for the empirical treatment of a wide range of bacterial infectious diseases. In scientific research, it is often used to inhibit the growth of Agrobacterium, especially in the embryogenic callus regeneration system, and can achieve good antibacterial and regenerative effects.
Aureobasidin A, also known as AbA, basifungin, and brachymycin A, is a cyclic ester peptide antibiotic isolated from the Aureobasidium pullulans No. R106 filamentous fungus. Aureobasidium A is a cyclic peptide and antifungal antibiotic. It is an inhibitor of inositol phosphorylated ceramide synthetase AUR1.
Antibiotics for transgenic plant screening
Screening agents are used for the screening and detection of transgenic plants, and the selection of such antibiotics can only be determined by the resistance genes carried by the transgenic plants (carrier resistance). Currently commonly used screening agents are kanamycin, G418 (geneticin), hygromycin B, spectinomycin, gentamycin and so on. Among them, kanamycin is the earliest widely used screening agent in plant genetic engineering. Its essence is aminoglycoside antibiotics to inhibit or interfere with the synthesis of some important proteins, and the concentration is generally not more than 50 mg/L. Hygromycin B is a strong cell growth screening agent and can also be highly toxic to many plants, so care should be taken to choose the appropriate screening agent to avoid the negative effects.
G418, an analogue of neomycin, kills cells by inhibiting genes for transposons Tn601 and Tn5, interfering with ribosome function and thus blocking protein synthesis. However, neomycin has no effect on eukaryotic cells, and G418 acts on both bacteria and eukaryotic cells, mainly for the selection of mammalian, plant or yeast cells. Its resistance gene is a dominant phenotype, and it is localized on Tn601 (903) and Tn5 transposons, and has been widely used in gene transfer, gene knockout, resistance screening and so on.
