Tissue Culture techniques of Flowers

1. The application value of flower tissue culture flower tissue culture is to separate a part of flower plants, such as stems, stem segments, leaves, flowers, immature embryos, etc., in aseptic test tube, combined with certain conditions such as nutrition, hormone, temperature, light and so on. Make it produce a complete plant. Because its conditions can be strictly controlled and grows rapidly, 1-2 months is a cycle, so it has important application value in the production of flowers and plants. Rapid mass propagation: it is widely used for some precious flowers which are difficult to breed and some flowers which are in urgent need of production in a short time. Orchids, chrysanthemums, gladiolus and other flowers use axillary buds to proliferate and get a large number of plants in a short time. African violets can produce adventitious buds through leaves and daffodils to produce adventitious buds to achieve the purpose of mass reproduction. In the aspect of flower breeding, lilies, irises and many other flowers can carry out distant hybridization. due to physiological metabolism and other reasons, hybrid embryos are often aborted at the early stage, so hybrid plants can not be obtained. However, the embryo culture in test tube can make it grow smoothly and obtain distant hybrids. In addition, many methods such as callus mutagenesis and pollen culture can be used for flower breeding. In the aspect of cultivating virus-free seedlings, a large number of flowers such as chrysanthemum, gladiolus, daffodils, tulips and dahlias propagate by asexual reproduction, and the virus is transmitted and accumulated from generation to generation, and the harm is becoming more and more serious. On the other hand, when the growing points of 0.1-0.5 mm in size were isolated, the virus-free seedlings were basically obtained. Therefore, this technique has been widely used in the cultivation of virus-free seedlings of flowers.

2. Requirements for laboratory and equipment of flower tissue culture Flower tissue culture is a new technology of modern industrial production of flowers, which is cultivated under the condition of artificial control, so it has certain requirements for laboratories and equipment. ① chemistry laboratory: mainly responsible for the task of preparing culture medium. It is required to have all kinds of chemical reagents, glassware, weighing balance and so on. ② washing room: mainly for glassware washing, requiring a tap water device and an oven for drying after washing. ③ sterilization room: mainly disinfect the culture medium and utensils. There should be a high-pressure sterilizer with water and power. ④ inoculation room: it is the place where flower materials are separated, sterilized, inoculated and transferred. It is required to be airtight, clean, neat and equipped with ultraviolet lamp, which can be sterilized at any time. Some can also be replaced by inoculation boxes or ultra-clean worktables. ⑤ culture room: is the place where flower materials are cultivated and grown. It requires cleanliness, good heat preservation, uniform room temperature, thermal insulation and fireproof performance. Equipment ① balance: used for weighing drugs and hormones when preparing culture medium. An ordinary balance is used for a large number of elements, and an analytical balance for trace elements and hormones. ② acidity meter: used to determine the pH of the culture medium. ③ autoclave: it is used for sterilization of culture medium and instruments. ④ oven: used for drying and sterilization of washed glassware. ⑤ distilled water manufacturing device: pure water is obtained for culture. ⑥ refrigerator: for storing mother liquor and plant materials. ⑦ inoculation box or ultra-clean workbench: an operating place for inoculation or transfer of plant materials. ⑧ air conditioner: used to control room temperature.

3. Requirements of culture medium for flower tissue culture medium is a very important substrate in flower plant tissue culture. At present, there are many kinds of media, but their main components are roughly the same, and the main components are water. There are also a large number of elements, trace elements, vitamins, growth regulators, sucrose and Agar. At present, MS medium is most widely used in flower tissue culture. Its composition is when preparing 1 liter (1000 ml) culture medium Add 1.65g ammonium nitrate, 1.9g potassium nitrate, 0.44g calcium chloride, 0.37g magnesium sulfate, 0.17g potassium dihydrogen phosphate, 0.83mg potassium iodide, 5.2mg boric acid, 22.3mg magnesium sulfate, 3.6mg zinc sulfate, 0.25mg sodium molybdate, 0.025 mg copper sulfate, 0.025 mg cobalt chloride, 27.8mg ferric sulfate, 30g sucrose and 7g Agar. Other growth regulators should be determined according to the type of flowers and the purpose of cultivation. The concentration of a large number of elements in MS medium is too high, so it is common to use the concentrations of a large number of elements in 1 beat 2 and 1 hand 4 as culture, so that the growth effect is good. Before preparing the culture medium, prepare the triangular flask, test tube, beaker, measuring tube, suction and other glassware and weigh the medicine in advance. During the preparation, the Agar was dissolved, and then various nutrient elements and sucrose deeply hydrolyzed in water were added, and then the pH of the medium was adjusted with sodium hydroxide or hydrochloric acid. In the future, it can be injected into the bottle and cover the bottle cap. The culture medium should be sterilized under high pressure. After cooling, it can be pre-cultured in the culture room for 3 days, if there is no miscellaneous bacteria pollution, the flower materials can be inoculated.

4. the process of flower tissue culture flower plant tissue culture is aseptic culture, that is, the material is required to be free of miscellaneous bacteria. When cutting flower materials from fields or greenhouses, the healthy and disease-free mother plants should be selected, and the parts with young and strong meristematic ability should be selected to facilitate growth. Although the materials have been selected, there are always a lot of miscellaneous bacteria on the outside. For this reason, surface sterilization should be carried out before inoculation. Usually rinse with tap water for more than ten minutes, and those with mud should be brushed away. After rinsing, soak in 70% alcohol for 10-15 seconds. Then rinse twice with sterile water (autoclaved distilled water), then soak in 10% bleach clarification solution for 20 minutes, and finally rinse with sterile water for 3-4 times. Some laundry detergent can be added to materials with furry hair that are not easily wetted and sterilized. The above operations should be carried out in aseptic environment conditions such as inoculation box or ultra-clean workbench. After surface sterilization of the material, use sterile filter paper to absorb the water. Then cut the desired part with a scalpel, usually a few millimeters in size, and cultivate virus-free seedlings less than 1 millimeter, and then inoculate the material on the culture bottle with an anatomical needle or gun tweezers. Tools should be dipped in 95% alcohol or sterilized by flame disinfection to avoid cross-contamination caused by bacteria. When operating, you should wear overalls and work caps, wash your hands in advance, and then try again with alcohol cotton.