Key Technical Points and Detailed Operation Methods of Flower Tissue Culture

1. Application Value of Flower Tissue Culture Flower tissue culture is the separation of a part of a flower plant, such as stems, stem segments, leaves, flowers, immature embryos, etc., and culturing them in a sterile test tube with certain nutrients, hormones, temperature, light and other conditions to produce complete plants. Because its conditions can be strictly controlled and growth is rapid, with 1-2 months as one cycle, it has important application value in the production of flower plants. In terms of rapid mass propagation: it is widely used for some rare and valuable flower varieties that are difficult to propagate and for some flowers urgently needed in large quantities within a short period. Orchids, chrysanthemums, gladioli and other flowers use axillary bud proliferation to obtain large numbers of plants in a short time. African violets can be induced through leaves, and narcissus through scales, to produce adventitious buds, thereby achieving the purpose of mass propagation. In flower breeding: lilies, irises and many other flowers can undergo distant hybridization, but due to reasons such as physiological metabolism, the hybrid embryos often abort early, so hybrid plants cannot be obtained. However, embryo culture in test tubes can allow them to grow smoothly and obtain distant hybrids. In addition, methods such as callus mutagenesis and pollen culture can also be used for flower breeding. In cultivating virus-free seedlings: a large number of flowers such as chrysanthemums, gladioli, narcissus, tulips and dahlias are propagated by vegetative propagation, and viruses are transmitted and accumulated from generation to generation, making the harm increasingly serious. By isolating 0.1-0.5 mm growing points of flower plants and culturing them, basically virus-free seedlings are obtained. Therefore, this technique has been widely used in the cultivation of virus-free flower seedlings.

2. Requirements of Flower Tissue Culture for Laboratories and Equipment Flower tissue culture is the cultivation of flowers under artificially controlled conditions and is a new technology for modern factory-style flower production, so it has certain requirements for laboratories and equipment. In terms of laboratories: ① Chemistry laboratory: mainly responsible for preparing culture medium. It requires various chemical reagents, various glassware, balances, etc. ② Washing room: mainly for washing glassware, requiring tap water facilities and also an oven so that items can be dried after washing. ③ Sterilization room: mainly for disinfecting culture medium and utensils. It needs an autoclave and must have a water source and power supply. ④ Inoculation room: a place for separating, disinfecting, inoculating and transferring flower materials. It requires being sealed, clean and tidy, equipped with ultraviolet lamps, and able to sterilize at any time. In some cases, an inoculation box or ultra-clean workbench can also be used instead. ⑤ Culture room: a place where flower materials are cultured and grown. It requires cleanliness, good heat preservation, uniform room temperature, and heat insulation and fireproof performance. In terms of equipment: ① Balance: used for weighing chemicals and hormones when preparing culture medium. Ordinary balances are used for macroelements; analytical balances are used for microelements and hormones. ② pH meter: used for measuring the pH of the culture medium. ③ Autoclave: used for sterilizing culture medium, instruments and utensils. ④ Oven: used for drying and sterilizing washed glassware. ⑤ Distilled water production device: to obtain pure water for culture use. ⑥ Refrigerator: used for storing stock solutions and plant materials. ⑦ Inoculation box or ultra-clean workbench: an operation site for inoculating or transferring plant materials. ⑧ Air conditioner: used for controlling room temperature.

3. Requirements of Flower Tissue Culture for Culture Medium Culture medium is a very important substrate in flower plant tissue culture. At present, many kinds are used, but their main components are broadly the same. The main component is water, and other components also include macroelements, microelements, vitamins, growth regulators, sucrose and agar, etc. At present, MS medium is the most widely used in flower tissue culture. Its composition is that when preparing 1 liter (1000 milliliters) of culture medium, add 1.65 grams of ammonium nitrate, 1.9 grams of potassium nitrate, 0.44 grams of calcium chloride, 0.37 grams of magnesium sulfate, 0.17 grams of potassium dihydrogen phosphate, 0.83 milligrams of potassium iodide, 5.2 milligrams of boric acid, 22.3 milligrams of magnesium sulfate, 3.6 milligrams of zinc sulfate, 0.25 milligrams of sodium molybdate, 0.025 milligrams of copper sulfate, 0.025 milligrams of cobalt chloride, 27.8 milligrams of ferric sulfate, 30 grams of sucrose and 7 grams of agar. Other growth regulators should be determined according to the type of flower and the purpose of culture. The concentration of macroelements in MS medium is too high, so 1/2 or 1/4 macroelement concentrations are often used for culture, and the growth effect is better in this way. Before preparing the culture medium, prepare Erlenmeyer flasks, test tubes, beakers, graduated cylinders, pipettes and other glassware, and weigh the chemicals in advance. During preparation, first dissolve the agar, then add the various nutrient elements and sucrose dissolved in water, then use sodium hydroxide or hydrochloric acid to adjust the pH of the culture medium, generally keeping it at about 5.7. After that, it can be dispensed into culture bottles and the bottle caps covered. After the culture medium is prepared, it must undergo autoclaving. After cooling, place it in the culture room for pre-culture for 3 days. Only if there is no contamination by miscellaneous microorganisms can inoculation of flower materials be carried out.

4. Process of Flower Tissue Culture Flower plant tissue culture is sterile culture, which means the cultured material is required not to carry miscellaneous microorganisms. When cutting flower materials from fields or greenhouses and other places, healthy mother plants free of diseases and pests should be selected, and young parts with strong meristematic ability should be taken to facilitate growth. Although the collected materials have been selected, there are still inevitably many miscellaneous microorganisms on the outside. For this reason, surface sterilization should be carried out before inoculation. Usually, first rinse with tap water for more than ten minutes, and brush off any soil. After rinsing clean, soak in 70% alcohol for 10-15 seconds for disinfection. Then rinse twice with sterile water (autoclaved distilled water), then soak in 10% clarified bleaching powder solution for 20 minutes for disinfection, and finally rinse 3-4 times with sterile water. For materials with fuzz that are not easily moistened and disinfected, a little washing powder can be added. The above operations should all be carried out under sterile environmental conditions such as an inoculation box or ultra-clean workbench. For materials that have undergone surface sterilization, use sterile filter paper to absorb the water. Then use a scalpel to cut the required part, usually several millimeters in size; for cultivating virus-free seedlings, it should be below 1 millimeter, and then use a dissecting needle or forceps to inoculate the material onto the culture bottle for culture. After the tools are used, they should immediately be dipped in 95% alcohol, or sterilized by flame sterilization, to avoid cross-contamination caused by tools carrying microorganisms. During operation, wear work clothes and a work cap, wash hands beforehand, and then wipe them once with an alcohol cotton pad.