Experimental principle
Flower tissue culture is the separation of a part of a flower plant body, such as stems, stem segments, leaves, flowers, immature embryos, etc., in a sterile test tube, and with certain nutrition, hormones, temperature, light and other conditions to make it complete Plant. Because its conditions can be strictly controlled and grow rapidly, 1-2 months is a cycle, so it has important application value in the production of flowers and plants.
In terms of rapid mass reproduction: it is widely used for some precious varieties of flowers that are difficult to reproduce and some flowers that are urgently needed in a short period of time. Orchids, chrysanthemums, gladiolus and other flowers use axillary buds to proliferate and obtain a large number of plants in a short time. African violets can achieve mass reproduction by inducing adventitious buds through the leaves and narcissus through the scales.
In terms of flower breeding: many flowers such as lily and iris can be remotely crossed. Due to physiological metabolism and other reasons, hybrid embryos are often aborted early, so hybrid plants cannot be obtained. The embryo culture in the test tube can make it grow smoothly and obtain distant hybrids. In addition, various methods such as callus mutagenesis and pollen culture can also be used for flower breeding.
In the cultivation of virus-free seedlings: a large number of flowers such as chrysanthemums, gladiolus, narcissus, tulips, and dahlias are propagated by asexual reproduction. However, the growth point of the 0.1-0.5 mm size of the flower plant is isolated, and the virus-free seedling is basically cultivated. Therefore, this technology has been widely used in the cultivation of flower-free virus seedlings.
Laboratory equipment
Laboratory
1) Chemical laboratory: mainly undertakes the task of preparing medium. It is required to have various chemical reagents, various glassware, and weighing scales.
2) Washing room: It is mainly used for washing glassware, which requires a tap water device and an oven for drying after washing.
3) Sterilization room: mainly sterilize the culture medium and utensils. Must have an autoclave, with water and power.
4) Inoculation room: It is the place where flower materials are separated, disinfected and inoculated and transferred. It is required to be sealed, clean, tidy, and equipped with ultraviolet lamps, which can be sterilized at any time. Some can also be replaced with inoculation boxes or ultra-clean workbenches.
5) Cultivation room: It is the place where flower materials are cultivated and grown. Requires cleanliness, good thermal insulation, uniform room temperature, and thermal insulation and fire resistance.
2. Equipment
1) Balance: For weighing medicines and hormones when preparing medium. Ordinary balances are used for a large number of elements; analytical balances are used for trace elements and hormones.
2) Acidity meter: for measuring the pH of the culture medium.
3) Autoclave: It is used for sterilization of culture medium and equipment.
4) Oven: used for drying and sterilizing washed glassware.
5) Distilled water manufacturing device: Obtain purified water for cultivation.
6) Refrigerator: for storing mother liquor and plant materials.
7) Inoculation box or ultra-clean workbench: an operation place for inoculating or transferring plant materials.
8) Air conditioner: for controlling room temperature.
Experimental Materials
The culture medium is a very important substrate in the tissue culture of flowers and plants. Currently, there are many kinds of applications, but the main components are roughly the same. The main component is water, and there are a large number of elements, trace elements, vitamins, growth regulators, sucrose and agar Wait.
At present, MS medium is the most widely used in flower tissue culture. Its composition is to add 1.65 g of ammonium nitrate, 1.9 g of potassium nitrate, 0.44 g of calcium chloride, 0.37 g of magnesium sulfate, 0.17 g of potassium dihydrogen phosphate, 0.83 mg of potassium iodide, and 5.2 of boric acid when preparing 1 liter (1000 ml) of medium Mg, magnesium sulfate 22.3 mg, zinc sulfate 3.6 mg, sodium molybdate 0.25 mg, copper sulfate 0.025 mg, cobalt chloride 0.025 mg, iron sulfate 27.8 mg, sucrose 30 g and agar 7 g. Other growth regulators should be determined according to the type of flower and the purpose of cultivation. The concentration of a large number of elements in the MS medium is too high. Therefore, 1/2 or 1/4 of the large element concentration is often used for cultivation, so that the growth effect is listed well.
Before preparing the medium, prepare flasks, test tubes, beakers, measuring cylinders, suction and other glassware, and weigh the medicines in advance. When preparing, first dissolve the agar, then add various nutrients and sucrose that are deeply dissolved in water, and then adjust the pH of the culture medium with sodium hydroxide or hydrochloric acid, generally around 5.7. Afterwards, it can be dispensed into the culture bottle and the cap is closed. The medium is prepared by autoclaving. After cooling, put it in the culture room for 3 days for pre-cultivation. Inoculate the flower material only if there is no contamination by bacteria.
Experimental procedure
1. Cut flower materials from fields or greenhouses, select a strong and disease-free mother plant, and take young and strong parts to facilitate growth.
2. Rinse with tap water for more than ten minutes. Brush off any mud. After rinsing, soak in 70% alcohol for 10-15 seconds to disinfect. Then rinse twice with sterile water (autoclaved distilled water), then soak in 10% bleached powder clear solution for 20 minutes for disinfection, and finally rinse with sterile water 3-4 times. Washing powder can be added to materials with fur that are not easy to wet and disinfect. The above operations should be carried out in sterile environment conditions such as inoculation boxes or ultra-clean workbenches. After sterilizing the surface, use sterile filter paper to absorb the water. Then use a scalpel to cut out the desired part, usually a few millimeters in size, and the virus-free vaccine should be less than 1 millimeter, and then use a scalpel or gun-type forceps to inoculate the material on the culture bottle. After the tool is used, it should be dipped in 95% alcohol or sterilized by flame sterilization to avoid cross contamination caused by the tool. Work clothes and work caps should be worn during operation, and hands should be washed in advance, and then wiped with alcohol cotton.
3. After inoculation, the flower material is put into the cultivation room for cultivation. The cultivation room is a place where flower materials are cultivated and grown, usually a few to ten square meters. The height is about 2 meters, and the space is small, which can save energy for temperature control. The culture material is put on the culture rack for cultivation. The culture rack can be made of wood or metal, with 4-5 layers, each layer is 40-50 cm high, and the fluorescent lamp is installed above. The shelf is about 1.2 meters long, consistent with the length of a 40-watt fluorescent lamp, and 80-90 cm wide. Two fluorescent lamps can be installed on each floor, so the illuminance during cultivation is about 3000 lux. Most of the temperature in the cultivation room adopts day and night constant temperature cultivation, which is kept at 25 ℃ ± 2 ℃, and some adopt day and night temperature-changing cultivation. The temperature at night can be lower, which should be determined according to the needs of flower growth. Fluorescent lighting for 12-16 hours per day.
4. Flower test tube seedlings generally provide good conditions for growth in various aspects. They generally have good growth and development and many root systems. However, the survival rate during transplanting is not high due to the lack of grasping its characteristics. This is because the test tube seedlings are cultivated under high humidity in the bottle, artificially provide carbon sources such as sucrose, and the flower materials are in a heterotrophic living state, which can be said to be much more delicate than the flowers grown in the greenhouse. Suddenly moved from the bottle to the soil to let it live an autotrophic life, often caused damage or even death due to too drastic changes. For this reason, when the flower test tube seedlings start to be removed, they should still be covered with a glass bottle or covered with a film bag (a few small holes are opened) and removed after 1 week. Spray conditions are more ideal. Shade for the first 7-10 days, then gradually see the sun. It is better to use half of sand and vermiculite as the transplanting matrix. It should be well drained and ventilated. Pouring nutrient solution every other day. Step by step exercise in this way, let it adapt to the environment. After 2-3 weeks, the seedlings can be moved to the cultivation soil and planted after acclimation.
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