Sterilization method for increasing tissue culture starting rate of Litsea coreana Levl. var. lanuginose (Migo) Yanget P. H. Huang explants
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A sterilization method and tissue culture technology are applied in the field of sterilization to increase the initiation rate of tissue culture of Leopard skin camphor explants, which can solve the problems of cumbersome steps, increased sterilization time, and easy browning, so as to reduce pollution rate, increase the start-up rate, facilitate the effect of promotion
Active Publication Date: 2019-12-31
CHONGQING ACADEMY OF FORESTRY SCI
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However, the pollution problem in plant tissue culture is one of the three major problems currently plaguing tissue culture work
During the process from the primary culture of explants to the subculture, due to the bacteria in the plant itself or improper operation methods, endophytic bacteria pollution, operation pollution, and environmental pollution are prone to occur. Once the tissue culture materials are contaminated, it is difficult to rescue. The general practice It is a waste of a lot of manpower, material and financial resources, and it will delay production and cause serious losses.
At present, the research on pollution control is mostly biased towards the control and physical prevention of antibiotics. Antibiotics have a certain degree of pertinence for pollution prevention and control, but procedures such as filtration and addition are required, and the steps are cumbersome; if the antibiotics are autoclaved with the medium, the sterilization The effect will decrease again, and the prevention and control effect is not ideal
If the conventional tissue culture sterilization method is followed, the contamination rate of the explants is high, and increasing the sterilization time can effectively reduce the contamination rate of the explants, but 1‰ mercury liter shock sterilization for 4 minutes will lead to different degrees of emergence of shoots of Cinnamomum camphora If the alcohol cleaning time is too long, browning will easily occur, and the body also contains a large amount of phenols, quinones and other substances, which are very prone to browning and reduce the start-up rate; if the sterilization time is reduced, the explants start up faster, But the pollution rate is very high
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Embodiment 1
[0034] (1) Step 1: In vivo disinfection. Check the weather forecast, select continuous sunny days, cut off the old leaves that were not born in the current year on the branches, leaving only the buds, and spray 1‰ carbendazim on the living body, once in the morning and once in the evening, for 3 consecutive days.
[0035] (2) Step 2: Cleaning of foreign matter on the surface. Take the shoots that have been sprayed with too much bacterazim, first rinse off the dust on the surface of the shoots with clean water, then put in 1‰ washing powder solution, and keep stirring until all the foreign matter attached to the surface of the shoots is removed, and then rinse with clean water .
[0036] (3) Step 3: In vitro disinfection. First peel off the young leaves on the surface of the shoots, then soak the shoots in 1% pvp sterile solution for 10 minutes, then transfer them to running water, rinse with large water for 10 minutes, then switch to small water for 20 minutes, and finally t...
Embodiment 2
[0043]Step 1: Choose sunny weather, cut off the old leaves that were not born in the same year on the branches of Maobaopi camphor, leaving only the buds, and spray 1‰ of carbendazim on the living body, once in the morning and in the evening.
[0044] All the other steps are the same as in Example 1.
[0045] The pollution and explant start-up within 30 days after inoculation were counted, and the statistics were made every 1 day.
Embodiment 3
[0047] (1) Step 1: Choose continuous sunny weather, cut off the old leaves that were not born in the current year on the branches of the leopard skin, leaving only the buds, and spray 1‰ of carbendazim on the living body, once in the morning and once in the evening, continuously Spray for 5 days.
[0048] All the other steps are the same as in Example 1.
[0049] The pollution and explant start-up within 30 days after inoculation were counted, and the statistics were made every 1 day.
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Abstract
The invention discloses a sterilization method for increasing the tissue culture starting rate of Litsea coreana Levl. var. lanuginose (Migo) Yanget P. H. Huang explants, and belongs to the technicalfield of tissue culture. The sterilization method includes the steps: (1) spraying carbendazim solution onto branch living bodies with tender shoots to sterilize the branch living bodies; (2) pickingthe tender shoots, cleaning up the tender shoots, placing the tender shoots into detergent powder liquid, stirring the detergent powder liquid and the tender shoots until foreign matters attached to the surfaces of the tender shoots are separated, and cleaning up the tender shoots for standby application; (3) stripping tender leaves on surface layers of the tender shoots, placing the tender shootsinto pvp (polyvinyl pyrrolidone) sterile solution, washing out the tender shoots with water, transferring the tender shoots into sterile water, rinsing the tender shoots for three times and drainingthe tender shoots for standby application; (4) transferring the drained tender shoots into a sterile operation desk, disinfecting the tender shoots with 75wt% alcohol for 10 seconds, rinsing the tender shoots, draining the water, transferring the tender shoots into oscillation liquid, disinfecting the tender shoots, rinsing the tender shoots for three times and then draining the water. The sterilization method can effectively reduce the contamination rate of Litsea coreana Levl. var. lanuginose (Migo) Yanget P. H. Huang and increase the starting rate.
Description
technical field [0001] The invention relates to the technical field of tissue culture, in particular to a sterilization method for improving the starting rate of tissue culture of camphor camphora explants. Background technique [0002] Litsea coreana Levl. var. lanuginose (Migo) Yanget P.H. Huang) is a small tree of the genus Zingiberaceae in the family Lauraceae, and is the main raw material plant for the ancient tea drink Eagle Tea in Southwest China. In recent years, because of its rich flavonoid antioxidants such as kaempferol and quercetin, Eagle tea has the effect of lowering blood sugar and blood lipids, and is purely natural, pollution-free, and unique in flavor. It has been favored by the market and performed relatively well. application value and economic value. [0003] Leopard skin camphor is a dioecious plant, the seeds contain substances that inhibit rooting, the natural seedling rate is extremely low, and the seeds of wild resources are difficult to reproduc...
Claims
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