CN110622861B - Sterilization method for improving tissue culture starting rate of cinnamomum camphora explants - Google Patents
Sterilization method for improving tissue culture starting rate of cinnamomum camphora explants Download PDFInfo
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Abstract
The invention discloses a sterilization method for improving the tissue culture starting rate of a cinnamomum pantherinum explant, belonging to the technical field of tissue culture, and the sterilization method comprises the following steps: (1) spraying carbendazim solution on the live branches with the tender buds for disinfection; (2) adopting tender shoots, cleaning, then putting the tender shoots into a washing powder liquid, stirring until foreign matters attached to the surfaces of the tender shoots are separated, and cleaning the tender shoots for later use; (3) firstly, stripping off a layer of tender leaves on the surface of the tender shoots, then putting the tender shoots into a pvp sterile solution for soaking, washing with water, then transferring into sterile water for rinsing for 3 times, and filtering to dry for later use; (4) transferring the dried tender shoots into a sterile operating table, sterilizing with 75 wt% alcohol for 10s, rinsing, filtering to remove water, sterilizing in shaking solution, rinsing for 3 times, and filtering to remove water. The sterilization method can effectively reduce the pollution rate of the cinnamomum pantherinum and improve the starting rate.
Description
Technical Field
The invention relates to the technical field of tissue culture, in particular to a sterilization method for improving the tissue culture starting rate of a cinnamomum camphora explant.
Background
The Pimenta dioica (Litseae reanana Levl. var. lanuginose (Migo) YangetP. H. Huang) is a small arbor of Litsea of Lauraceae, and is a main raw material plant of ancient tea, Litsea coreana in southwest. In recent years, the eagle tea has the effects of reducing blood sugar and blood fat due to the fact that the eagle tea is rich in flavonoid antioxidant substances such as kaempferol and quercetin, has the characteristics of pure nature, no pollution, unique flavor and the like, is deeply favored by the market, and shows higher application value and economic value.
The cinnamomum leoparum is a heterotrophic plant of male and female, the seeds contain substances for inhibiting rooting, the natural seedling rate is extremely low, and the seeds of wild resources have high propagation difficulty and large genetic differentiation. And because the tissue contains a large amount of polyphenol compounds, the seedlings are difficult to root during cutting and raising, so the tissue culture rapid propagation technology is one of the main methods for solving the artificial propagation of the cinnamomum leoparum. However, the pollution problem in plant tissue culture is one of three problems which plague the tissue culture work at present. In the process from primary culture to secondary culture of explants, endophyte pollution, operation pollution and environmental pollution are easy to occur due to the fact that plants carry bacteria or operation methods are improper, and once the tissue culture materials are polluted, the tissue culture materials are difficult to rescue. At present, the research of pollution control is mostly biased to the control and physical precautionary measures of antibiotics, and the antibiotics have certain pertinence to pollution control, but need to be filtered, added and other procedures, and have complicated steps; if the antibiotic is sterilized with the culture medium under high pressure, the sterilization effect will be reduced, and the prevention and control effect is not ideal. If the conventional tissue culture sterilization method is adopted, the explant has high pollution rate, the explant pollution rate can be effectively reduced by increasing the sterilization time, but 1 per thousand of mercuric chloride is vibrated and sterilized for 4min, so that the young bud of the cinnamomum pantherinum is inactivated in different degrees, browning is easy to occur after the alcohol cleaning time is overlong, and in addition, a large amount of phenols, quinones and other substances are contained in the explant, so that the browning is easy to occur, and the starting rate is reduced; if the sterilization time is reduced, the explant is started quickly, but the pollution rate is extremely high. Therefore, a sterilization method for improving the tissue culture starting rate of the cinnamomum camphora and reducing the pollution rate is urgently needed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a sterilization method for improving the tissue culture starting rate of a cinnamomum pantherinum explant, which can effectively improve the starting rate of the cinnamomum pantherinum tissue culture, reduce the pollution rate and improve the survival rate.
The invention solves the technical problems by the following technical means:
a sterilization method for improving the tissue culture starting rate of a cinnamomum camphora explant comprises the following steps:
(1) spraying carbendazim solution on the live branches with the tender buds for disinfection;
(2) adopting tender shoots, cleaning, then putting the tender shoots into a washing powder liquid, stirring until foreign matters attached to the surfaces of the tender shoots are separated, and cleaning the tender shoots for later use;
(3) firstly, stripping off a layer of tender leaves on the surface of the tender shoots, then putting the tender shoots into a pvp sterile solution for soaking, washing with water, then transferring into sterile water for rinsing for 3 times, and filtering to dry for later use;
(4) transferring the dried tender shoots into a sterile operating platform, sterilizing for 10s by using 75 wt% alcohol, rinsing, draining, transferring into a shaking solution for sterilization, rinsing for 3 times, draining, and transferring to a starting culture medium.
Further, 1 ‰ carbendazim is sprayed in step (1), and the spraying is carried out once in the morning and at night for 3 days continuously.
Furthermore, the concentration of the washing powder liquid in the step (2) is 1 per mill.
Further, the specific operation of the step (3) is as follows: firstly, stripping off tender leaves on a layer of tender bud surface, then transferring the tender bud into a 1% pvc sterile solution for soaking for 10min, then transferring into flowing water, washing for 30min with water, finally transferring into sterile water for rinsing for 3 times, washing with water, optionally washing with big water for 10min, then transferring into small water for washing for 20min, wherein the flow rates of the big water and the small water are the routine selection flow rates of a person skilled in the art.
Further, in the step (4), the shaking solution is prepared by mixing 1% of pvp and 1% of mercuric chloride solution according to the volume ratio of 1: 100.
Further, after the disinfection, rinsing and filtering of the shaking solution in the step (4), spraying a microcrystalline antibacterial agent on the tender shoots, wherein the microcrystalline antibacterial agent contains 2-5 wt% of sodium chloride, 2-3 wt% of microcrystalline particles and the balance of water, vermiculite is used as a core in the microcrystalline particles, and picromerite crystals are wrapped outside the microcrystalline particles.
Further, the grain size of the microcrystal grains is 1-1.5 mu m.
The microcrystalline granule comprises vermiculite, cupric chloride, sodium chloride, and schoenite. The vermiculite is monoclinic system, is flaky, has an interlayer domain, has a larger specific surface area, and simultaneously has certain ion exchange capacity and stronger adsorbability, but the calcium ion exchange capacity in the vermiculite is smaller, and the vermiculite is not easy to directly replace copper ions, so through sodium modification, the copper ions are used for exchanging out sodium ions with large ion exchange capacity, namely, the copper ions with bactericidal effect are intercalated and enter, germs in the tissue culture process of the cinnamomum pantherinum can be eliminated, and the start rate is improved. After the schoenite and the vermiculite are mixed, the vermiculite is used as a crystal nucleus for recrystallization, so that the fertilizer can be slowly released, and the survival rate of tissue culture is further improved.
Further, the preparation method of the microcrystalline antibacterial agent comprises the following steps:
A. heating vermiculite with the particle size of 1-1.5 mu m to 480-500 ℃, taking out after 30min, cooling, adding the cooled vermiculite into 5 wt% hydrochloric acid solution, stirring for 2-3h at 70 ℃, taking out, heating again to 300-350 ℃, taking out after 20min, and cooling for later use;
after high-temperature treatment, vermiculite expands, the distance between layers is increased, hydrochloric acid solution can conveniently enter, negative charges of the vermiculite structure layer can be effectively reduced after hydrochloric acid treatment, and as the vermiculite absorbs water again in the hydrochloric acid solution, high-temperature treatment is needed again to ensure that the vermiculite expands again, the distance between layers is increased, the dispersity is improved, and ion exchange is facilitated;
B. mixing the vermiculite obtained in the step (1) with 5 wt% of sodium chloride solution, heating to 70 ℃ after uniformly stirring, stirring for reacting for 4 hours, performing centrifugal separation, heating the precipitate to 250-300 ℃, taking out after 40 minutes, cooling to 90-110 ℃, adding 0.5-1 wt% of copper chloride solution, adjusting the pH to 7.5-8, stirring for reacting for 4-5 hours at 50-60 ℃, taking out, calcining for 20 minutes at 170 ℃, cleaning with deionized water, drying in the air to obtain copper ion type vermiculite, and cooling for later use;
under the condition of 70 ℃, calcium ions in the vermiculite are replaced by sodium ions in a sodium chloride solution, then the vermiculite is heated again, the vermiculite expands, hydrogen is more easily ionized in a weak alkali environment, the surface of the vermiculite is negatively charged, at the moment, the copper ions are more easily replaced by the sodium ions, and then the copper ions are calcined, so that unreacted copper chloride is dehydrated and is convenient to remove.
C. Dissolving schoenite in deionized water, heating to 60 ℃ to prepare a schoenite saturated solution, then adding copper ion type vermiculite and ethylene glycol, uniformly mixing, heating to 60-80 ℃, keeping the temperature, stirring at the speed of 300rpm for 2h, standing, cooling for 8-10h, heating to 250 ℃ at the speed of 5 ℃/min, introducing air, preserving the heat for 1h, and then taking out to obtain microcrystalline particles;
the saturated schoenite can be recrystallized under the natural evaporation, the schoenite crystal can grow the vermiculite that step B obtained as the crystal nucleus into the micrite granule, and the slowly in-process that dissolves of schoenite crystal, the vermiculite that has the copper ion also slowly exposes, when providing potash fertilizer, magnesium fertilizer, kills the bacterium that has bred on the explant, guarantees the survival rate.
D. And (3) storing the microcrystalline particles in 2-5 wt% of sodium chloride solution to obtain the microcrystalline antibacterial agent.
Further, in the step B, the volume ratio of the sodium chloride solution to the copper chloride solution is 3: 1.
Further, in the step C, 8g of copper ion type vermiculite and 0.05ml of ethylene glycol were added to 100ml of the schoenite saturated solution.
The invention has the following beneficial effects:
1. the sterilization method can effectively reduce the pollution rate of the cinnamomum pantherinum and improve the starting rate.
2. The microcrystalline antibacterial agent can be used for continuously disinfecting and sterilizing the cinnamomum pantherinum explant in the culture process of the cinnamomum pantherinum explant, the starting rate is improved, and the picromerite can be used as a fertilizer to provide nutrients for the growth of the explant.
3. The tissue culture method of the invention is simple and convenient, and is convenient for popularization
Detailed Description
The present invention will be described in detail with reference to specific examples below:
example 1:
(1) step 1: and (4) living body disinfection. And inquiring weather forecast, selecting continuous sunny days, cutting off old leaves which are not grown in the current year on branches, only leaving tender buds, spraying 1 per mill of carbendazim on living bodies, and continuously spraying for 3 days in the morning and at night.
(2) And 2, cleaning foreign matters on the surface. Adopting the tender shoots sprayed with the carbendazim, firstly washing dust on the surfaces of the tender shoots with clear water, then adding 1 per mill of washing powder liquid, continuously stirring until all foreign matters attached to the surfaces of the tender shoots are separated, and then rinsing with clear water.
(3) And 3, sterilizing in vitro. Firstly, stripping off tender leaves on a layer of tender bud surface, then putting the tender bud into 1% pvc sterile solution for soaking for 10min, then transferring into flowing water, washing for 10min with big water, then washing for 20min with small water, finally transferring into sterile water for fully rinsing for 3 times, and filtering for later use. The flow rates of the large and small water are those conventionally employed by those skilled in the art.
(4) And 4, step 4: and (5) performing sterile operation. Transferring the dried tender shoots into a sterile operating table, disinfecting the tender shoots with 75 wt% of alcohol for 10s, rinsing the tender shoots, draining the water, transferring the tender shoots into a shaking solution, mixing the shaking solution with 1 per mill of pvp and 1 per mill of mercuric chloride solution according to a volume ratio of 1:100, disinfecting the shaking solution for 5min, and rinsing the shaking solution for 3 times with sterile clear water to drain the water.
(5) And 5: and (5) switching. Inoculating the sterilized tender shoots into a hairy leopard cinnamomum camphora starting culture medium, inoculating 1 seed in each bottle, and inoculating 50 bottles.
Preparation of the Start Medium (the same preparation method of the Start Medium was used in all examples of the invention):
a starter culture medium is prepared, wherein each liter of the starter culture medium contains MS culture medium, 6mg/L riboflavin, 2mg/L6-BA, 0.1mg/L LNAA, 30mg/L sucrose and 6.5mg/L agar, and water is used as a solvent, and the balance is water.
And counting the contamination and explant starting within 30 days after inoculation, and counting every 1 day.
Example 2
Step 1: selecting clear weather, cutting off old leaves which are not grown in the current year on branches of the cinnamomum camphora, only leaving tender buds, and spraying 1 per mill of carbendazim on living bodies, wherein the young leaves are used for killing the old leaves, and the young leaves are used for killing the old leaves once in the morning and at night.
The rest of the procedure was the same as in example 1.
And counting the contamination and explant starting within 30 days after inoculation, and counting every 1 day.
Example 3
(1) Step 1: selecting continuous clear weather, cutting off old leaves of the branches of the cinnamomum camphora which are not grown in the current year, only leaving tender buds, spraying 1 per mill of carbendazim on the living body, and continuously spraying for 5 days in the morning and at night.
The rest of the procedure was the same as in example 1.
And counting the contamination and explant starting within 30 days after inoculation, and counting every 1 day.
Example 4
Steps 1, 2, 4, 5 as in example 1.
(3) And step 3: and (5) sterilizing in vitro. Firstly, stripping off tender leaves on a layer of tender bud surface, then putting the tender bud into 1% pvc sterile solution for soaking for 10min, then transferring into flowing water, washing for 3min with big water, then washing for 10min with small water, finally transferring into sterile water for fully rinsing for 3 times, and filtering for later use. The flow rates of the large and small water are those conventionally employed by those skilled in the art.
And counting the contamination and explant starting within 30 days after inoculation, and counting every 1 day.
Example 5
Steps 1, 2, 4, 5 as in example 1.
(3) And step 3: and (5) sterilizing in vitro. Firstly, stripping off tender leaves on a layer of tender bud surface, then putting the tender bud into 1% pvc sterile solution for soaking for 10min, then transferring into flowing water, washing for 30min with big water, then washing for 1h with small water, finally transferring into sterile water for fully rinsing for 3 times, and filtering for later use. The flow rates of the large and small water are those conventionally employed by those skilled in the art.
And counting the contamination and explant starting within 30 days after inoculation, and counting every 1 day.
Example 6
Steps 1, 2, 5, as in example 1.
(3) And 3, sterilizing in vitro. Peeling off tender leaves on the surface of tender shoots, transferring into flowing water, washing with big water for 10min, washing with small water for 20min, rinsing in sterile water for 3 times, and filtering to remove water. The flow rates of the large and small water are those conventionally employed by those skilled in the art.
(4) And 4, step 4: and (5) performing sterile operation. Transferring the dried tender shoots into a sterile operating table, sterilizing with 75 wt% alcohol for 10s, rinsing, draining, transferring into a solution containing 1 ‰ of mercuric chloride, shaking for sterilizing for 5min, rinsing with sterile clear water for 3 times, and draining.
In the period, the tender shoots are not put into a 1% pvc sterile solution for soaking for 10min, and 1% of pvc is not added into the shaking solution.
And counting the contamination and explant starting within 30 days after inoculation, and counting every 1 day.
Statistical examples 1-6 the experimental data are shown in table 1:
the contamination rate is the number of contaminated bottles/inoculated bottles × 100%
Explant priming rate ═ number of shoots growing or proliferating bottles/number of uncontaminated bottles × 100%
The start-up situation is good: the bud grows high or proliferates, and is yellow green and strong;
the starting situation is better: high or multiplied bud, white bud color, slender bud;
the start-up situation is generally: the bud is not obvious in length, does not proliferate, and has no obvious change or the color of the bud turns white.
TABLE 1
Example 7:
the preparation method of the microcrystalline antibacterial agent adopted in the sterilization method for improving the tissue culture starting rate of the cinnamomum pantherinum explant comprises the following steps:
A. heating 20g of vermiculite with the particle size of 1-1.3 mu m to 480 ℃, preserving heat for 30min, taking out, cooling to room temperature, adding the cooled vermiculite into 5 wt% hydrochloric acid solution, stirring at 70 ℃ for 3h, taking out, heating again to 350 ℃, preserving heat for 20min, taking out, and cooling for later use; in the two heating processes, the heating rate is 15 ℃/min;
B. mixing the vermiculite obtained in the step (1) with 150ml of 5 wt% sodium chloride solution, uniformly stirring, heating to 70 ℃, preserving heat, stirring, reacting for 4 hours, centrifugally separating, heating the precipitate to 300 ℃, preserving heat for 40 minutes, taking out, cooling to 90 ℃, adding 50ml of 1 wt% copper chloride solution, adjusting the pH to about 7.6, heating in a water bath to 50 ℃, stirring, reacting for 4 hours, taking out, preserving heat, calcining for 20 minutes at 170 ℃, cleaning with deionized water, drying in the air to obtain copper ion type vermiculite, and cooling for later use;
C. dissolving schoenite in deionized water, heating to 60 ℃ to prepare a schoenite saturated solution, then taking 250ml of schoenite saturated solution, adding 20g of the copper ion type vermiculite obtained in the step B and 0.125ml of ethylene glycol, uniformly mixing, heating to 60 ℃, stirring at a constant temperature of 300rpm for 2h, standing, cooling, standing for 8h, heating to 250 ℃ at a speed of 5 ℃/min, introducing air, keeping the temperature for 1h, and then taking out to obtain microcrystalline particles with the particle size of about 2.1 mu m;
D. and (3) storing the microcrystalline particles in 2 wt% sodium chloride solution to obtain the microcrystalline antibacterial agent.
Example 8:
the preparation method of the microcrystalline antibacterial agent adopted in the sterilization method for improving the tissue culture starting rate of the cinnamomum pantherinum explant comprises the following steps:
A. heating 40g of vermiculite with the particle size of 1.3-1.5 mu m to 500 ℃, preserving heat for 30min, taking out, cooling to room temperature, adding into 5 wt% hydrochloric acid solution, stirring at 70 ℃ for 2h, taking out, heating to 300 ℃ again, preserving heat for 20min, taking out, and cooling for later use; in the two heating processes, the heating rate is 15 ℃/min;
B. mixing the vermiculite obtained in the step (1) with 200ml of 5 wt% sodium chloride solution, uniformly stirring, heating to 70 ℃, preserving heat, stirring, reacting for 4 hours, centrifugally separating, heating the precipitate to 250 ℃, preserving heat for 40 minutes, taking out, cooling to 110 ℃, adding 66.7ml of 1 wt% copper chloride solution, adjusting the pH to about 7.8, heating in a water bath to 60 ℃, stirring, reacting for 5 hours, taking out, preserving heat, calcining for 20 minutes at 170 ℃, cleaning with deionized water, drying in the air to obtain copper ion type vermiculite, and cooling for later use;
C. dissolving schoenite in deionized water, heating to 60 ℃ to prepare a schoenite saturated solution, then taking 500ml of schoenite saturated solution, adding 40g of the copper ion type vermiculite obtained in the step B and 0.25ml of ethylene glycol, uniformly mixing, heating to 80 ℃, stirring at a constant temperature and a speed of 300rpm for 2h, standing, cooling, standing for 10h, heating to 250 ℃ at a speed of 5 ℃/min, introducing air, keeping the temperature for 1h, and then taking out to obtain microcrystalline particles with a particle size of about 2.5 mu m;
D. and (3) storing the microcrystalline particles in a 5 wt% sodium chloride solution to obtain the microcrystalline antibacterial agent.
Experiment 1:
(1) step 1: selecting clear weather, cutting off old leaves which are not grown in the current year on branches of the cinnamomum camphora, only leaving tender buds, and spraying 1 per mill of carbendazim on living bodies, wherein the young leaves are used for being used in the morning and the evening;
the rest of the steps 2-4 are the same as example 1, and then the microcrystalline antibacterial agent prepared in example 7 is sprayed on the tender shoots, wherein the microcrystalline antibacterial agent contains 2 wt% of sodium chloride, 2 wt% of microcrystalline particles and the balance of water.
(5) And 5: and (5) switching. Inoculating the sterilized tender shoots into a cinnamomum koehirae starting culture medium, inoculating 1 in each bottle, inoculating 50 bottles, counting the pollution condition and the explant starting condition within 30 days after inoculation, and counting once every 1 day.
Experiment 2:
(1) step 1: selecting clear weather, cutting off old leaves which are not grown in the current year on branches of the cinnamomum camphora, only leaving tender buds, and spraying 1 per mill of carbendazim on living bodies, wherein the young leaves are used for being used in the morning and the evening;
the rest of the steps 2-4 are the same as example 1, and then the microcrystalline antibacterial agent prepared in example 8 is sprayed on the tender shoots, wherein the microcrystalline antibacterial agent contains 5 wt% of sodium chloride, 3 wt% of microcrystalline particles and the balance of water.
(5) And 5: and (5) switching. Inoculating the sterilized tender shoots into a cinnamomum koehirae starting culture medium, inoculating 1 in each bottle, inoculating 50 bottles, counting the pollution condition and the explant starting condition within 30 days after inoculation, and counting once every 1 day.
In comparison with example 2, the statistical results are shown in table 2:
TABLE 2
As can be seen by comparing examples 1-6 with experiments 1-2:
1. the comparative example 1/2/3, proper amount of carbendazim sprayed on living bodies can effectively help the cinnamomum camphora leoparum reduce the pollution rate, the time for pollution to begin to appear after inoculation is also delayed, the pollution rate is gradually reduced along with the increase of the spraying times, but the explant is influenced by the excessive spraying. Example 1 works best.
2. Compared with the 1/4/5 example, the running water washing is helpful for reducing the contamination rate of the Cinnamomum pantherinum explants and improving the starting rate, but the time is too short, the effect is not good, the time is too long, the switching cost is increased, compared with the example 1 example, the contamination rate is only reduced by 4%, the time of contamination after inoculation is only prolonged by 2 days, the treatment time is 2 times, and the in vitro disinfection treatment of the example 1 is the best.
3. 1% of pvp sterile solution is added for low-temperature soaking for 10min or 1 ‰ of pvp is added into shaking solution, which can effectively help to reduce contamination rate of endophyte, and helps to improve the starting rate of the cinnamomum pantherinum.
4. In comparative example 2, experiment 1 and experiment 2, after the microcrystalline antibacterial agent is sprayed, the contamination rate is obviously reduced, the time for contamination occurrence in inoculation is prolonged, although contamination occurs, the starting rate is obviously increased, and the growth condition is good.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims. The techniques, shapes, and configurations not described in detail in the present invention are all known techniques.
Claims (4)
1. A sterilization method for improving the tissue culture starting rate of a cinnamomum camphora explant is characterized by comprising the following steps:
(1) spraying 1 ‰ carbendazim solution on branch living body with tender bud for sterilizing, and continuously spraying for 3 days in the morning and evening;
(2) taking tender shoots, cleaning, then putting the tender shoots into 1 per mill of washing powder liquid, stirring until foreign matters attached to the surfaces of the tender shoots are separated, and cleaning for later use;
(3) firstly, stripping off tender leaves on a layer of tender bud surface, then transferring the tender buds into a 1% pvc sterile solution for soaking for 10min, then transferring into flowing water, washing for 10min with big water, then washing for 20min with small water, finally rinsing for 3 times in sterile clear water, and filtering for later use;
(4) transferring the filtered tender shoots into a sterile operation table, disinfecting for 10s by using 75 wt% alcohol, rinsing, filtering to remove water, transferring into a shaking solution, mixing the shaking solution by using 1 thousandth of pvp and 1 thousandth of mercury bichloride according to a volume ratio of 1:100 to prepare the mixture, disinfecting for 5min in the shaking solution, rinsing for 3 times, filtering to remove water, spraying a microcrystalline antibacterial agent to the tender shoots, wherein the microcrystalline antibacterial agent contains 2-5 wt% of sodium chloride, 2-3 wt% of microcrystalline particles and the balance of water, vermiculite is used as a core in the microcrystalline particles, picromerite crystals are coated outside the microcrystalline particles, and the particle size of the microcrystalline particles is 2-3 mu m;
(5) inoculating the sterilized tender shoots into a starting culture medium of a cinnamomum komarovii, wherein each liter of the starting culture medium contains MS culture medium, 6mg/L riboflavin, 2mg/L6-BA, 0.1mg/L LNAA, 30mg/L sucrose and 6.5mg/L agar, water is used as a solvent, and the balance is water.
2. The sterilization method for improving the tissue culture starting rate of the cinnamomum camphora presl explant according to claim 1, wherein the microcrystalline antibacterial agent is prepared by the following steps:
A. heating vermiculite with the particle size of 1-1.5 mu m to 480-500 ℃, taking out after 30min, cooling, adding the cooled vermiculite into 5 wt% hydrochloric acid solution, stirring for 2-3h at 70 ℃, taking out, heating again to 300-350 ℃, taking out after 20min, and cooling for later use;
B. mixing the vermiculite obtained in the step A with 5 wt% of sodium chloride solution, heating to 70 ℃ after uniformly stirring, stirring for reacting for 4 hours, performing centrifugal separation, heating the precipitate to 250-300 ℃, taking out after 40 minutes, cooling to 90-110 ℃, adding 0.5-1 wt% of copper chloride solution, adjusting the pH to 7.5-8, stirring for reacting for 4-5 hours at 50-60 ℃, taking out, calcining for 20 minutes at 170 ℃, cleaning with deionized water, drying in the air to obtain copper ion type vermiculite, and cooling for later use;
C. dissolving schoenite in deionized water, heating to 60 ℃ to prepare a schoenite saturated solution, then adding copper ion type vermiculite and ethylene glycol, uniformly mixing, heating to 60-80 ℃, keeping the temperature, stirring at the speed of 300rpm for 2h, standing, cooling for 8-10h, heating to 250 ℃ at the speed of 5 ℃/min, introducing air, preserving the heat for 1h, and then taking out to obtain microcrystalline particles;
D. and (3) storing the microcrystalline particles in 2-5 wt% of sodium chloride solution to obtain the microcrystalline antibacterial agent.
3. The sterilization method for increasing the tissue culture initiation rate of the cinnamomum camphora presl explant according to claim 2, wherein in the step B, the volume ratio of the sodium chloride solution to the copper chloride solution is 3: 1.
4. The sterilization method for improving the tissue culture initiation rate of the cinnamomum pantherinum explant according to claim 3, wherein in the step C, 8g of copper ion type vermiculite and 0.05ml of ethylene glycol are added per 100ml of schoenite saturated solution.
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