阅读说明：本技术 一种提高毛豹皮樟外植体组织培养启动率的灭菌方法 (Sterilization method for improving tissue culture starting rate of cinnamomum camphora explants ) 是由 戴前莉 卢敏 朱恒星 黄飞逸 黄馨 于 2019-10-15 设计创作，主要内容包括：本发明公开了一种提高毛豹皮樟外植体组织培养启动率的灭菌方法,属于组织培养技术领域,所述灭菌方法的步骤如下：(1)带嫩芽的枝条活体上喷洒多菌灵溶液进行消毒；(2)采取嫩芽,清洗干净,随后放入洗衣粉液中,搅拌至嫩芽表面附着异物脱离,清洗干净,待用；(3)先剥离掉嫩芽表面一层的嫩叶,随后将嫩芽放入pvp无菌溶液中浸泡,用水冲洗干净后转入无菌水中漂洗3遍,滤干待用；(4)将滤干的嫩芽转入无菌操作台,用75wt％酒精消毒10s后漂洗干净、滤干水分,转入震荡液中消毒,随后漂洗3次,滤干水分即可。本发明的灭菌方法能有效降低毛豹皮樟的污染率,提高启动率。(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.)

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 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.

2. The sterilization method for improving the tissue culture initiation rate of the cinnamomum pantherinum explant according to claim 1, wherein 1 ‰ carbendazim is sprayed in step (1), and the spraying is performed once in the morning and once in the evening for 3 days.

3. The sterilization method for improving the tissue culture initiation rate of the cinnamomum pantherinum explant according to claim 2, wherein the concentration of the laundry powder liquid in the step (2) is 1 ‰.

4. The sterilization method for improving the tissue culture starting rate of the cinnamomum camphora presl explant according to claim 3, wherein the specific operation of the step (3) is as follows: firstly, stripping off tender leaves on a layer of the surface of the tender shoots, then transferring the tender shoots into a 1% pvc sterile solution for soaking for 10min, then transferring into flowing water, washing for 30min by using water, and finally transferring into sterile clear water for rinsing for 3 times.

5. The sterilization method for improving the tissue culture initiation rate of the cinnamomum pantherinum explant according to claim 4, wherein in the step (4), the shaking solution is prepared by mixing 1% pvp and 1% mercuric chloride solution according to a volume ratio of 1: 100.

6. The sterilization method for improving the tissue culture initiation rate of the cinnamomum koehmannii explant according to any one of claims 1 to 5, characterized in that after the tissue culture initiation rate of the cinnamomum koehmannii explant is subjected to shake liquid sterilization, rinsing and drying, a microcrystalline antibacterial agent is sprayed on 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.

7. The sterilization method for improving the tissue culture initiation rate of the cinnamomum camphora presl explant according to claim 6, wherein the crystallite particles have a particle size of 2-3 μm.

8. The sterilization method for improving the tissue culture starting rate of the cinnamomum camphora presl explant according to claim 7, 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 (1) with 5 wt% of sodium chloride solution, heating to 70 ℃ after uniformly stirring, stirring for reacting for 4h, performing centrifugal separation, heating the precipitate to 250-300 ℃, taking out after 40min, cooling to 90-110 ℃, adding 0.5-1 wt% of copper chloride solution, adjusting the pH to 7.5-8, stirring for reacting for 4-5h at 50-60 ℃, taking out, calcining for 20min 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.

9. The sterilization method for increasing the tissue culture initiation rate of the cinnamomum camphora presl explant according to claim 8, wherein in the step B, the volume ratio of the sodium chloride solution to the copper chloride solution is 3: 1.

10. The sterilization method for improving the tissue culture initiation rate of the cinnamomum pantherinum explant according to claim 9, 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.

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: