Disinfection method of pinellia ternate explant and tissue culture method of pinellia ternate

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a disinfection method of a pinellia ternate explant and a tissue culture method of pinellia ternate. The disinfection method comprises the following steps: putting a pinellia ternate explant into ethanol which contains plant essential oil and has the volume fraction of 70-80%, disinfecting for 30-70 seconds, then disinfecting in mercuric chloride with the mass fraction of 0.08-0.13%, and washing off the residual mercuric chloride on the surface of the explant after mercuric chloride disinfection is finished, so as to obtain the disinfected explant; wherein the volume fraction of the plant essential oil is 3%-7%; the plant essential oil is selected from one or more of lemongrass essential oil, oregano essential oil, folium eucalypti essential oil, cinnamon essential oil and mint essential oil. The disinfection method provided by the invention can effectively reduce the damage caused by direct contact of mercuric chloride to plant stem tip cells, increase the germination rate of Xiaozhou pinellia tuber stem tip culture, and reduce the pollution rate of Xiaozhou pinellia tuber virus-free seedlings.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a method for disinfecting a pinellia explant and a method for culturing the pinellia tissue. Background Art

[0002] Pinellia ternata (Thunb.) Breit. is the dried tuber of a perennial herbaceous plant of the genus Pinellia in the family Araceae. It is also known as Sanxingcao, Diwen, and Shoutian. This product is spherical, some are slightly oblique, and the diameter is 0.7-1.6cm. The surface is white or light yellow, with a sunken stem scar on the top and densely covered with pockmarked root scars around; the bottom is blunt and smooth. The texture is solid, the cross section is white, and it is rich in powder. It has a slight smell, a spicy taste, numbing the tongue and piercing the throat. It is a commonly used Chinese herbal medicine in traditional Chinese medicine. The dried tuber has the effects of drying dampness and resolving phlegm, lowering adverse reactions and stopping vomiting, and eliminating lumps and dispersing knots. Modern pharmacological studies have found that Pinellia also has anti-tumor, anti-inflammatory, antibacterial, and anti-epileptic pharmacological effects. Among them, the pinellia produced in Xi County, Xinyang, Henan Province is large, white, powdery, and has good efficacy. It is said that 9g can replace 15g of pinellia from other places. It is a treasure among similar medicinal materials and is called "Xi Pinellia". The earliest record of Xi Pinellia was in the "Xi County Chronicles" in the fourth year of Jiaqing in the Qing Dynasty (1799); in 1914, Xi Pinellia participated in the International Commodity Fair held in San Francisco, USA; after 1949, Xi Pinellia was a key export medicinal material; the 1979 edition of "Cihai" listed Pinellia as a specialty of Xi County. Traditional Xi Pinellia is mainly collected from the wild, but due to excessive collection of Xi Pinellia, the wild Xi Pinellia resources in Xinyang are gradually exhausted. In order to meet the clinical needs of Xi Pinellia, the artificial cultivation of Xi Pinellia is expanding, but the source of Xi Pinellia is short. In production, small tubers, stem tips, leaves and other explants are often used for propagation, but it is found in production that laboratory tissue culture is extremely susceptible to bacterial and fungal infections.

[0003] At present, the conventional method of disinfecting the stem tip of Pinellia ternata tissue culture in the laboratory is to disinfect with 75% ethanol for 30s, disinfect with 0.1% mercuric chloride for 15min, and rinse with sterile water for 15min after disinfection. The above methods have disadvantages. Mercuric chloride is a powerful disinfectant with the advantages of thorough disinfection effect, short disinfection time and high efficiency. However, the stem tip of Pinellia ternata is a very fragile part. Mercuric chloride is a highly toxic reagent, which can easily cause irreversible damage to the stem tip of Pinellia ternata during the disinfection process, directly causing the stem tip to stop growing for a long time after inoculation, which affects the benefits of artificial cultivation. Therefore, finding a mild disinfection method to disinfect Pinellia ternata explants has become a research hotspot. Summary of the invention

[0004] In order to solve the defect in the prior art that long-term mercuric chloride disinfection will affect the stem tip of Pinellia ternata and cause damage, the present invention provides a method for disinfecting Pinellia ternata explants and a method for culturing Pinellia ternata tissue.

[0005] The first aspect of the present invention provides a method for disinfecting a pinellia explant, comprising: placing the pinellia explant in ethanol containing 70% to 80% of plant essential oil by volume for disinfection for 30s to 70s, and then disinfecting in mercuric chloride with a mass fraction of 0.08% to 0.13% to obtain a disinfected explant;

[0006] Wherein, the volume fraction of the plant essential oil is 3% to 7%;

[0007] The plant essential oil is selected from one or more of lemongrass essential oil, oregano essential oil, eucalyptus essential oil, cinnamon essential oil and peppermint essential oil.

[0008] In some embodiments of the present invention, the ethanol is used for disinfection for 25 seconds to 35 seconds.

[0009] In some embodiments of the present invention, the time for disinfection using the mercuric chloride is 10 minutes to 20 minutes.

[0010] In some embodiments of the present invention, the explant is rinsed with sterile water to wash away the mercuric chloride remaining on the surface of the explant. In some embodiments of the present invention, the time for rinsing with sterile water is 10 minutes to 25 minutes.

[0011] In some embodiments of the present invention, after the skin of the Pinellia explant is peeled off, dust and dirt are washed off and then disinfected using the ethanol.

[0012] In some embodiments of the present invention, the Pinellia explant is a tuber or leaf of Pinellia.

[0013] The second aspect of the present invention provides a method for tissue culture of Pinellia ternata, comprising the step of sterilizing Pinellia ternata explants using the sterilization method to obtain sterilized explants; inoculating the sterilized explants into a culture medium for culture to obtain callus tissue, and then inducing the callus tissue to root and germinate to obtain tissue culture seedlings.

[0014] In some embodiments of the present invention, the culture conditions are light intensity of 1800lx to 2200lx, light duration of 12h / d to 16h / d, and temperature of 24°C to 26°C.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] In the present invention, Pinellia ternata from Xi County, Xinyang City, Henan Province is used as a test material, natural plant essential oil is combined with 75% ethanol for disinfection, and then the essential oil is placed in 0.1% mercuric chloride for disinfection, which can effectively reduce the damage caused by direct contact of mercuric chloride to plant stem tip cells, provide a mild disinfection method, and at the same time, increase the germination rate of Pinellia ternata stem tip culture and reduce the contamination rate of Pinellia ternata virus-free seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a comparison chart of the contamination rate and survival rate of each group.

[0018] Figure 2 This is a graph showing the effect of different oregano essential oil disinfection times on the germination rate of the stem tip of Pinellia ternata.

[0019] Figure 3 This is a graph showing the effect of different mercuric chloride disinfection times on the germination rate of the stem tip of Pinellia ternata.

[0020] Figure 4 This is a graph showing the effect of different sterile water washing times on the germination rate of the stem tip of Pinellia ternata.

[0021] Figure 5 This is a graph showing the effects of different oregano essential oil concentrations on the germination rate of the stem tip of Pinellia ternata.

[0022] Figure 6 The disinfection time of oregano essential oil was 30s, the disinfection time of 0.1% mercuric chloride was 15min, the rinsing time of sterile water was 10min, and the concentration of oregano essential oil was 5% for the tissue culture seedlings of Pinellia ternata.

[0023] Figure 7 The sterilization time of oregano essential oil was 30s, the sterilization time of 0.1% mercuric chloride was 15min, the sterile water was rinsed for 10min, and the oregano essential oil concentration was 3%.

[0024] Figure 8 The Pinellia ternata is contaminated by Rhizopus fungi.

[0025] Fig. 9 The Pinellia ternata is contaminated by Aspergillus niger fungus.

[0026] Fig.10 Pinellia ternata contaminated by Penicillium bacteria.

[0027] Fig.11 To stop the bacterial contamination of Pinellia ternata, the water is in a polluted state.

[0028] Fig.12 To prevent bacterial contamination of Pinellia ternata, it appears in the form of oily bubbles.

[0029] Fig.13 These are yellow colonies caused by bacterial contamination of Pinellia ternata.

[0030] Fig.14 These are white colonies caused by bacterial contamination of Pinellia ternata.

[0031] Fig.15 Bacterial contamination appears as milky white colonies. DETAILED DESCRIPTION

[0032] The present invention is described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0033] 1. Experimental Materials and Instruments

[0034] 1. Experimental Materials

[0035] Table 1 Materials and reagents

[0036]

[0037] Note: Xinyang Xibanxia was identified by Associate Professor Liang Lixiang as the dried tuber of Pinellia ternata, a plant of the Araceae family.

[0038] 2. Experimental instruments

[0039] Table 2 Experimental instruments

[0040] name model factory Electric pressure steam sterilizer XFS-50CA Zhejiang Xinfeng Medical Equipment Co., Ltd. Double clean bench SW-CJ-2D Zhejiang Sujing Purification Equipment Co., Ltd. Stereo microscope SX-5AP Shanghai Optical Instrument Factory No.1 Electronic analytical balance (0.001g) WT3003 Hangzhou Wante Weighing Instrument Co., Ltd. Acidity meter PHSJ-4F Jinan Welling Medical Equipment Co., Ltd. Purified water machine UPT-60 Suzhou Jiazhou Purification Equipment Co., Ltd. Hot air circulation oven CT-C-0 Nanjing Novi Drying Equipment Co., Ltd. Multifunctional induction cooker C21-Simple101 Guangdong Midea Household Electrical Appliances Manufacturing Co., Ltd. Vaccination equipment sterilizer YH-JZ-II Shanghai Yuhan Biotechnology Co., Ltd. Ozone disinfection machine CBR-3-5g(FL-805N) Shenzhen Feili Electric Technology Co., Ltd.

[0041] Embodiment 1:

[0042] 1. Test methods

[0043] (1) Preparation of sterile MS medium

[0044] Use an electronic balance to weigh 6.5 g of agar, 4.74 g of MS medium, and 30 g of white sugar, and set aside. Add 1000mL of purified water into the pot, pour the weighed agar into the pot, turn on medium-low heat, stir continuously during heating, then pour the weighed MS culture medium powder into the pot, continue heating and stirring, after boiling, pour the weighed white sugar into the pot, continue heating until all materials in the pot are dissolved and the culture medium is clear and transparent, stop heating, measure the pH of the culture medium in the pot with an acidity meter, add 0.1mol / L sodium hydroxide solution if the pH is low, add 0.1mol / L hydrochloric acid solution if the pH is high, adjust the pH to 6.0, cool slightly, add 1mL of 1mg / mL 6-BA and 0.1mL of 0.1mg / mL NAA to the culture medium, stir evenly, divide into culture bottles, about 50mL per bottle, sterilize in an electric pressure steam sterilizer, the sterilization conditions are 0.1Mpa, 121℃, 30min, take out, cool and set aside. Before each experiment, place the lab coat, sterile water, sterile paper, tweezers, scalpels, dissecting needles and operating instruments in an electric pressure steam sterilizer for disinfection.

[0045] (2) Pretreatment of the stem tip of Pinellia ternata

[0046] Select healthy and intact tubers of Pinellia ternata, peel off the skin, rinse the surface dust, soil and other substances with drinking water, cut off the stem tip as the explant for plant tissue culture, put it in a wide-mouth bottle, rinse it several times with drinking water, add a little detergent, shake it gently for 30 minutes, rinse it repeatedly with drinking water, rinse it three times with sterile water, put it in a clean bench with ultraviolet sterilization, and set it aside.

[0047] (3) Disinfection of stem tips

[0048] Disinfect the washed and ready-to-use stem tips of Pinellia ternata according to Table 3. After each disinfection with mercuric chloride, rinse the stem tips with sterile water for 3 to 4 times. Inoculate 60 bottles for each disinfection method.

[0049] Table 3 Disinfection methods

[0050] deal with Disinfection methods Process A 75% ethanol disinfection for 30 seconds, 0.1% mercuric chloride disinfection for 15 minutes Treatment B 5% lemongrass essential oil in 75% ethanol for 30 seconds, 0.1% mercuric chloride for 15 minutes Process C 5% oregano essential oil in 75% ethanol for 30 seconds, 0.1% mercuric chloride for 15 minutes Processing D 5% eucalyptus essential oil in 75% ethanol for 30 seconds, 0.1% mercuric chloride for 15 minutes Process E 5% cinnamon essential oil in 75% ethanol for 30 seconds, 0.1% mercuric chloride for 15 minutes Processing F 5% peppermint essential oil in 75% ethanol for 30 seconds, 0.1% mercuric chloride for 15 minutes

[0051] (4) Vaccination

[0052] Place the prepared sterile paper, sterile culture medium, and the disinfected stem tip of Pinellia ternata in the clean bench, turn on the ultraviolet lamp and fan of the clean bench, and put the sterilized tweezers, scalpels, dissecting needles and other instruments into the inoculation instrument sterilizer to heat to about 280°C for 35 minutes. Hang the lab coat behind the first door of the sterile room for ultraviolet disinfection, put disposable medical gloves, disposable medical hoods, disposable medical masks, and 75% ethanol under the ultraviolet lamp of the transfer window for disinfection, turn on the ozone disinfector and ultraviolet lamp in the main room of the sterile room, and turn on the air conditioner and fan. Wait for the disinfection to be completed, quickly open the first door of the sterile room, turn off the air conditioner and fan, put on the lab coat, quickly press the door handle with your elbow to open the door, go through the second and third doors, spray the whole body with 75% ethanol for disinfection, open the transfer window, take out the corresponding tools, disinfect them with 75% ethanol and wear them, spray the clean bench work panel and handles with 75% ethanol, turn on the lighting and intermediate ventilation gear, lift the clean bench door 15 cm upward with both hands, then spray the hands and arms for disinfection, enter the clean bench with both hands, quickly light the alcohol lamp, wipe the surface of the clean bench with an alcohol cotton ball, move the heated and disinfected tweezers, scalpel, dissecting needle and other tools into a beaker filled with 75% ethanol for cooling, and after completing the above work, rinse each group of shoot tips with sterile water for 15 minutes, rinse the first bottle for 5 minutes, the second bottle for 3 minutes, the third bottle for 3 minutes, the fourth bottle for 2 minutes, and the fifth bottle for 2 minutes. Use tweezers to gently clamp the stem tip of Pinellia ternata rinsed with sterile water to the surface of sterile paper to absorb dry water, then gently peel off the outer layer of the stem tip under a stereo microscope, remove the inner stem tip, cut off the excess part around it with a scalpel, and gently clamp the stem tip with tweezers to inoculate it into a sterile culture bottle containing culture medium. Note: (1) After each inoculation operation is completed, the used tweezers, dissecting needles, scalpels, etc. must be placed in 75% ethanol for disinfection before use. Each time the above tools are used, they must be heated in the outer flame of an alcohol lamp until the ethanol evaporates and the surface is dry. (2) After opening the lid of the sterile bottle, the bottle mouth should be vertically facing downwards to reduce contamination. (3) The inoculation operation should be performed within 5 cm of the outer flame of the alcohol lamp to reduce contamination.

[0053] Place the inoculated stem tip sterile culture bottle in the Pinellia ternata tissue culture room. The culture conditions are light intensity 2000lx, light time 12h / d, temperature (25±1)℃, and disinfect the Pinellia ternata tissue culture room with an ozone sterilizer for 35 minutes every other day.

[0054] 2. Measurement methods and indicators

[0055] During the tissue culture of the pinellia stem apex, the growth and contamination of the pinellia stem apex in the sterile culture bottle were observed every day. After 7 days, the contamination rate of virus-free seedlings and the germination rate of the stem apex were calculated. The calculation formula was:

[0056] Contamination rate of virus-free seedlings: (number of contaminations by a single disinfection method / 100) × 100%

[0057] Shoot tip germination rate: (single disinfection method germination number / 100) × 100%

[0058] 3. Statistical analysis

[0059] IBM SPSS statistics 26.0 data analysis software was used to perform variance analysis and significant difference analysis on the experimental data, and EXCEL 2010 was used to organize the data.

[0060] Table 4 Variance homogeneity test

[0061]

[0062]

[0063] Note: “-” means there is no such item.

[0064] According to the variance homogeneity test in Table 4, it can be seen that the significance is > 0.05, indicating that the variance is homogeneous and the one-way analysis of variance can be used.

[0065] Table 5 ANOVA

[0066]

[0067] Note: “-” means there is no such item.

[0068] The results of variance analysis in Table 5 showed that the contamination rate of virus-free seedlings was significant at 0.003<0.05, and the survival rate of shoot tips was significant at 0.004<0.05. The analysis showed that the shoot tip tissue culture of Pinellia ternata was treated with the disinfection method in Table 1, and there was statistical significance between the groups in the contamination rate of virus-free seedlings and the germination rate of shoot tips.

[0069] Depend on Figure 1 From the comparison of the contamination rate and stem tip germination rate of each group of virus-free seedlings, it can be seen that the use of different essential oils has a greater impact on the germination rate of the stem tip tissue culture of Pinellia ternata, the germination rate is C>B>D=E>F>A, and the contamination rate is A>F>E=D>B>C. The results show that the disinfection effect of oregano essential oil on the bacteria and fungi of the stem tip of Pinellia ternata is better than that of lemongrass essential oil, eucalyptus essential oil, cinnamon essential oil, and peppermint essential oil, and the combination of adding essential oils has better bactericidal effect than 75% ethanol and 0.1% mercuric chloride.

[0070] 4. Optimization of the method for disinfecting the stem tip of Pinellia ternata by orthogonal test

[0071] (1) Single factor test

[0072] 1) Oregano essential oil disinfection time

[0073] The disinfection time of mercuric chloride was fixed at 15min, the washing time of sterile water was 15mim, and the concentration of oregano essential oil was 5%. The disinfection was carried out under the conditions of oregano essential oil disinfection time of 10s, 30s, 50s, 70s, and 90s respectively. The effect of different oregano essential oil disinfection time on the germination rate of the stem tip of Pinellia ternata was investigated. The results are shown in Figure 2 . The results showed that with the increase of oregano essential oil disinfection time, the germination rate of the stem tip of Pinellia ternata showed a trend of first increasing and then decreasing. Since oregano essential oil was disinfected with 75% ethanol, the stem tip cells of Pinellia ternata may be destroyed and gradually die with the increase of disinfection time in 75% ethanol. The germination rate of the stem tip of Pinellia ternata was the highest when the oregano essential oil disinfection time was 30s and 50s, and the survival rate was higher when the oregano essential oil disinfection time ranged from 30s to 70s. In order to save time in subsequent experiments, the oregano essential oil disinfection time was fixed at 30s.

[0074] 2) Mercuric chloride disinfection time

[0075] The disinfection time of oregano essential oil was fixed at 30s, the washing time of sterile water was 15min, the concentration of oregano essential oil was 5%, and the disinfection time was 5min, 10min, 15min, 20min, and 25min under 0.1% mercuric chloride. The effect of different mercuric chloride disinfection time on the germination rate of the stem tip of Pinellia ternata was investigated. The results are shown in Figure 3 The results showed that with the increase of mercuric chloride disinfection time, the germination rate of the stem tip of Pinellia ternata changed greatly, showing a trend of first increasing and then decreasing. It may be that the short immersion time of 0.1% mercuric chloride is not enough to completely kill the bacteria. The longer the disinfection time, the more serious the damage to the stem tip, resulting in the weaker ability of the stem tip to resist the damage of pathogens, the gradually increased contamination rate, and the lower the survival rate. When the disinfection time is 15 minutes, the survival rate of the stem tip of Pinellia ternata is the highest, so the disinfection time range of 0.1% mercuric chloride is selected as 10 minutes to 20 minutes.

[0076] 3) Sterile water flushing time

[0077] The disinfection time of oregano essential oil was fixed at 30s, the disinfection time of mercuric chloride was 15min, and the concentration of oregano essential oil was 5%. After each disinfection operation, the stem tip of Pinellia ternata was rinsed with sterile water for 5min, 10min, 15min, 20min, and 25min respectively. The effect of different sterile water rinsing times on the germination rate of the stem tip of Pinellia ternata was investigated. The results are shown in Figure 4The results showed that as the sterile water washing time increased, the germination rate of the stem tip of Pinellia ternata showed a trend of first increasing and then stabilizing. The germination rate of the stem tip of Pinellia ternata was the highest when the sterile water washing time was 10min, 20min, and 25min, which may be because the residual mercuric chloride liquid had been washed away by sterile water washing for 10min. In order to save time in subsequent experiments, the sterile water washing time was fixed at 10min.

[0078] 4) Oregano essential oil concentration

[0079] The disinfection time of oregano essential oil was fixed at 30s, the disinfection time of mercuric chloride was 15min, and the rinsing time of sterile water was 10min. The stem tips of Pinellia ternata were disinfected at oregano essential oil concentrations of 1%, 3%, 5%, 7%, 9%, and 11%, respectively. The effects of different oregano essential oil concentrations on the germination rate of Pinellia ternata were investigated. The results are shown in Table 1. Figure 5 . The results showed that with the increase of oregano essential oil concentration, the survival rate of Pinellia ternata tissue culture showed a trend of first increasing and then decreasing. It may be because low concentration of oregano essential oil is not enough to kill bacteria or fungi on the surface of the stem tip, and high concentration of oregano essential oil inhibits the growth of the stem tip, making it difficult to germinate for a long time. When the concentration of oregano essential oil is 5%, the germination rate of Pinellia ternata stem tip is the highest, so the concentration range of oregano essential oil is selected to be 3% to 7%.

[0080] (2) Orthogonal test

[0081] According to the results of single factor investigation, the evaluation indicators were the contamination rate and survival rate of tissue culture, and the disinfection time of ethanol oregano essential oil, dilution concentration, mercuric chloride disinfection time, and sterile water washing time were selected as the main factors. 9 (3 4 ) was used to conduct a four-factor three-level orthogonal test design. The orthogonal test factor levels are shown in Table 6, the orthogonal test intuitive analysis is shown in Table 7, and the variance analysis is shown in Table 8.

[0082] Table 6 Orthogonal test factor level table

[0083] factor A B C D Level 1 30 10 10 3 Level 2 50 15 15 5 Level 3 70 20 20 7

[0084] Table 7 Orthogonal test intuitive analysis table

[0085]

[0086]

[0087] α is the significance level of variance analysis in orthogonal experimental design. When α is 0.05, variance analysis is performed with oregano essential oil concentration (D) as the error, as shown in Table 8.

[0088] Table 8 Analysis of variance

[0089] factor Sum of Squared Deviations Degrees of Freedom F Ratio F critical value Oregano essential oil disinfection time (A) 0.222 2 1.000 19.000 Mercuric chloride disinfection time (B) 6.889 2 31.032 19.000 Sterile water flushing time (C) 5.556 2 25.027 19.000 Oregano essential oil concentration (D) 0.889 2 4.005 19.000 error 0.220 2

[0090] The orthogonal test intuitive analysis table in Table 7 and the variance analysis in Table 8 show that factors B and C are more significant, and B>C. The order of influence of each factor on the disinfection effect of the stem tip is B>C>D>A, that is, mercuric chloride disinfection time>sterile water washing time>oregano essential oil concentration>oregano essential oil disinfection time. The best disinfection combination obtained by the orthogonal test is A 2 B 3 C 1 D 1 That is, the disinfection time of oregano essential oil is 50s, the disinfection time of mercuric chloride is 20min, the sterile water washing time is 10min, and the oregano essential oil concentration of 3% is the best disinfection combination for the stem tip of Pinellia ternata.

[0091] (3) Repeated validation test results

[0092] According to the optimal disinfection process conditions of Pinellia ternata A 2 B 3 C 1 D 1 , 150 stem tips of Pinellia ternata were taken and three groups of parallel verification tests were carried out. The results are shown in the table. The germination rates of the stem tips of Pinellia ternata were measured to be 92%, 94% and 90% respectively, and the average value of the three measurements was 92%, with an RSD of 2.17%. The contamination rates of the stem tips of Pinellia ternata were measured to be 8%, 6% and 10% respectively, and the average value of the three measurements was 8%.

[0093] Table 9 Verification test results

[0094]

[0095]

[0096] Note: “-” means there is no such item.

[0097] The results of the study showed that the optimal disinfection solution was A 2 B 3 C 1 D 1 , that is, oregano essential oil disinfection for 50s, mercuric chloride disinfection time for 20min, sterile water washing for 10min, oregano essential oil concentration of 3%, the average germination rate of Pinellia stem tips reached 90%, growth was rapid and the survival state of the stem tips was good. After 7 days of observation, the average contamination rate of Pinellia virus-free seedlings was 10%.

[0098] Explant sterilization is one of the key factors for the success of plant tissue culture. Therefore, finding a fast and efficient explant sterilization method is crucial to improving the efficiency and success rate of plant tissue culture. Commonly used disinfectants include sodium hypochlorite, calcium hypochlorite, hydrogen peroxide, mercuric chloride, etc. Among them, the sterilization effect of mercuric chloride is widely recognized, which is consistent with the conclusion of this experiment.

[0099] Explant disinfection is an important part of the plant tissue culture process. During the inoculation of the shoot tips of Pinellia ternata, the infection of the shoot tips of Pinellia ternata by bacteria or fungi was very obvious, which seriously affected the survival rate of tissue culture. In the long-term experiment, it was found that the tissue culture of the shoot tips of Pinellia ternata can grow from the original several millimeters to about 4 cm in about 7 days. The tissue culture of the shoot tips of Pinellia ternata can reduce the viruses accumulated in the asexual reproduction of Pinellia ternata several times, and it is currently a commonly used detoxification method for Pinellia ternata. The shoot tips of Pinellia ternata were disinfected through the above strict disinfection procedures, and the shoot tips of Pinellia ternata grew well in sterile bottles. The experiment also experienced many failures. When exploring the concentration of essential oil disinfection, the number of failures was relatively large. The shoot tips inoculated in the sterile bottle did not grow for 7 days. The reason for this was that although high concentrations of essential oils can inhibit and kill bacteria or fungi on the surface of Pinellia ternata, high concentrations of essential oils may also inhibit the growth of Pinellia ternata cells. Finally, the dilution concentration of the essential oil was controlled at about 5%, with good sterilization effect and good growth (such as Figure 6 , Figure 7 ).

[0100] After many failed experiments and empirical analysis, plant explants are not only susceptible to foreign bacteria during tissue culture, but also contaminated again 7 days after inoculation. This is often caused by contamination from endophytes. Endophytes are commonly found in plant tissues and are difficult to completely eliminate using conventional disinfection methods. When plant tissues are cultured in vitro, there are no symptoms within 3 to 5 days of inoculation, but colonies continue to appear during the subsequent culture process. Bacteria such as Bacillus and Pseudomonas and Rhizopus (such as Figure 8 ), Aspergillus niger (such as Fig. 9 ), Penicillium (such as Fig.10 ), yeast and other fungi are the main contaminants. Bacterial contamination is manifested by obvious water stains around the explants or on the surface of the culture medium (such as Fig.11 ), oily bubbles (such as Fig.12 ) or shrunken and yellow, white, or milky white (such as Fig.13 , Fig.14 , Fig.15 ) and other colored colonies. Therefore, exploring a method for explant disinfection that can effectively kill or reduce plant endophytes can also increase its germination rate.

[0101] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0102] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for disinfecting a Pinellia explant, characterized in that: include: The explants of Pinellia ternata are placed in ethanol containing 70% to 80% of plant essential oil by volume for disinfection for 30s to 70s, and then disinfected in mercuric chloride with a mass fraction of 0.08% to 0.13%, and the mercuric chloride remaining on the surface of the explants is washed off after the disinfection with mercuric chloride to obtain the disinfected explants; Wherein, the volume fraction of the plant essential oil is 3% to 7%; The plant essential oil is selected from one or more of lemongrass essential oil, oregano essential oil, eucalyptus essential oil, cinnamon essential oil and peppermint essential oil.

2. The disinfection method according to claim 1, characterized in that: The time for disinfection using the ethanol is 25s to 35s.

3. The disinfection method according to claim 1, characterized in that: The time for disinfection using the mercuric chloride is 10 minutes to 20 minutes.

4. The disinfection method according to claim 1, characterized in that: Rinse with sterile water to wash away any residual mercuric chloride on the surface of the explants.

5. The disinfection method according to claim 4, characterized in that: The time for sterile water flushing is 10 minutes to 25 minutes.

6. The disinfection method according to claim 1, characterized in that: After the outer skin of the pinellia explant is peeled off, dust and dirt are washed off and then disinfected with the ethanol.

7. The disinfection method according to claim 1, characterized in that: The pinellia explant is the tuber or leaf of pinellia.

8. A method for tissue culture of Pinellia ternata, characterized in that: The Pinellia explant is disinfected using the disinfection method as described in any one of claims 1 to 7 to obtain a disinfected explant; the disinfected explant is inoculated into a culture medium for culture to obtain callus tissue, and then the callus tissue is induced to root and germinate to obtain a tissue culture seedling.

9. The tissue culture method according to claim 8, wherein The culture conditions are as follows: light intensity of 1800lx to 2200lx, light duration of 12h / d to 16h / d, and temperature of 24°C to 26°C.

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