A tissue culture method for Chinese bayberry

Abstract

The present invention relates to the technical field of plant tissue culture, and particularly relates to a tissue culture method for Chinese bayberry. The method includes steps of explant selection, explant disinfection, bud induction culture, rooting culture and acclimatization and transplantation. Among them, the explant disinfection combined with the bud induction culture method effectively solves the problems of difficult disinfection and easy browning of Chinese bayberry explants. The tissue culture method for Chinese bayberry of the present invention can rapidly obtain a large number of Chinese bayberry seedlings, and the Chinese bayberry seedlings grow well with a high transplantation survival rate, and can be applied to the large-scale aseptic propagation of Chinese bayberry.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and specifically relates to a tissue culture method for Myrica rubra. Background Art

[0002] Myrica rubra (scientific name: Myrica rubra Sieb. et Zucc., English name: China Bayberry) is the most characteristic fruit tree species in China. The fruit of Myrica rubra has a unique flavor, is rich in nutrients, and contains substances such as polysaccharides and polyphenols, with high nutritional and economic value. Due to the long juvenile period and difficult rooting of Myrica rubra, grafting is commonly used for the propagation of superior Myrica rubra varieties in current production. However, although grafting propagation has the advantage of maintaining the excellent characteristics of fruit trees, in recent years, diseases of Myrica rubra have occurred frequently, and due to the certain incubation period of pathogenic microorganisms, the traditional grafting propagation method is more likely to exacerbate the spread of the disease, which has caused serious harm to several major Myrica rubra production areas. Therefore, it is particularly important to carry out aseptic propagation through scientific means.

[0003] Plant tissue culture refers to the process of culturing embryos, organs, tissues, protoplasts, etc. of plants in vitro under sterile and artificially controlled environmental conditions using artificial culture to regenerate and develop into complete plants. Plant tissue culture has significant advantages in plant propagation and breeding, such as: small occupied space, not restricted by regions and seasons; virus elimination of crops; short culture cycle; no variation, and can maintain the excellent characteristics of the original mother plant; and so on.

[0004] Myrica rubra belongs to woody plants. Due to its perennial nature and high degree of lignification, it is difficult to disinfect the stem segments of woody plants. Chinese Patent No. 201010603448.4 records: "The new leaves, bud tips, etc. of Myrica rubra are sensitive to sterilizing agents such as alcohol, sodium hypochlorite, and mercuric chloride, and are extremely easy to cause damage to the explants during the sterilization process. While old branches are often difficult to sterilize. Although they are not as sensitive as new leaves and bud tips, they are also easily damaged if the sterilization time is slightly longer, and if the time is short, it will lead to contamination." Therefore, how to sterilize the explants is a major problem faced by the tissue culture of Myrica rubra. In addition, during the tissue culture process of Myrica rubra, we found that the explants are prone to browning during the culture process and ultimately lead to the corruption and death of Myrica rubra tissue. Summary of the Invention

[0005] In view of the above problems, the present invention provides a tissue culture method for Myrica rubra, which reduces the contamination rate in the culture of Myrica rubra explants to less than 5%, and at the same time, significantly reduces tissue browning and effectively improves the success rate of tissue culture of Myrica rubra.

[0006] The object of the present invention is achieved through the following technical solutions:

[0007] A tissue culture method for Chinese bayberry, the method comprising the following steps;

[0008] (1) Selection of explants: Take new branches of Chinese bayberry, remove the leaves and retain the axillary buds, and cut them into explants 8 - 12 cm long;

[0009] (2) Disinfection of explants: Put the explants obtained in step (1) into a laminar flow hood for disinfection treatment;

[0010] (3) Bud induction culture: Cut off both ends of the explants after disinfection treatment in step (2), and cut to obtain stem segments 2 - 5 cm long with axillary buds, and inoculate the stem segments into the bud induction medium;

[0011] (4) Rooting culture: Cut off the axillary buds of the stem segments after bud induction culture in step (3) and inoculate them into the rooting medium for rooting culture;

[0012] (5) Acclimatization and transplantation: When the adventitious roots grow to 2 - 4 cm in step (4) during rooting culture, acclimatize the new seedlings and transplant them into the soil;

[0013] Among them, the specific process of the disinfection treatment includes: Disinfect the explants with 75% alcohol for 25 - 35 s, then rinse with sterile water 2 times or more, disinfect with 15% sodium hypochlorite for 10 - 20 min, and rinse with sterile water 3 times or more;

[0014] The bud induction medium contains 200 - 300 μL / L ProClin preservative, 0.8 - 1.2 g / L polyvinylpyrrolidone (PVP), 0.3 - 0.6 g / L ascorbic acid (LAA), and 0.8 - 1.2 g / L activated carbon (AC).

[0015] ProClin preservative is a new generation of high - efficiency biological preservative, including ProClin150, 200, 300, 5000, and its active ingredients are mainly 2 - methyl - 4 - isothiazolin - 3 - one (MCI) and 5 - chloro - 2 - methyl - 4 - isothiazolin - 3 - one (CMCI). ProClin preservative has broad - spectrum antibacterial activity, can inhibit the growth of microorganisms such as bacteria, fungi, and yeasts for a relatively long time; at the same time, it can maintain the activity of enzymes in the system and can exert a good antibacterial effect at a low concentration of 6 - 30 ppm. It is a bacteriostatic agent dedicated to in vitro diagnostic reagents (IVD). At present, there are no relevant research reports on the use of ProClin preservative in plant tissue culture, and there are no relevant reports on the application of ProClin preservative in the tissue culture of Chinese bayberry.

[0016] During the research process of this application, it was unexpectedly found that adding 200-300 μL / L of ProClin preservative to the bud induction medium could effectively reduce the contamination of explants. Combining with the described disinfection treatment method, the contamination rate of explants could be controlled below 5%. Adding 0.8-1.2 g / L of polyvinylpyrrolidone (PVP), 0.3-0.6 g / L of ascorbic acid (LAA), and 0.8-1.2 g / L of activated carbon (AC) to the bud induction medium could effectively reduce the browning rate of explants, and the browning rate was below 10%. In addition, under the conditions of disinfection and bud induction culture of the explants, the survival rate of the explants was high and the growth was good.

[0017] In some embodiments, the formula of the bud induction medium is: WPM medium + 0.5 g / L ascorbic acid (LAA) + 1.0 g / L polyvinylpyrrolidone (PVP) + 1.0 g / L activated carbon (AC) + 0.5 mg / L 6-benzyladenine (6-BA) + 0.04 mg / L naphthaleneacetic acid (NAA) + 250 μL / L ProClin preservative + 6 g / L agar (Gel) with pH = 5.2.

[0018] In some embodiments, the formula of the rooting medium is: 1 / 2MS medium + 0.5 g / L ascorbic acid (LAA) + 1.0 g / L polyvinylpyrrolidone (PVP) + 1.0 g / L activated carbon (AC) + 0.5 mg / L indolebutyric acid (IBA) + 6 g / L agar (Gel) with pH = 5.2.

[0019] In some embodiments, after 18-25 days of bud induction culture, when the axillary buds grow to 2-5 cm, the axillary buds are cut for rooting culture.

[0020] In some embodiments, the ProClin preservative is ProClin300.

[0021] In some embodiments, the conditions of the bud induction culture include: the light intensity is 1800-2500 Lx, and the light cycle is 14-16 h / d; the temperature is 28°C ± 2°C during light, and the temperature is 26°C ± 2°C when there is no light.

[0022] In some embodiments, the conditions of the rooting culture are the same as those of the bud induction culture.

[0023] In some embodiments, the steps of acclimatizing the newly germinated seedlings include: gradually opening the lids of the tissue culture bottles and spraying clear water every day; when all the lids are opened, wash the root medium of the Myrica rubra seedlings clean and transplant them into the soil.

[0024] In some embodiments, the explants are temporarily stored in a 0.5 g / L ascorbic acid (LAA) solution after disinfection.

[0025] Compared with the prior art, the present invention has the following technical advantages: (1) The explant disinfection method of the Myrica rubra tissue culture method of the present invention combined with the use of ProClin preservative in the bud induction medium greatly reduces the contamination rate of explants, effectively solving the problems of difficult sterilization and easy contamination faced by Myrica rubra tissue culture. (2) The present invention uses sodium hypochlorite to replace the traditional mercuric chloride disinfectant, which has low toxicity and avoids the harm of mercuric chloride disinfectant to users and the environment. (3) The Myrica rubra tissue culture method of the present invention effectively solves the problem of explant browning. The Myrica rubra seedlings obtained by the Myrica rubra tissue culture method of the present invention grow well and have a high transplanting survival rate, and can be applied to the large-scale aseptic propagation of Myrica rubra. Brief Description of the Drawings

[0026] Figure 1 Photo of bud induction culture of Myrica rubra explants in Group 4 in Example 1;

[0027] Figure 2 Photo of bud induction culture of Myrica rubra explants in Group 8 in Example 1;

[0028] Figure 3 Process of Myrica rubra bud induction and adventitious root generation. A is the bud induction stage, and axillary buds can be seen germinating; B is the axillary buds growing larger, and the bud induction process has been completed; C is cutting the buds of this size and transplanting them into the rooting medium; D has taken root. Detailed Embodiments

[0029] The technical solutions of the present invention are further described below through specific embodiments. The specific embodiments do not represent limitations on the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention. The following embodiments can be referenced, borrowed, or combined with each other. For the same terms, similar features, and similar implementation steps in different embodiments, they will not be described repeatedly.

[0030] The main reagent sources used in the following embodiments of the present application are as follows:

[0031] WPM medium: from Beijing Coolaber Technology Co., Ltd.

[0032] MS medium: from Beijing Coolaber Technology Co., Ltd.

[0033] LAA: L-ascorbic acid, from Sangon Biotech (Shanghai) Co., Ltd.

[0034] PVP: polyvinylpyrrolidone, from Sangon Biotech (Shanghai) Co., Ltd.

[0035] AC: activated carbon, from Kangpeisi Biology.

[0036] 6-BA: 6-benzyladenine, from Sangon Biotech (Shanghai) Co., Ltd.

[0037] NAA: α-naphthaleneacetic acid, from Sangon Biotech (Shanghai) Co., Ltd.

[0038] IBA: indolebutyric acid or indole-3-butyric acid, from Sangon Biotech (Shanghai) Co., Ltd.

[0039] Gel: plant gel, from Sigma. When used, adjust the pH to 5.2 with 1M potassium hydroxide.

[0040] Example 1. Research on the prevention of Myrica rubra explant contamination

[0041] To solve the problems of difficult disinfection and easy contamination of Myrica rubra explants, this application conducted a detailed study on the method for preventing Myrica rubra explant contamination, as follows.

[0042] 1. Test method

[0043] Select healthy and disease-free Myrica rubra new branches with good growth, remove the leaves without damaging the axillary buds, cut them into 10-cm stem segments with axillary buds, and rinse them with running water for 30 min to remove the dust on the surface of the explants to obtain the explants. Treat them according to the following method (3 bottles for the same treatment, about 30 in each bottle):

[0044] Group 1: Immerse the explants in 75% alcohol for 30 s and then rinse them twice with sterile water. Cut off both ends of the explants, cut them into 3 parts to obtain 3-cm stem segments with axillary buds, and inoculate the stem segments into bud induction medium 1.

[0045] Group 2: Immerse the explants in 75% alcohol for 30 s and then rinse them twice with sterile water, soak them in 5% sodium hypochlorite for 15 min, and rinse them three times with sterile water. Take out the explants, cut off both ends, cut them into 3 parts to obtain 3-cm stem segments with axillary buds, and inoculate the stem segments into bud induction medium 1.

[0046] Group 3: Immerse the explants in 75% alcohol for 30 s and then rinse them twice with sterile water, soak them in 10% sodium hypochlorite for 15 min, and rinse them three times with sterile water. Take out the explants, cut off both ends, cut them into 3 parts to obtain 3-cm stem segments with axillary buds, and inoculate the stem segments into bud induction medium 1.

[0047] Group 4: Immerse the explants in 75% alcohol for 30 s and then rinse them twice with sterile water, soak them in 15% sodium hypochlorite for 15 min, and rinse them three times with sterile water. Take out the explants, cut off both ends, cut them into 3 parts to obtain 3-cm stem segments with axillary buds, and inoculate the stem segments into bud induction medium 1.

[0048] Group 5: Soak the explants in 75% alcohol for 30 s, then rinse them twice with sterile water. Soak them in 20% sodium hypochlorite for 15 min and rinse three times with sterile water. Take out the explants, cut off both ends, cut them into three parts to obtain stem segments with axillary buds and 3 cm in length, and inoculate the stem segments into Bud Induction Medium 1.

[0049] Group 6: Soak the explants in 75% alcohol for 30 s, then rinse them twice with sterile water. Cut off both ends of the explants, cut them into three parts to obtain stem segments with axillary buds and 3 cm in length, and inoculate the stem segments into Bud Induction Medium 2.

[0050] Group 7: Soak the explants in 75% alcohol for 30 s, then rinse them twice with sterile water. Soak them in 10% sodium hypochlorite for 15 min and rinse three times with sterile water. Take out the explants, cut off both ends, cut them into three parts to obtain stem segments with axillary buds and 3 cm in length, and inoculate the stem segments into Bud Induction Medium 2.

[0051] Group 8: Soak the explants in 75% alcohol for 30 s, then rinse them twice with sterile water. Soak them in 15% sodium hypochlorite for 15 min and rinse three times with sterile water. Take out the explants, cut off both ends, cut them into three parts to obtain stem segments with axillary buds and 3 cm in length, and inoculate the stem segments into Bud Induction Medium 2.

[0052] Among them, the formula of Bud Induction Medium 1 is: WPM medium + 0.5 g / L LAA + 1.0 g / L AC + 0.5 mg / L 6 - BA + 0.04 mg / L NAA + 6 g / L pH = 5.2 Gel. The formula of Bud Induction Medium 2 is: WPM medium + 0.5 g / L LAA + 1.0 g / L AC + 0.5 mg / L 6 - BA + 0.04 mg / L NAA + 250 μL / L ProClin300 + 6 g / L pH = 5.2 Gel.

[0053] After culturing for 7 days, observe and count the contamination status of the explants. The contamination rate = the number of contaminated explants / the total number of explants × 100. The culture conditions include: the light intensity is 2000 Lx ± 200 Lx, the light cycle is 14 - 16 h / d; the temperature is 28°C ± 2°C during light, and 26°C ± 2°C when there is no light.

[0054] 2. Test Results and Analysis

[0055] The test results are as follows in the table:

[0056] Table 1 Contamination of Myrica rubra Explants under Different Disinfection Conditions

[0057] Group Total number of explants Number of contaminated explants Contamination rate (%) Group 1 90 61 67.8 Group 2 93 32 34.4 Group 3 87 26 30.0 Group 4 84 14 16.7 Group 5 96 18 18.8 Group 6 93 20 21.5 Group 7 87 6 6.9 Group 8 84 2 2.4

[0058] As can be seen from the results in the table: (1) Comparing the results of Groups 1 to 5, using sodium hypochlorite for disinfection can significantly reduce the contamination rate. When the concentration of sodium hypochlorite is 15%, the lowest contamination rate is 16.7%, indicating that the optimal disinfection concentration of sodium hypochlorite for treating Myrica rubra explants is 15%. (2) Comparing the results of Group 1 and Group 6, using Bud Induction Medium 2 can greatly reduce the contamination rate, indicating that adding ProClin300 to the bud induction medium can play a good role in preventing contamination. (3) Comparing Groups 1 to 8, the contamination rates of Groups 7 and 8 are significantly lower than those of other groups. Especially, the contamination rate of Group 8 is the lowest, at 2.4%, and the contamination rate is lower than 5%. This shows that using sodium hypochlorite treatment combined with adding ProClin300 can play a better role in resisting explant contamination, especially under the condition of using 15% sodium hypochlorite treatment combined with adding ProClin300, the effect is optimal.

[0059] It was found in the study that the explants in the above-mentioned Groups 1 to 8 all showed browning phenomenon after cultivation, such as Figure 1 (Group 4) and Figure 2 (Group 8), that is, there is a browning problem whether using Bud Induction Medium 1 or Bud Induction Medium 2.

[0060] Example 2. Study on anti-browning of Myrica rubra explants

[0061] In order to solve the problem that Myrica rubra explants are prone to browning, this application conducted a comparative study on the bud induction medium.

[0062] 1. Test method

[0063] Select healthy and disease-free, well-growing new branches of Myrica rubra, remove the leaves and keep the axillary buds undamaged, cut them into 10-cm stem segments with axillary buds, rinse them with running water for 30 minutes to remove the dust on the surface of the explants, and obtain the explants. Disinfect the explants with 75% alcohol for 30 s, then rinse them twice with sterile water, soak them in 15% sodium hypochlorite for 15 minutes, and rinse them three times with sterile water. Take out and cut off both ends of the explants, and cut them into 3 parts to obtain stem segments about 3 cm long with axillary buds. Inoculate these stem segments into different bud induction media respectively, denoted as Group 9: Bud Induction Medium 2; Group 10: Bud Induction Medium 3; Group 11: Bud Induction Medium 4; Group 12: Bud Induction Medium 5; Group 13: Bud Induction Medium 6 (3 bottles for the same treatment, about 15 in each bottle)

[0064] The formula of Bud Induction Medium 2 is the same as that in Example 1.

[0065] The formula of Bud Induction Medium 3 is: WPM medium + 0.5 g / L LAA + 1.0 g / L PVP + 0.5 mg / L 6 - BA + 0.04 mg / L NAA + 250 μL / L ProClin300 + 6 g / L pH = 5.2 Gel.

[0066] The formula of bud induction medium 4 is: WPM medium + 1.0 g / L AC + 0.5 mg / L 6-BA + 0.04 mg / L NAA + 250 μL / L Proclin 300 + 6 g / L pH = 5.2 Gel.

[0067] The formula of bud induction medium 5 is: WPM medium + 1.0 g / L PVP + 1.0 g / L AC + 0.5 mg / L 6-BA + 0.04 mg / L NAA + 250 μL / L Proclin 300 + 6 g / L pH = 5.2 Gel.

[0068] The formula of bud induction medium 6 is: WPM medium + 0.5 g / L LAA + 1.0 g / L PVP + 1.0 g / L AC + 0.5 mg / L 6-BA + 0.04 mg / L NAA + 250 μL / L Proclin 300 + 6 g / L pH = 5.2 Gel.

[0069] After culturing for 7 days, observe and count the browning situation of the explants. Browning rate = number of browning explants / total number of explants × 100. The culture conditions include: light intensity of 2000 Lx ± 200 Lx, light cycle of 14 - 16 h / d; temperature of 28°C ± 2°C during light and 26°C ± 2°C without light.

[0070] 2. Test results

[0071] The statistical results of the browning rate are as follows in the table:

[0072] Table 2 Browning rate of Myrica rubra explants under different bud induction media

[0073]

[0074] From the data in the table, it can be seen that the browning rates from high to low are: Group 11 > Group 9 > Group 10 > Group 12 > Group 13. The browning rate of Group 13 is the lowest, at 6.5%. Being lower than 10% indicates that the bud induction medium of Group 13 has the best effect on inhibiting the browning of Myrica rubra explants.

[0075] Yangmei belongs to woody plants. During the in vitro culture process of woody plants, a large amount of polyphenols such as tannins will be produced, which is likely to cause the browning of explants. Adding activated carbon to the culture medium can adsorb oxidizing substances, thereby inhibiting browning. Polyvinylpyrrolidone (PVP) is a synthetic water-soluble polymer compound that can form complexes with polyphenols (such as tannins), thereby reducing the amount of polyphenols and achieving the effect of inhibiting browning. From the above results, it can also be seen that the effect of group 12 is better than that of group 11, indicating that the effect of inhibiting the browning of Yangmei explants is better when PVP and AC are used together. It may be that AC and PVP respectively achieve a synergistic effect through adsorption and complexation, which can further reduce the polyphenol concentration and inhibit browning. It should be noted that the browning rate of group 13 is the lowest, indicating that when LAA, PVP and AC are used in combination, the effect of inhibiting the browning rate is the best. The browning reaction is divided into enzymatic browning and non-enzymatic browning. Enzymatic browning is the reaction process in which phenolase catalyzes phenols to form quinones and their polymers under aerobic conditions, and it is also the main factor leading to the browning of explants. Non-enzymatic browning refers to the phenomenon that substances such as phenols are oxidized under non-enzymatic conditions and show a significant color change and tend to deepen, which is mainly caused by environmental stress. The reason why the combination of PVP, AC and LAA inhibits browning optimally may be: PVP complexes with polyphenols to reduce the polyphenol content; AC adsorbs quinone substances; LAA is an antioxidant that can reduce the Cu 2+ to Cu + , and can inhibit the activity of phenol oxidase. PVP, AC and LAA play a synergistic role in inhibiting browning from three aspects: reducing the substrate of the browning reaction, reducing the product of the browning reaction and reducing the enzyme activity. At the same time, as a vitamin, LAA itself also plays an oxidation-reduction role in the electron transfer system in plants, can capture free radicals in plants, promote plant metabolism, regulate plant growth, and may also enhance the plant's own anti-browning ability. The specific reasons still need further research.

[0076] Example 3. A method for tissue culture of Yangmei

[0077] (1) Selection of explants

[0078] Select healthy and disease-free, well-growing new branches of Yangmei, remove the leaves and ensure that the axillary buds are not damaged, cut them into 10-cm stem segments with axillary buds, and rinse them with running water for 30 minutes to remove the dust on the surface of the explants.

[0079] (2) Disinfection of explants

[0080] Transfer the above-cleaned explants to a clean bench for disinfection. The explants are disinfected with 75% alcohol for 30 s, then washed twice with sterile water, and then disinfected with 15% sodium hypochlorite for 15 min, and rinsed 5 times with sterile water. The disinfected explants are temporarily immersed in a 0.5 g / L LAA sterile solution for standby (the storage time does not exceed two days).

[0081] (3) Bud induction culture

[0082] Trim both ends of the disinfected explants and divide them into 3 stem segments about 3 cm long with axillary buds. Inoculate these stem segments into the bud induction medium. Place the stem segments flat on the medium to induce the germination of axillary buds.

[0083] The formula of the bud induction medium is: WPM medium + 0.5 g / L LAA + 1.0 g / L PVP + 1.0 g / L AC + 0.5 mg / L 6 - BA + 0.04 mg / L NAA + 250 μL / L Proclin300 + 6 g / L Gel (pH 5.2).

[0084] The culture conditions are: light intensity is 2000 Lx ± 200 Lx, light cycle is 14 - 16 h / d; the temperature during light (daytime) is 28℃ ± 2℃, and the temperature during non - light (nighttime) is 26℃ ± 2℃.

[0085] After culturing for 7 days, the axillary buds can be seen germinating with the naked eye. After 14 days, the axillary buds germinate. Continue to culture until 21 days, and the axillary buds grow to about 3 cm.

[0086] (4) Rooting culture

[0087] Cut the axillary buds about 3 cm high in step (3) and inoculate them into the rooting medium, with the bud tip facing up and the stem base inserted into the medium about 0.5 cm to induce the formation of adventitious roots.

[0088] The formula of the rooting medium is: 1 / 2 MS medium + 0.5 g / L LAA + 1.0 g / L PVP + 1.0 g / L AC + 0.5 mg / L IBA (indole - 3 - butyric acid) + 6 g / L Gel (pH 5.2).

[0089] The culture conditions are the same as in step (3). After culturing for 28 days, adventitious roots can be seen breaking through the epidermis of the stem segment with the naked eye.

[0090] (5) Acclimatization and transplantation

[0091] When the adventitious roots of the newly - born seedlings in step (4) grow to about 3 cm long, gradually open the lid of the tissue culture bottle (about 1 / 4 each time), and spray clear water every day to maintain the humidity. When the lid is fully opened, wash the medium from the roots of the Myrica rubra seedlings and transplant them into the nutrient soil.

[0092] The culture conditions are: light intensity is 2000 Lx ± 200 Lx, light cycle is 14 - 16 h / d; the temperature during light (daytime) is 28℃ ± 2℃, the temperature during non - light (nighttime) is 26℃ ± 2℃, and the humidity is 90% ± 2%.

[0093] During the cultivation process, it was found that although browning of Myrica rubra mainly occurred in the bud induction culture stage, there was also less browning phenomenon in the rooting culture stage. Adding LAA, PVP, and AC to the rooting medium could also play a good role in anti-browning.

[0094] The growth status of explants during the tissue culture process of Myrica rubra is as Figure 3 shown. It can be seen that Myrica rubra seedlings with good growth status can be obtained by the above method. The obtained Myrica rubra seedlings were transplanted into the soil, and the survival rate reached 87.5%.

[0095] It should be noted that the above embodiments only exemplarily explain the present invention. General adjustments or equivalent replacements are made to the various condition parameters in the above embodiments, and the effects after adjustment or equivalent replacement are the same. The adjusted or equivalently replaced solutions are still within the scope of this application. For example, ProClin300 in the embodiment is modified to other ProClin preservatives, such as ProClin150, ProClin200, or ProClin5000; the concentrations of LAA, PVP, 6-BA, NAA, Gel, etc. in the culture medium are simply adjusted, or some conventional components are added to the culture medium.

Claims

1. A tissue culture method for Chinese bayberry, characterized in that, The method comprises the following steps; (1) Selection of explants: Take the newly grown branches of Chinese bayberry, remove the leaves and retain the axillary buds, and cut them into explants with a length of 8-12 cm; (2) Disinfection of explants: Put the explants obtained in step (1) into a clean bench for disinfection treatment; (3) Bud induction culture: Cut off both ends of the explants after disinfection treatment in step (2), and cut to obtain a stem segment with axillary buds and a length of 2-5 cm, and inoculate the stem segment into the bud induction medium; (4) Rooting culture: Cut off the axillary buds of the stem segments after bud induction culture in step (3) and inoculate them into the rooting medium for rooting culture; (5) Acclimatization and transplantation: When the adventitious roots grow to 2-4 cm during the rooting culture in step (4), acclimatize the newly grown seedlings and transplant them into the soil; Among them, the specific process of the disinfection treatment includes: Disinfect the explants with 75% alcohol for 25-35 s, then rinse them with sterile water for 2 times or more, disinfect them with 15% sodium hypochlorite for 10-20 min, and rinse them with sterile water for 3 times or more; The bud induction medium contains 200-300 μL / L ProClin preservative, 0.8-1.2 g / L polyvinylpyrrolidone, 0.3-0.6 g / L ascorbic acid and 0.8-1.2 g / L activated carbon.

2. The method according to claim 1, characterized in that The formula of the bud induction medium is: WPM medium + 0.5 g / L ascorbic acid + 1.0 g / L polyvinylpyrrolidone + 1.0 g / L activated carbon + 0.5 mg / L 6-benzyladenine + 0.04 mg / L naphthaleneacetic acid + 250 μL / L Proclin preservative + 6 g / L agar, pH = 5.

2.

3. The method according to claim 1, wherein The formula of the rooting medium is: 1 / 2MS medium + 0.5 g / L ascorbic acid + 1.0 g / L polyvinylpyrrolidone + 1.0 g / L activated carbon + 0.5 mg / L indole-3-butyric acid + 6 g / L agar, pH = 5.

2.

4. The method according to claim 1, wherein After 18-25 days of bud induction culture, when the axillary buds grow to 2-5 cm, cut off the axillary buds for rooting culture.

5. The method according to claim 1, characterized in that The ProClin preservative is ProClin300.

6. The method according to claim 1, wherein The conditions for the bud induction culture include: The light intensity is 1800-2500 Lx, and the light cycle is 14-16 h / d; The temperature during light is 28°C ± 2°C, and the temperature without light is 26°C ± 2°C.

7. The method according to claim 6, wherein The conditions for the rooting culture are the same as those for the bud induction culture.

8. The method according to claim 1, wherein The steps for acclimatizing the newly grown seedlings include: Gradually open the lids of the tissue culture bottles and spray clean water every day; When all the lids are opened, wash the root medium of the Chinese bayberry seedlings clean and transplant them into the soil.

9. The method according to claim 1, wherein The explants are temporarily stored in a 0.5 g / L ascorbic acid solution after disinfection.

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