Method for disinfecting stems with buds of dogwood and rapidly inducing calluses from axillary buds

Abstract

The invention provides a method for disinfecting stems with buds of dogwood and rapidly inducing calluses from axillary buds. According to the method, dogwood seedlings are taken, the dogwood seedlings are transplanted into a planting pot for pre-culture, and in the pre-culture process, carbendazim liquid medicine is adopted to conduct root irrigation on the dogwood seedlings; taking an explant from a new shoot of the dogwood, wherein the explant is a stem section with buds; and sequentially disinfecting the explant by adopting an ethanol solution and an HgCl2 solution to obtain the disinfected explant. Then, inoculating the disinfected explants into a first culture medium, and carrying out first tissue culture to obtain sterile seedlings with germinated axillary buds; or inoculating the axillary buds into a second culture medium, and performing second tissue culture to obtain the callus directly induced by the axillary buds. The method is simple to operate, short in required time, high in axillary bud germination rate and excellent in callus induction effect, and provides technical support for dogwood gene function research and molecular breeding.

Description

Technical Field

[0001] The present invention belongs to the field of tissue culture of Cornus officinalis, and particularly relates to a method for disinfecting the stem segments with buds of Cornus officinalis and rapidly inducing callus from axillary buds. Background Art

[0002] Cornus officinalis, as a traditional precious Chinese medicinal material, has important research value. Cornus officinalis, which uses dried mature pulp as medicine, not only plays a role in the traditional field, but also has the potential to treat chronic kidney diseases and diabetes, and can delay aging, enhance immunity, control fat and sugar metabolism, etc. Especially in terms of neuroprotection, Cornus officinalis shows significant biological activities against Alzheimer's disease, brain injury, Parkinson's disease, etc., and provides a new direction for the research and development of anti-cancer drugs.

[0003] The existing disinfection method for the culture of Cornus officinalis is to disinfect the tender stems of Cornus officinalis with 0.1% mercuric chloride solution + Tween 80 for 14 - 16 minutes. Since Tween 80 will cause the normal physiological processes of plants, resulting in slow growth and even withering and falling off of leaves, the survival rate will be relatively low; since the stem segments of Cornus officinalis with axillary buds usually show lignification, the germination rate of axillary buds will be relatively low; since there are villi at the petiole of the stem segments with buds, it is impossible to completely disinfect, resulting in a relatively high contamination rate. In addition, in existing research, there is no study showing that axillary buds after disinfection can rapidly induce the formation of callus.

[0004] In view of this, it is necessary to provide a method for disinfecting the stem segments with buds of Cornus officinalis and rapidly inducing callus from axillary buds to solve the technical problem of how to obtain explants for the culture of Cornus officinalis and the rapid formation of callus from axillary buds; further solve the technical problem of how to improve the survival rate and germination rate after inoculation of explants; and / or, how to improve the callus induction rate when axillary buds rapidly form callus. Summary of the Invention

[0005] The main purpose of the present invention is to provide a method for disinfecting the stem segments with buds of Cornus officinalis and rapidly inducing callus from axillary buds, aiming to solve the above technical problems.

[0006] To achieve the above purpose, the present invention provides a disinfection method for the stem segments with buds of Cornus officinalis, including the steps of:

[0007] S1, taking the seedlings of Cornus officinalis; transplanting the seedlings of Cornus officinalis into a planting pot for pre-cultivation, and during the pre-cultivation process, irrigating the roots of the seedlings of Cornus officinalis with carbendazim liquid medicine;

[0008] S2, taking explants from the newly sprouted shoots of Cornus officinalis, the explants being stem segments with buds; using ethanol solution and HgCl 2The explant is disinfected with the solution in sequence to obtain the disinfected explant.

[0009] Furthermore, the seedlings of Cornus officinalis are taken from field soil; the seedlings of Cornus officinalis are the seedlings germinated from Cornus officinalis seeds in the same year; the substrate in the planting pot contains perlite, vermiculite, peat soil, and black soil.

[0010] Furthermore, the carbendazim solution is a 300 - 400 - fold solution of 25 ± 1% carbendazim wettable powder;

[0011] After the pre - culture for 10 ± 1 d, the root irrigation is carried out, and after the root irrigation, the pre - culture is carried out for 15 ± 1 d, and then the explant is taken.

[0012] Furthermore, the length of the explant is 3 - 5 cm.

[0013] Furthermore, the volume concentration of the ethanol solution is 70 - 76%, the mass concentration of the HgCl 2 solution is 0.09 - 0.11%, the disinfection time of the explant in the ethanol solution is 25 - 35 s, and the disinfection time of the explant in the HgCl 2 solution is 11 - 12 min; after the disinfection of the explant is completed, the explant is rinsed with sterile water.

[0014] The present invention also provides a cultivation method of Cornus officinalis, including: obtaining the disinfected explant by using the disinfection method of the Cornus officinalis bud - bearing stem segment as described above; then, inoculating the disinfected explant into the first culture medium and carrying out the first tissue culture to obtain the sterile seedlings with axillary bud germination.

[0015] Furthermore, the first culture medium includes MS culture medium; the first culture medium contains the first plant growth regulator;

[0016] The first plant growth regulator includes 6 - BA, and the concentration of 6 - BA in the first culture medium is 1 - 2 mg / L; or, the first plant growth regulator includes 6 - BA and NAA, the concentration of 6 - BA in the first culture medium is 1 - 2 mg / L, and the concentration of NAA in the first culture medium is 0.1 - 1.0 mg / L.

[0017] Furthermore, the first tissue culture includes: after inoculating the explant into the first culture medium, dark - culturing at 27 - 29 °C for 10 - 20 h, and then transferring to the light condition for culturing for at least 5 d to obtain the sterile seedlings with axillary bud germination; wherein, when culturing under the light condition, the culture temperature is 28 - 32 °C, the light intensity is 2200 - 2300 lx, and the daily light time is 10 - 16 h / d.

[0018] The present invention also provides a method for rapidly inducing callus from axillary buds of Cornus officinalis, comprising: obtaining sterilized explants by using the sterilization method of the Cornus officinalis stem segments with buds as described above; then taking axillary buds from the explants;

[0019] Inoculating the axillary buds into a second culture medium and performing secondary tissue culture to obtain callus directly induced from the axillary buds.

[0020] Furthermore, the second culture medium comprises an MS culture medium; the second culture medium contains a second plant growth regulator;

[0021] The second plant growth regulator comprises KT and 2,4-D, the concentration of KT in the second culture medium is 1.5 - 2.0 mg / L, and the concentration of 2,4-D in the second culture medium is 0.8 - 1.0 mg / L;

[0022] The secondary tissue culture comprises: after inoculating the explants into the second culture medium, performing dark culture at 27 - 29 °C for 2 - 5 d, and then transferring to light conditions for culturing for at least 5 d to obtain axenic seedlings with axillary buds germinated; wherein, when culturing under light conditions, the culture temperature is 28 - 32 °C, the light intensity is 2200 - 2300 lx, and the daily light duration is 10 - 16 h / d.

[0023] Compared with the prior art, the present invention has at least the following advantages:

[0024] The operation of the present invention is simple, the required time is short, the axillary bud germination rate is high, and the callus induction effect is excellent, providing technical support for the research on the gene function of Cornus officinalis and molecular breeding. The present invention obtains explants for the culture of Cornus officinalis and the rapid formation of callus from axillary buds, improving the survival rate and germination rate after inoculation of the explants; reducing the browning rate and contamination rate after inoculation of the explants; reducing the browning rate of axillary buds when rapidly forming callus from axillary buds; and improving the callus induction rate when rapidly forming callus from axillary buds.

[0025] In the present invention, there is no seasonal limitation on materials, and the potted seedlings of Cornus officinalis can shoot throughout the year in the greenhouse as explants; due to the serious toxicity of the field seedlings of Cornus officinalis, there is still a problem of contamination after axillary bud germination, and the contamination rate can reach 100%; while the present invention perfectly solves this problem by transplanting field seedlings. The contamination rate of the present invention is only 6.58%, the germination rate is 93.3%, and the germination time is short; in the present invention, the axillary buds will not die after germination, solving the problem of the toxicity of the disinfectant to the stem segments of Cornus officinalis with buds; in the present invention, the axillary buds can directly induce callus without waiting for the axillary buds to germinate; in the present invention, the axillary buds can induce callus within 20 d. The traditional regeneration process requires the use of axenic seedling leaves, and the breeding cycle is long, greatly shortening the culture time. Description of the Drawings

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0027] Figure 1 For Example 1, the overall growth status of the virus-free pot seedlings after detoxification under the condition of irrigating the roots with a 400-fold solution of 25% carbendazim wettable powder; the results show that the overall growth of the virus-free dogwood seedlings is more vigorous;

[0028] Figure 2 For Example 1, the growth status of individual seedlings of the virus-free pot seedlings after detoxification under the condition of irrigating the roots with a 400-fold solution of 25% carbendazim wettable powder; the results show that the growth of individual virus-free pot seedlings is more vigorous;

[0029] Figure 3 For Example 2, the contaminated condition of the axillary buds of the virus-unremoved field seedlings with bud-bearing stem segments that did not germinate (without root irrigation detoxification, and the bud-bearing stem segments of dogwood were disinfected with 75% alcohol for 0.5 min and 0.1% mercuric chloride for 11 min in sequence); the results show that the main reason for contamination is endophytic bacteria;

[0030] Figure 4 For Example 2, the contaminated condition of the axillary buds of the virus-unremoved field seedlings with bud-bearing stem segments that germinated for 5 days (without root irrigation detoxification, and the bud-bearing stem segments of dogwood were disinfected with 75% alcohol for 0.5 min and 0.1% mercuric chloride for 11 min in sequence); the results show that due to endophytic bacteria, it is still impossible to disinfect completely after germination;

[0031] Figure 5 For Example 2, the contaminated condition of the axillary buds of the virus-unremoved field seedlings with bud-bearing stem segments that germinated for 20 days (without root irrigation detoxification, and the bud-bearing stem segments of dogwood were disinfected with 75% alcohol for 0.5 min and 0.1% mercuric chloride for 11 min in sequence); the results show that it is still impossible to completely prevent endophytic bacteria contamination after axillary bud germination;

[0032] Figure 6 For Example 2, the contaminated condition of the axillary buds of the virus-free pot seedlings with bud-bearing stem segments that germinated for 5 days (with root irrigation detoxification, and the bud-bearing stem segments of dogwood were disinfected with 75% alcohol for 0.5 min and then with 0.1% mercuric chloride for 11 min); the results show no contamination;

[0033] Figure 7For Example 2, it shows the bacterial contamination status of axillary buds of the virus-free seedlings with buds in the potted plants after 10 days of germination (root irrigation for virus elimination, and the stem segments with buds of Cornus officinalis were disinfected with 75% alcohol for 0.5 min and then with 0.1% mercuric chloride for 11 min); the result shows no contamination;

[0034] Figure 8 For Example 4, it shows the bacterial contamination and growth status of axillary buds of the virus-free seedlings with buds in the potted plants after being disinfected and inoculated into the MS + 2.0 mg / L 6-BA + 1.0 mg / L NAA medium for 20 days of germination; the result shows no contamination and extremely vigorous growth;

[0035] Figure 9 For Example 5, it is a photo of the axillary buds inoculated into the callus induction medium (MS + 1.5 mg / L KT + 0.8 mg / L 2,4-D) for 5 days; the result shows that the bud bodies have started to swell;

[0036] Figure 10 For Example 5, it is a photo of the axillary buds inoculated into the callus induction medium (MS + 1.5 mg / L KT + 0.8 mg / L 2,4-D) for 10 days; the result shows that the axillary buds germinate and callus appears on the surface;

[0037] Figure 11 For Example 5, it is a photo of the axillary buds inoculated into the callus induction medium (MS + 1.5 mg / L KT + 0.8 mg / L 2,4-D) for 15 days; the result shows that the axillary buds are completely induced into callus;

[0038] Figure 12 It is an overall photo of the growth of the callus of Cornus officinalis of the present invention for 15 days; the result shows that the callus induction effect is extremely good.

[0039] The realization, functional characteristics and advantages of the object of the present invention will be further described in conjunction with the embodiments with reference to the accompanying drawings. Specific Embodiments

[0040] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0041] Moreover, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement it. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope required by the present invention.

[0042] When numerical ranges are given in the embodiments, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention, based on the understanding of those skilled in the art of the prior art and the description of the present invention, any method, device, and material similar or equivalent to the prior art described in the embodiments of the present invention can also be used to implement the present invention.

[0043] In the present invention, the 1 / 2MS, MS, B5, and WPM basal media were all purchased from Solarbio Science & Technology Co., Ltd.; the product numbers of the 1 / 2MS, MS, B5, and WPM basal media are M8520, M8527, B8830, and LB0180 respectively. In the present invention, the pH of each medium was adjusted to 5.8 and then sterilized; in the present invention, sterile water was obtained by autoclaving at 121 °C for 30 min.

[0044] In the present invention, 6-BA refers to 6-benzylaminopurine; NAA refers to 2-(1-naphthyl)acetic acid; KT refers to 6-furfurylaminopurine; 2,4-D refers to 2,4-dichlorophenoxyacetic acid.

[0045] In the present invention, the survival rate refers to the number of shoot segments with buds after 15 days of germination / the total number of inoculations; the contamination rate refers to the number of contaminated shoot segments with buds after germination / the total number of inoculations; the germination rate refers to the number of shoot segments with buds after germination / the total number of inoculations; the browning rate refers to the number of browned ones / the total number of inoculations; the callus induction rate refers to the total number of callus formed / the total number of axillary buds inoculated.

[0046] It should be understood that during the tissue culture process of Cornus officinalis, contamination (bacterial and fungal contamination) and browning are the primary challenges. Contamination often stems from the difficulty in inactivating the bacteria carried by field seedlings or incomplete sterilization of explant materials, and browning often results from over-disinfection. The contamination and browning of explants seriously hinder the growth of tissue-cultured seedlings and even lead to the failure of the whole batch. In addition, the treatment of explants is also crucial, as poor explants will reduce the regeneration ability and tissue culture efficiency. At the same time, the precise adjustment of the culture medium formula and the strict control of culture conditions, such as light, temperature, humidity, and ventilation conditions, all have a significant impact on the growth rate and quality of tissue-cultured seedlings and need to be carefully regulated to ensure the success of the culture.

[0047] The present invention provides a disinfection method for shoot segments with buds of Cornus officinalis, comprising the steps of:

[0048] S1, taking the seedlings of Cornus officinalis; transplanting the seedlings of Cornus officinalis into a planting pot for pre-culture, and during the pre-culture process, drenching the seedlings of Cornus officinalis with a carbendazim solution.

[0049] In the present invention, the seedlings of Cornus officinalis are taken from field soil, and the seedlings of Cornus officinalis are field seedlings; the seedlings of Cornus officinalis are the seedlings germinated from the seeds of Cornus officinalis in the current year. Specifically, the present invention uses the seedlings sown in the field in the previous year of Cornus officinalis as materials.

[0050] In the present invention, the planting pot contains a substrate, and the substrate in the planting pot includes perlite, vermiculite, peat soil, and black soil; the specific substrate ratio used in the present invention is perlite:vermiculite:peat soil:black soil = 1:1:1:6. When transplanting in the present invention, after cleaning the roots of the field seedlings (the seedlings of Cornus officinalis) and drying them, transplant the field seedlings into the planting pot with the substrate. The carbendazim liquid medicine is a 300 - 400 times liquid medicine of 25 ± 1% carbendazim wettable powder, further a 350 - 400 times liquid medicine of 25 ± 1% carbendazim wettable powder, specifically 400 times liquid medicine. When irrigating the roots in the present invention, it is necessary to avoid the leaves to prevent the leaves from being corroded.

[0051] Specifically, the transplantation and detoxification of Cornus officinalis can be understood as follows: In June - July, take the seedlings germinated from the seeds of Cornus officinalis in the current year as materials, dig them out from the field with soil, wash the roots clean with slow running water, and then place them in a bucket filled with tap water to prevent the seedlings from dying due to water loss. Add the prepared substrate (perlite:vermiculite:peat soil:black soil = 1:1:1:6) to the flower pot. After transplanting into the flower pot, irrigate the roots of the potted seedlings with a 300 - 400 times liquid medicine of 25% carbendazim wettable powder.

[0052] In the present invention, after the pre - culture for 10 ± 1 d, the root irrigation is carried out, and after the root irrigation, the pre - culture for 15 ± 1 d is carried out, and then the explant is taken.

[0053] S2, Take an explant from the newly sprouted shoots of Cornus officinalis, and the explant is a bud - bearing stem segment after removing the leaves; use an ethanol solution and HgCl 2 solution to disinfect the explant in sequence to obtain the disinfected explant.

[0054] In the present invention, the length of the explant is 3 - 5 cm; the newly sprouted shoot is a semi - lignified newly sprouted shoot. When obtaining the bud - bearing stem segment, the petiole is left on the bud - bearing stem segment during the pruning process.

[0055] In the present invention, the volume concentration of the ethanol solution is 70 - 76%, further 74 - 76%, specifically 75%; the mass concentration of the HgCl 2 solution is 0.09 - 0.11%, specifically 0.1%; the disinfection duration of the explant in the ethanol solution is 25 - 35 s, specifically 30 s; the disinfection duration of the explant in the HgCl 2The disinfection duration in the solution is 11 - 12 min, further 11 - 11.5 min, specifically 11 min; after completing the disinfection of the explant, the explant is rinsed with sterile water. The disinfection process of the present invention is soaking, and continuous shaking is required during the soaking process.

[0056] The present invention also provides a cultivation method of Cornus officinalis, which is characterized by including: obtaining a disinfected explant by using the disinfection method of the Cornus officinalis bud-bearing stem segment as described above; then, inoculating the disinfected explant into a first culture medium and performing first tissue culture to obtain a sterile seedling with axillary bud germination.

[0057] The first culture medium includes 1 / 2MS or MS culture medium, preferably MS culture medium; the first culture medium contains a first plant growth regulator.

[0058] The first plant growth regulator includes 6-BA, and the concentration of 6-BA in the first culture medium is 1 - 2 mg / L, further 1.9 - 2.0 mg / L; or, the first plant growth regulator includes 6-BA and NAA, the concentration of 6-BA in the first culture medium is 1 - 2 mg / L (further 1.9 - 2.0 mg / L), and the concentration of NAA in the first culture medium is 0.1 - 1.0 mg / L or 0.5 - 1.0 mg / L (further 0.9 - 1.0 mg / L); for example, the first culture medium containing the first plant growth regulator is MS + 1.9 - 2.0 mg / L 6-BA + 0.9 - 1.0 mg / L NAA, specifically MS + 2.0 mg / L 6-BA + 1.0 mg / L NAA.

[0059] The first tissue culture includes: after inoculating the explant into the first culture medium, dark culturing at 28 ± 1 °C for 10 - 20 h, and then transferring to light conditions for culturing for at least 5 d or culturing for 5 - 25 d (further 15 - 20 d or 15 - 16 d) to obtain a sterile seedling with axillary bud germination; wherein, when culturing under light conditions, the culture temperature is 28 - 32 °C, the light intensity is 2200 - 2300 lx, and the daily light time is 10 - 16 h / d.

[0060] The present invention first uses the Cornus officinalis field seedlings as materials, transplants them into pots and adds carbendazim to reduce the fungal content of the seedlings themselves, and then disinfects the treated seedlings with 75% ethanol, HgCl 2 After disinfection, they are inoculated into a culture medium containing a plant growth regulator for cultivation, successfully solving the problems of difficult disinfection of the Cornus officinalis bud-bearing stem segment and difficult germination of axillary buds.

[0061] The present invention also provides a method for rapidly inducing callus from axillary buds of Cornus officinalis, comprising: obtaining a disinfected explant by using the disinfection method of the Cornus officinalis stem segment with buds as described above; then taking axillary buds from the explant; inoculating the axillary buds into a second culture medium and performing a second tissue culture to obtain callus directly induced from the axillary buds.

[0062] In the present invention, the second culture medium comprises an MS medium; the second culture medium contains a second plant growth regulator; the second plant growth regulator comprises KT and 2,4-D, the concentration of KT in the second culture medium is 1.5 - 2.0 mg / L (further 1.5 - 1.6 mg / L), and the concentration of 2,4-D in the second culture medium is 0.8 - 1.0 mg / L (further 0.8 - 0.9 mg / L); for example, the second culture medium containing the second plant growth regulator is MS + 1.5 - 1.6 mg / L KT + 0.8 - 0.9 mg / L 2,4-D, specifically MS + 1.5 mg / L KT + 0.8 mg / L 2,4-D.

[0063] The second tissue culture comprises: after inoculating the explant into the second culture medium, performing dark culture at 28 ± 1°C for 2 - 5 d, and then transferring it to light conditions for culturing for at least 5 d or culturing for at least 10 d or culturing for 10 - 20 d (further 10 - 15 d) to obtain a sterile seedling with axillary bud germination; wherein, when culturing under light conditions, the culture temperature is 28 - 32°C, the light intensity is 2200 - 2300 lx, and the daily light time is 10 - 16 h / d.

[0064] The present invention rapidly induces callus from disinfected axillary buds of Cornus officinalis, with a high callus induction rate and an extremely high proliferation efficiency; this method is simple to operate, requires a short time, has a high axillary bud germination rate, and an excellent callus induction effect, providing technical support for the research on the gene function of Cornus officinalis and molecular breeding.

[0065] The following are specific examples of the present invention:

[0066] Example 1

[0067] Detoxification comparison of Cornus officinalis:

[0068] Transplanting of field seedlings of Cornus officinalis: Taking the seedlings germinated from Cornus officinalis seeds in the same year in June - July as materials, digging them out of the field with soil, rinsing the roots clean with slow running water, and placing them in a bucket filled with tap water to prevent the seedlings from dying due to water loss; adding the prepared substrate (perlite: vermiculite: peat soil: black soil = 1:1:1:6) to a flower pot (planting pot), and transplanting the Cornus officinalis seedlings into the flower pot to obtain potted seedlings.

[0069] Virus detoxification of transplanted Macrocarpium officinale seedlings after emergence in the field: After culturing the potted seedlings under light conditions for 10 days, use root irrigation agents to irrigate the potted seedlings; after the root irrigation is completed, continue to culture for 15 days, and then take explants from the newly sprouted shoots of Macrocarpium officinale. The explants are stem segments with buds after removing the leaves, and the length is controlled at 3 - 5 cm.

[0070] Disinfect the explants with a 0.1% concentration of HgCl 2 solution for 10 minutes, and after disinfection, inoculate them into the MS + 2.0 mg / L 6 - BA medium, and culture them in the dark at 28°C for 15 hours, then transfer them to light conditions for culture. The culture temperature under light conditions is 30°C, the light intensity for culture is 2200 lx, and the light culture time is 14 h / d. After culturing for 15 days, count the axillary bud contamination rate and survival rate (see Table 1 for details). There are 10 bottles for each treatment, and it is repeated 3 times.

[0071] In this example, the root irrigation agents used for root irrigation are respectively 100, 200, 300, 400, 500 - fold liquid medicines (mass multiples) of 25% carbendazim wettable powder; potassium permanganate with concentrations of 0.05%, 0.1%, 0.15%, 0.2%, 0.25% (mass concentrations); 400, 600, 800, 1000, 1200 - fold liquid medicines (mass multiples) of mancozeb.

[0072] Referring to the comparative analysis of virus detoxification of different test agents shown in Table 1, it can be found that: after transplanting Macrocarpium officinale to flowerpots, using 400 - fold liquid medicine of 25% carbendazim wettable powder in the potted seedlings, the survival rate of sterile seedlings is 86.67%. In addition, as a display of the potted seedlings after virus detoxification, refer to Figure 1-2 shown, under the condition of virus detoxification by irrigating with 400 - fold liquid medicine of 25% carbendazim wettable powder, the overall growth of the Macrocarpium officinale seedlings after virus detoxification is vigorous.

[0073] Table 1 Effects of different virus - detoxification methods on the germination of stem segments with buds

[0074]

[0075] Note: Different capital letters after the data in the table indicate significant differences at the 0.05 level between groups, and different lowercase letters after the data in the table indicate significant differences at the 0.05 level within groups; the same below.

[0076] Example 2

[0077] I. Disinfection comparison of explants:

[0078] Transplanting of Macrocarpium officinale field seedlings: In June or July, take the seedlings germinated from Macrocarpium officinale seeds in the same year as the material. Dig them out of the field with soil attached, rinse the roots clean with slow running water, and place them in a bucket filled with tap water to prevent the seedlings from dying due to water loss. Add the prepared substrate (perlite: vermiculite: peat soil: black soil = 1:1:1:6) to the flower pot (planting pot), and transplant the Macrocarpium officinale seedlings into the flower pot to obtain potted seedlings.

[0079] Virus detoxification of Macrocarpium officinale field seedlings after transplantation: After culturing the potted seedlings under light conditions for 10 days, drench the roots of the potted seedlings with a 400-fold solution (mass multiple) of 25% carbendazim wettable powder. After the drenching is completed, continue to culture for 15 days, and then take explants from the newly sprouted shoots of Macrocarpium officinale. The explants are stem segments with buds after removing the leaves, and the length is controlled at 3 - 5 cm.

[0080] Disinfection treatment of explants: After separately disinfecting the explants with 75% ethanol solution (volume concentration) for 0.5 min, 1 min, and 1.5 min respectively, conduct disinfection separately according to the following treatments: continue to disinfect with 5% NaClO solution (mass concentration) for 10 min, 15 min, 20 min, and 25 min; disinfect with 0.1% HgCl 2 solution (mass concentration) for 9 min, 10 min, 11 min, and 12 min. The disinfection process is soaking, and shake continuously during the soaking process to ensure thorough disinfection. After the disinfection is completed, rinse with sterile water.

[0081] Inoculate the disinfected explants into the MS + 2.0 mg / L 6 - BA medium, and count the contamination rate and germination rate after culturing for 15 days (see Table 2 for details). Conduct dark culture at 28°C for 15 h, and then transfer to light conditions for culture. The culture temperature under light conditions is 30°C, the light intensity for culture is 2200 lx, and the light culture time is 14 h / d. There are 30 bottles for each treatment, and repeat 3 times.

[0082] Referring to the comparative analysis of different disinfection methods shown in Table 2, it can be found that: after disinfecting the Macrocarpium officinale stem segments with buds with 75% alcohol for 0.5 min and then with 0.1% mercuric chloride for 11 min, the contamination rate of sterile seedlings after 15 days is only 6.58%, and the germination rate after 15 days reaches 93.3%.

[0083] Table 2 Effects of different disinfection methods on the germination of stem segments with buds

[0084]

[0085]

[0086] II. Control of non - virus - detoxified field seedlings:

[0087] The non-detoxified field seedlings were used as the control group for treatment; specifically, compared with the first part of this example, root irrigation was not carried out, and the stem segments with buds of Cornus officinalis were disinfected with 75% alcohol for 0.5 min and then with 0.1% mercuric chloride for 11 min in sequence.

[0088] See Figure 3-5 For understanding, the non-detoxified field seedlings cannot prevent endophyte contamination.

[0089] See Figure 6-7 As shown, in the first part of this example, root irrigation was used for detoxification, and after the stem segments with buds of Cornus officinalis were disinfected with 75% alcohol for 0.5 min, they were then disinfected with 0.1% mercuric chloride for 11 min, which can avoid endophyte contamination.

[0090] Example 3

[0091] Comparison of basal media:

[0092] Transplantation of Cornus officinalis field seedlings: In June to July, the seedlings germinated from Cornus officinalis seeds in the same year were used as materials. They were dug out of the field with soil, and after the roots were washed clean with slow running water, they were placed in a bucket filled with tap water to prevent the seedlings from dying due to water loss; the prepared substrate (perlite: vermiculite: peat soil: black soil = 1:1:1:6) was added to the flower pot (planting pot), and the Cornus officinalis seedlings were transplanted into the flower pot to obtain potted seedlings.

[0093] Detoxification after transplantation of Cornus officinalis field seedlings: After the potted seedlings were cultured under light conditions for 10 d, they were irrigated with a 400-fold solution (mass multiple) of 25% carbendazim wettable powder; after the root irrigation was completed, they were cultured for another 15 d, and then explants were taken from the newly sprouted shoots of Cornus officinalis. The explants were stem segments with buds after the leaves were cut off, and the length was controlled at 3 - 5 cm.

[0094] Disinfection treatment of explants: The explants were disinfected with 75% alcohol (volume concentration) for 0.5 min and then with 0.1% mercuric chloride for 11 min. The disinfection process was soaking, and the explants were continuously shaken during the soaking process to ensure thorough disinfection. After disinfection was completed, they were rinsed with sterile water.

[0095] Effect of different basal media on axillary bud germination: The disinfected explants (stem segments with buds) were inoculated into basal media containing 2.0 mg / L 6 - BA, and the germination rate, contamination rate, and browning rate after 15 d of culture were counted (see Table 3 for details); the basal media were 1 / 2MS, WPM, MS, and B5 respectively.

[0096] In this example, it was first cultured in the dark at 28 °C for 15 h, and then transferred to the light condition for culture. The culture temperature under the light condition was 30 °C, the light culture intensity was 2200 lx, and the light culture time was 14 h / d. To prevent cross-contamination of explants, there were 30 bottles for each treatment, with 3 replicates.

[0097] Referring to the comparative analysis in Table 3, the basal medium finally determined in this example was the MS basal medium.

[0098] Table 3 Effects of different basal media on the germination of shoot segments with buds

[0099]

[0100] Example 4

[0101] Comparison of plant growth regulators:

[0102] The disinfected explants (shoot segments with buds) obtained in the same way as in Example 3 were inoculated into the MS medium containing plant growth regulators, and the germination time was counted, and the browning rate and germination rate after 15 d of culture were counted (see Table 4 for details); the plant growth regulators and concentrations in the medium were 6-BA (1, 2, 3 mg / L) and NAA (0, 0.1, 0.5, 1.0 mg / L) respectively.

[0103] In this example, it was first cultured in the dark at 28 °C for 15 h, and then transferred to the light condition for culture. The culture temperature under the light condition was 30 °C, the light culture intensity was 2200 lx, and the light culture time was 14 h / d. To prevent cross-contamination of explants, there were 30 bottles for each treatment, with 3 replicates.

[0104] Referring to Table 4 for understanding, the preferably axillary bud germination medium of the present invention finally confirmed in this example was: MS + 2.0 mg / L 6-BA + 1.0 mg / L NAA, and the shortest time for all germination (except for the cases where germination could not occur) was 15 d.

[0105] Referring to Figure 8 As shown, after the axillary buds of the shoot segments with buds of the potted virus-free seedlings were disinfected and inoculated into the MS + 2.0 mg / L 6-BA + 1.0 mg / L NAA medium, contamination could be avoided and the growth was vigorous.

[0106] Table 4 Effects of different plant growth regulators on axillary bud germination

[0107]

[0108] Example 5

[0109] Effects of different basal media and plant growth regulators on axillary bud-induced callus:

[0110] Obtain the disinfected explants (stem segments with buds) as in Example 3. Take the axillary buds from the disinfected explants and inoculate them into 1 / 2MS, WPM, MS, and B5 media containing plant growth regulators respectively, and count the browning rate and callus induction rate of the axillary buds after 15 days of culture (see Table 5 for details). Different concentrations of plant growth regulators, KT (1, 1.5, 2.0 mg / L) and 2,4-D (0.6, 0.8, 1.0 mg / L), are added to the media respectively.

[0111] In this example, first, dark culture is carried out at 28 °C for 3 days, and then it is transferred to light conditions for culture. The culture temperature under light conditions is 30 °C, the light intensity for culture is 2200 lx, and the light culture time is 14 h / d. To prevent cross-contamination of axillary buds, there are 30 bottles for each treatment, and it is repeated 3 times.

[0112] Table 5 Effects of different basal media and plant growth regulators on callus induction from axillary buds

[0113]

[0114] From the above content, it can be seen that the optimal medium formula for inducing embryogenic callus from axillary buds of Cornus officinalis is: MS + 1.5 mg / L KT + 0.8 mg / L 2,4-D. In addition, as shown in Figure 9-12 , when the axillary buds are inoculated into the callus induction medium (MS + 1.5 mg / L KT + 0.8 mg / L 2,4-D), the axillary buds can be induced into callus.

[0115] In the above technical solution of the present invention, the above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. All equivalent structural transformations made under the technical concept of the present invention by using the content of the specification and drawings of the present invention, or directly / indirectly applied in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A method for disinfecting a stem segment of Cornus officinalis with buds, characterized in that: Includes steps: S1, taking a seedling of Cornus officinalis; transplanting the seedling of Cornus officinalis into a planting pot for pre-cultivation, and during the pre-cultivation process, irrigating the roots of the seedling of Cornus officinalis with a carbendazim solution; S2, taking an explant from a new shoot of the Cornus officinalis, wherein the explant is a stem segment with buds; The explant is sterilized by using an ethanol solution and a HgCl2 solution in sequence to obtain a sterilized explant.

2. The method for disinfecting the stem segments of Cornus officinalis with buds according to claim 1, characterized in that: The seedlings of Cornus officinalis are taken from field soil; the seedlings of Cornus officinalis are seedlings germinated in the current year from seeds of Cornus officinalis; the matrix in the planting pot contains perlite, vermiculite, peat soil and black soil.

3. The method for disinfecting the stem segments of Cornus officinalis with buds according to claim 1, characterized in that: The carbendazim liquid is 300-400 times the liquid of 25±1% carbendazim wettable powder; The root irrigation is performed after the pre-culture for 10±1 days, and the pre-culture is performed for 15±1 days after the root irrigation, and then the explants are taken.

4. The method for disinfecting the stem segments of Cornus officinalis with buds according to claim 1, characterized in that: The length of the explant is 3-5 cm.

5. The method for disinfecting the stem segments of Cornus officinalis with buds according to claim 1, characterized in that: The volume concentration of the ethanol solution is 70-76%, the mass concentration of the HgCl2 solution is 0.09-0.11%, the disinfection time of the explant in the ethanol solution is 25-35s, and the disinfection time of the explant in the HgCl2 solution is 11-12min; after the disinfection of the explant is completed, the explant is rinsed with sterile water.

6. A method for cultivating Cornus officinalis, characterized in that: include: The sterilized explant is obtained by using the sterilization method of the cornus officinalis stem segment with buds as described in any one of claims 1 to 5; Then, the sterilized explant is inoculated into a first culture medium and subjected to a first tissue culture to obtain sterile seedlings with axillary buds.

7. The method for cultivating Cornus officinalis according to claim 6, characterized in that: The first culture medium comprises MS culture medium; the first culture medium contains a first plant growth regulator; The first plant growth regulator includes 6-BA, and the concentration of 6-BA in the first culture medium is 1-2 mg / L; or, the first plant growth regulator includes 6-BA and NAA, and the concentration of 6-BA in the first culture medium is 1-2 mg / L, and the concentration of NAA in the first culture medium is 0.1-1.0 mg / L.

8. The method for cultivating Cornus officinalis according to claim 6, characterized in that: The first tissue culture comprises: inoculating the explant into the first culture medium, culturing in the dark at 27-29° C. for 10-20 hours, and then transferring to light conditions for culturing for at least 5 days to obtain sterile seedlings with axillary buds; wherein, when culturing under light conditions, the culture temperature is 28-32° C., the light intensity is 2200-2300 lx, and the daily light time is 10-16 h / d.

9. A method for rapidly inducing callus from axillary buds of Cornus officinalis, characterized in that: include: The sterilized explant is obtained by using the sterilization method of the cornus officinalis stem segment with buds as described in any one of claims 1 to 5; Then, axillary buds are taken from the explants; The axillary buds are inoculated into a second culture medium and subjected to a second tissue culture to obtain callus tissues directly induced by the axillary buds.

10. The method for rapidly inducing callus from axillary buds of Cornus officinalis according to claim 9, characterized in that: The second culture medium comprises MS culture medium; the second culture medium contains a second plant growth regulator; The second plant growth regulator includes KT and 2,4-D, the concentration of KT in the second culture medium is 1.5-2.0 mg / L, and the concentration of 2,4-D in the second culture medium is 0.8-1.0 mg / L; The second tissue culture comprises: inoculating the explant into the second culture medium, culturing in the dark at 27-29° C. for 2-5 days, and then transferring to light conditions for culturing for at least 5 days to obtain sterile seedlings with axillary buds; wherein, when culturing under light conditions, the culture temperature is 28-32° C., the light intensity is 2200-2300 lx, and the daily light time is 10-16 h / d.

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