Tissue culture and rapid propagation seedling method for Japanese polygala

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a method for tissue culture and rapid propagation of polygala japonica. Comprising the following steps: sterilizing and disinfecting explants; carrying out primary culture on the sterilized and disinfected explant; carrying out multiplication culture on primary cluster buds; carrying out rooting culture on rootless tissue culture seedlings; domesticating and transplanting rooting tissue culture seedlings. A primary culture medium, a proliferation culture medium and a rooting culture medium are reasonably designed according to different growth regulator formulas by taking a stem section with buds of polygala japonica as an explant material and are respectively used for performing clumpy bud induction, clumpy bud proliferation and rooting induction on the explant material, the total culture process only needs about 100 days, the breeding efficiency is high, and the survival rate is high. The inductivity, the tufted bud multiplication coefficient and the rooting rate of the explant tufted buds all reach a relatively high level, the cultured tissue culture seedlings are good in quality and high in transplanting survival rate, the tissue culture and rapid propagation effects of the polygala japonica are good, and a solid foundation is laid for realizing industrialized production of the polygala japonica.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for rapid propagation of *Gynostemma pentaphyllum* seedlings through tissue culture. Background Technology

[0002] Polygala tenuifolia is a perennial herb belonging to the genus Polygala in the family Polygalaceae. It is distributed in Northeast, North, Southwest China and the Yangtze River basin, and grows mainly on hillsides and field ridges at altitudes of 800-2100 meters. It prefers a warm and humid climate and is tolerant of strong light. Internally, it is used to relieve phlegm and cough, promote blood circulation and reduce swelling, and detoxify and relieve pain. Externally, it is used to treat injuries from falls and blows, boils and carbuncles, snake and insect bites, etc.

[0003] The propagation methods of *Gynostemma pentaphyllum* are seed propagation and rhizome propagation. Seeds mature in late summer and early autumn and can be sown immediately after harvesting. Seedlings emerge in the following spring, about one and a half months later, and should be transplanted on a cloudy or rainy day or in the afternoon. Rhizome propagation can be carried out by transplanting in spring and autumn. It can be seen that seed propagation spans two seasons and has a long propagation cycle. In addition, seed propagation depends on natural germination and is greatly affected by uncontrollable factors such as the climate (such as temperature and humidity), soil conditions, and pests and diseases of the year, resulting in a low and unstable germination rate. Although rhizome propagation is slightly faster than seed propagation, it depends on the tillering ability of the mother plant. The number of rhizomes that can be tillered by a single plant is limited, resulting in a low propagation coefficient, which cannot meet the requirements of large-scale seedling production.

[0004] Tissue culture can induce a large number of callus or adventitious buds in a short time using tiny explants under sterile and controlled conditions. It has a fast propagation speed and short propagation cycle, which can provide strong support for the seedling needs of large-scale planting of Guazijin, construction of medicinal raw material bases or ecological restoration projects, and promote its industrialization. However, no tissue culture propagation methods for Guazijin have been reported so far. Summary of the Invention

[0005] To address the aforementioned technical deficiencies, this invention presents a method for rapid propagation of Guazi Jin tissue culture seedlings. By rationally preparing the culture medium and controlling the disinfection and light-based rooting process, the induction rate, proliferation coefficient, and rooting rate of explant shoots can reach high levels. The resulting tissue culture seedlings are of high quality, have a high transplant survival rate, and reduce production costs.

[0006] A method for rapid propagation of melon seedlings through tissue culture includes the following steps: Step S1: Sterilization and disinfection of explants. Cut the stem segments with buds from the Guazi Jin plant, clean them, and then sterilize and disinfect them to obtain sterilized and disinfected explants. Step S2: Primary culture of sterilized explants. Sterilized explants are inoculated into primary culture medium to induce axillary bud germination and obtain primary clustered buds. The primary culture medium consists of MS medium + 1 mg / L 6-BA + 0.05 mg / L NAA + 30 mg / L sucrose + 6 g / L agar. Step S3: proliferative culture of the primary cluster shoots, the primary cluster shoots are cut into 2-3 cm stem segments, soaked in 20-30 mg / L 3,6-dihydroxybenzylsucrose solution for 8-10 minutes, inoculated in the proliferative culture medium for culture, to obtain rootless tissue culture seedlings, the proliferative culture medium is composed of MS medium+0.1 mg / L ZT+1-1.5 mg / L 6-BA+0.01-0.03 mg / L NAA+30 mg / L sucrose+6 g / L agar; Step S4: rooting culture of the rootless tissue culture seedlings, the rootless tissue culture seedlings are inoculated into the rooting culture medium for bottle rooting culture, to obtain rooted tissue culture seedlings, the rooting culture medium is composed of 1 / 2MS medium+0.5 mg / L IBA+300-500 mg / L activated carbon+30 mg / L sucrose+6 g / L agar; Step S5: acclimatization and transplanting of the rooted tissue culture seedlings, the rooted tissue culture seedlings are first closed-bottle acclimatized for 7 days, then open-bottle acclimatized, transplanted into the seedling hole tray for culture to survival, and further transplanted into the field.

[0007] Further, step S1 specifically comprises the following steps: S1.1: in spring, a healthy and disease-free Cucurbita pepo plant is cut into 5-6 cm stem segments with buds, immersed in a soap solution for 25-30 min of oscillation cleaning, rinsed clean with tap water, then immersed in Tween-80 for 5-8 min of oscillation, and then washed under running water for 0.5-1 h to obtain clean stem segments with buds; S1.2: salicylic acid and metformin hydrochloride are placed in a container, sterile water is added and stirred until completely dissolved to obtain an explant cleaning solution, the salicylic acid concentration in the explant cleaning solution is 1-1.5 g / L, and the metformin hydrochloride concentration is 2-3 g / L; S1.3: the clean stem segments with buds are placed in a clean bench, first subjected to 40-45 s of disinfection treatment with 70-75% ethanol, rinsed 4-5 times with sterile water, then immersed in a 1% carbendazim solution for 6-8 min of sterilization and disinfection treatment, rinsed 4-5 times with sterile water again, then placed in a 0.05% mercury sulfate solution for 4-5 min of shaking sterilization, and finally rinsed 4-5 times with the explant cleaning solution to obtain sterilized and disinfected explants.

[0008] Further, step S2 specifically comprises the following steps: S2.1: the sterilized and disinfected explants are inoculated into the primary culture medium, and dark culture is performed at a temperature of 25-27℃ for 7 days; S2.2: the temperature is kept unchanged, and 25 days of light and dark alternating culture is performed under the condition of 10 h of daily light, and the light intensity is 2500-3000 lx, to obtain primary cluster shoots.

[0009] Further, step S3 specifically comprises the following steps: S3.1: cutting the primary cluster shoots into 2-3 cm stem segments, soaking in a 3,6-dicinnamoyl sucrose solution of 20-30 mg / L for 8-10 minutes, and then inoculating into the proliferation culture medium; S3.2: alternating light and dark culture for 35 days at 25-27℃, with 12 hours of daily light at an intensity of 2500-3000 lx, to obtain rootless tissue culture seedlings.

[0010] Further, step S4 specifically comprises the following steps: S4.1: inoculating the rootless tissue culture seedlings into culture bottles containing rooting culture medium, and culturing under red-blue composite light, with a culture temperature of 25-27℃, a light intensity of 2000-2500 lx, and 12 hours of daily light, for 20 days; S4.2: then switching to white light culture, keeping the culture temperature unchanged, with a light intensity of 2500-3000 lx and 10 hours of daily light, for 20 days, to obtain rooted tissue culture seedlings.

[0011] Further, in step S4.1, the red light spectrum in the red-blue composite light is (1-1.5):1, with a red light wavelength of 680-700 nm and a blue light wavelength of 400-420 nm.

[0012] Further, step S5 specifically comprises the following steps: S5.1: moving the culture bottles containing the rooted tissue culture seedlings to an outdoor shaded canopy for hardening-off for 7 days, with a shading degree of 60-70%, then opening the bottle mouth and placing for another 7 days, and frequently rotating the bottle body to ensure uniform light exposure; S5.2: then transplanting to nursery plug culture for survival, and further transplanting to the field.

[0013] Further, the culture medium in the nursery plug in step S5.2 is composed of peat soil: vermiculite: perlite at a mass ratio of 3:1:1.

[0014] The beneficial effects are: 1, the present application takes the bud stem section of melon seed gold as explant material, and reasonably designs the primary culture medium (MS medium + 1 mg / L of 6-BA + 0.05 mg / L of NAA + 30 mg / L of sucrose + 6 g / L of agar), the proliferation culture medium (MS medium + 0.1 mg / L of ZT + 1-1.5 mg / L of 6-BA + 0.01-0.03 mg / L of NAA + 30 mg / L of sucrose + 6 g / L of agar) and the rooting culture medium (1 / 2MS medium + 0.5 mg / L of IBA + 300-500 mg / L of activated carbon + 30 mg / L of sucrose + 6 g / L of agar) with different growth regulator formulations, which are respectively used for cluster bud induction, cluster bud proliferation and rooting induction of the explant material, and the total culture process only needs about 100 days, the propagation efficiency is high, and before the proliferation culture, the explant material is soaked in 3,6-diacetyl sucrose solution, which can adhere to the surface of the stem section, act as a high-quality carbon source and play the role of a signal molecule to stimulate cluster bud growth, greatly promote the proliferation of melon seed gold cluster buds, through experimental verification, the induction rate of melon seed gold explant cluster buds, cluster bud proliferation coefficient and rooting rate all reach a high level, the quality of the tissue culture seedlings cultivated is good, the survival rate of transplanting is high, and the melon seed gold tissue culture and rapid propagation effect is good, which lays a solid foundation for realizing the factory production of melon seed gold.

[0015] 2, the present application first disinfects the clean stem section with 70-75% ethanol, rinses and then immerses in 1% carbendazim for sterilization and disinfection, and then is placed in 0.05% mercuric chloride solution for shaking sterilization, and finally is rinsed clean with a sterile aqueous solution of salicylic acid and metformin hydrochloride, in this process, the combination of carbendazim and mercuric chloride solution for treating the explant can greatly reduce the pollution rate, and then rinsing with the sterile water containing salicylic acid and metformin hydrochloride, the salicylic acid can effectively complex and remove mercuric chloride, and the metformin hydrochloride can further provide sterilization effect, so as to reduce the explant contamination rate and improve the survival rate, and the two can also adhere to the surface of the explant, synergistically improve the absorption and utilization of the plant to the growth hormone in the primary culture medium, and the subsequent cluster bud induction rate, thereby reducing the production cost of melon seed gold tissue culture seedlings.

[0016] 3, in the process of rooting culture, first irradiate and culture the explant with red and blue composite light with an intensity of 2000-2500 lx for 20 days, and then culture the explant with white light with an intensity of 2500-3000 lx for 20 days, so that the rooting rate of the obtained rooting tissue culture seedlings is high, the number of roots is large, and the stability is good, which can further improve the survival rate of melon seed gold tissue culture seedlings, thereby reducing the production cost of tissue culture seedlings. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a photo for the explant of melon seed gold cultured in the primary culture medium to axillary bud germination.

[0018] Figure 2 This is a photo of the first generation of clustered shoots of Guazijin buds cultured on a proliferation medium for 20 days.

[0019] Figure 3 This is a photo of the first generation of clustered shoots of Guazijin buds cultured on a proliferation medium for 35 days.

[0020] Figure 4 This is a photo of Guazijin rootless tissue culture seedlings taken out after 40 days of rooting culture.

[0021] Figure 5 This is a photo of the rooted tissue culture seedlings of Guazijin after hardening and transplanting. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: A method for rapid propagation of melon seedlings through tissue culture, comprising the following steps:

[0024] S1: Sterilization and disinfection of explants S1.1: In spring, cut healthy, disease-free Guazi Jin plants into 5cm stem segments with buds, immerse them in soapy water and shake to clean for 25 minutes, rinse them with tap water, immerse them in Tween-80 and shake for 5 minutes, and then rinse them under running water for 0.5 hours to obtain clean stem segments with buds. S1.2: Place salicylic acid and metformin hydrochloride in a container, add sterile water and stir until completely dissolved to obtain explant washing solution. The concentration of salicylic acid in the explant washing solution is 1 g / L and the concentration of metformin hydrochloride is 2 g / L. S1.3: Place the clean stem segments with buds in a clean bench, first disinfect them with 70% ethanol for 40 seconds, rinse them three times with sterile water, then immerse them in 1% carbendazim solution for 6 minutes for sterilization, rinse them four times with sterile water, then place them in 0.05% mercuric chloride solution for 4 minutes for shaking sterilization, and finally rinse them four times with explant cleaning solution to obtain sterilized explants.

[0025] S2: Primary culture of sterilized explants S2.1: Sterilized explants were inoculated into primary culture medium, which consisted of MS medium + 1 mg / L 6-BA + 0.05 mg / L NAA + 30 mg / L sucrose + 6 g / L agar. The culture was carried out in the dark at 25°C for 7 days. S2.2: Keep the temperature unchanged, and carry out 25 days of light and dark alternating culture under the condition of 10 hours of daily light and 2500lx of light intensity, to obtain the primary cluster shoots.

[0026] S3: Proliferation culture of the primary cluster shoots S3.1: Cut the primary cluster shoots into 2cm stem segments, immerse them in 20mg / L 3,6-dicinnamoyl sucrose solution for 8 minutes, and then inoculate them into the proliferation culture medium composed of MS medium + 0.1mg / L ZT + 1mg / L 6-BA + 0.01mg / L NAA + 30mg / L sucrose + 6g / L agar; S3.2: Carry out 35 days of light and dark alternating culture under the condition of 25℃, 12 hours of daily light and 2500lx of light intensity, to obtain the rootless tissue culture seedlings.

[0027] S4: Rooting culture of the rootless tissue culture seedlings S4.1: Inoculate the rootless tissue culture seedlings into the culture bottle containing the rooting culture medium composed of 1 / 2MS medium + 0.5mg / L IBA + 300mg / L activated carbon + 30mg / L sucrose + 6g / L agar, and culture under the red and blue composite light, wherein the red and blue composite light has a red spectrum:blue spectrum ratio of 1:1, the red light wavelength is 680nm, the blue light wavelength is 400nm, the culture temperature is 25℃, the light intensity is 2000lx, the daily light time is 12 hours, and the culture time is 20 days; S4.2: Then change to white light culture, keep the culture temperature unchanged, the light intensity is 2500lx, the daily light time is 10 hours, and the culture time is 20 days, to obtain the rooted tissue culture seedlings.

[0028] S5: Acclimatization and transplanting of the rooted tissue culture seedlings S5.1: Move the culture bottle containing the rooted tissue culture seedlings to the outdoor shaded awning, close the bottle for 7 days, then open the bottle mouth, and place it for another 7 days, and frequently rotate the bottle body to ensure uniform light reception during the placement; S5.2: Then transplant it to the seedling culture hole tray with a culture medium composed of peat soil:vermiculite:perlite in a mass ratio of 3:1:1, and then transplant it to the field.

[0029] Example 2: A method for tissue culture and rapid propagation of Celastrus aculeatus, comprising the following steps:

[0030] S1: Sterilization of the explants S1.1: In spring, cut the healthy Celastrus aculeatus plant without diseases and pests into 5.5cm stem segments with buds, shake and wash them in soap solution for 28 minutes, rinse them clean with tap water, then immerse them in Tween-80 and shake for 7 minutes, and then rinse them under running water for 0.8 hours, to obtain clean stem segments with buds; S1.2: Put salicylic acid and metformin hydrochloride into a container, add sterile water and stir until completely dissolved to obtain explant washing solution, the concentration of salicylic acid in the explant washing solution is 1.2 g / L, and the concentration of metformin hydrochloride is 2.5 g / L; S1.3: Put the clean stem segments with buds into a clean bench, first sterilize with 73% ethanol for 43 s, then rinse with sterile water for 4 times, then immerse in 1% carbendazim solution for sterilization and disinfection treatment for 7 min, then rinse with sterile water for 4 times, then immerse in 0.05% mercury solution for shaking sterilization for 4.5 min, and finally rinse with the explant washing solution for 5 times to clean, to obtain sterilized explants.

[0031] S2: Primary culture of sterilized explants S2.1: Inoculate the sterilized explants into the primary culture medium, which is composed of MS medium + 1 mg / L 6-BA + 0.05 mg / L NAA + 30 mg / L sucrose + 6 g / L agar, and culture in the dark at 26°C for 7 days; S2.2: Keep the temperature unchanged, and carry out light and dark alternating culture for 25 days under the condition of 10 h of daily light and light intensity of 2650 lx, to obtain primary cluster buds.

[0032] S3: Proliferation culture of primary cluster buds S3.1: Cut the primary cluster buds into 2.5 cm stem segments, immerse in 25 mg / L 3,6-dicinnamoyl sucrose solution for 9 min, and then inoculate into the proliferation culture medium, which is composed of MS medium + 0.1 mg / L ZT + 1.2 mg / L 6-BA + 0.02 mg / L NAA + 30 mg / L sucrose + 6 g / L agar; S3.2: Carry out light and dark alternating culture for 35 days under the condition of 26°C, 12 h of daily light, and light intensity of 2700 lx, to obtain rootless tissue culture seedlings.

[0033] S4: Rooting culture of rootless tissue culture seedlings S4.1: Inoculate the rootless tissue culture seedlings into a culture bottle containing rooting culture medium, which is composed of 1 / 2MS medium + 0.5 mg / L IBA + 400 mg / L activated carbon + 30 mg / L sucrose + 6 g / L agar, and culture under red and blue composite light, wherein the red light spectrum is 1.2:1 of the blue light spectrum, the red light wavelength is 690 nm, and the blue light wavelength is 410 nm, the culture temperature is 26°C, the light intensity is 2250 lx, the daily light time is 12 h, and the culture time is 20 days; S4.2: Then change to white light culture, keep the culture temperature unchanged, the light intensity is 2750 lx, the daily light time is 10 h, and the culture time is 20 days, to obtain rooted tissue culture seedlings.

[0034] S5: acclimatization and transplanting of rooting tissue culture seedlings S5.1: move the culture bottle containing the rooting tissue culture seedlings to an outdoor shaded canopy for 7 days of bottle hardening, with a shading degree of 65%, then open the bottle mouth, and place it for another 7 days, and frequently rotate the bottle body to ensure uniform light exposure; S5.2: then transplant to a nursery plug culture with a culture medium composed of peat soil: vermiculite: perlite at a mass ratio of 3:1:1 to survive, and then transplant to the field.

[0035] Example 3: a method for tissue culture and rapid propagation of Cynanchum komarovii, comprising the following steps:

[0036] S1: sterilization and disinfection of explants S1.1: cut the Cynanchum komarovii plant growing healthily without pests and diseases into 6 cm stem segments with buds in spring, shake clean in soap solution for 30 min, rinse clean with tap water, shake in Tween-80 for 8 min, then rinse under running water for 1 h to obtain clean stem segments with buds; S1.2: place salicylic acid and metformin hydrochloride in a container, add sterile water and stir until completely dissolved to obtain an explant cleaning solution, the salicylic acid concentration in the explant cleaning solution is 1.5 g / L, and the metformin hydrochloride concentration is 3 g / L; S1.3: place the clean stem segments with buds in the clean bench, first sterilize with 75% ethanol for 45 s, rinse 5 times with sterile water, then immerse in 1% carbendazim solution for 8 min of sterilization and disinfection treatment, rinse 5 times with sterile water, then shake in a 0.05% mercury solution for 5 min of sterilization, and finally rinse 5 times with the explant cleaning solution to obtain sterilized and disinfected explants.

[0037] S2: primary culture of sterilized and disinfected explants S2.1: inoculate the sterilized and disinfected explants into the primary culture medium, which is composed of MS medium + 1 mg / L 6-BA + 0.05 mg / L NAA + 30 mg / L sucrose + 6 g / L agar, and incubate in the dark at 27°C for 7 days; S2.2: keep the temperature unchanged, and perform 25 days of light and dark alternating culture under the condition of 10 h of daily light with an illumination intensity of 3000 lx to obtain primary cluster buds.

[0038] S3: proliferation culture of primary cluster buds S3.1: The primary cluster shoots are cut into 3 cm stem segments, soaked in a 3,6-dihydroxyacetyl sucrose solution at 30 mg / L for 10 minutes, inoculated into a proliferation culture medium composed of MS medium + 0.1 mg / L ZT + 1.5 mg / L 6-BA + 0.03 mg / L NAA + 30 mg / L sucrose + 6 g / L agar, and cultured; S3.2: The light and dark alternating culture is carried out at 27℃ for 35 days under the condition of 12 hours of daily illumination at an illumination intensity of 3000 lx, to obtain rootless tissue culture seedlings.

[0039] S4: Rooting culture of the rootless tissue culture seedlings S4.1: The rootless tissue culture seedlings are inoculated into a culture bottle containing a rooting culture medium composed of 1 / 2MS medium + 0.5 mg / L IBA + 500 mg / L activated carbon + 30 mg / L sucrose + 6 g / L agar, and cultured under red and blue composite light, wherein the red light spectrum: blue light spectrum is 1.5:1, the red light wavelength is 700 nm, the blue light wavelength is 420 nm, the culture temperature is 27℃, the illumination intensity is 2500 lx, the daily illumination time is 12 hours, and the culture time is 20 days; S4.2: Then, white light culture is adopted, the culture temperature is kept unchanged, the illumination intensity is 3000 lx, the daily illumination time is 10 hours, and the culture time is 20 days, to obtain rooted tissue culture seedlings.

[0040] S5: Acclimatization and transplanting of the rooted tissue culture seedlings S5.1: The culture bottle containing the rooted tissue culture seedlings is moved to an outdoor shaded awning for 7 days of bottle hardening, the shading degree is 70%, then the bottle mouth is opened, and the bottle is placed for another 7 days, and the bottle body is frequently rotated to ensure uniform light reception; S5.2: Then, the seedlings are transplanted into a seedling culture tray composed of peat soil: vermiculite: perlite at a mass ratio of 3:1:1 to survive, and then transplanted into a field.

[0041] Comparative Example 1: A method for tissue culture and rapid propagation of Cucurbita pepo seedlings, which is different from Example 1 in that the carbendazim solution in step S1.3 is replaced by a sodium hypochlorite solution with a concentration of 2.5%, and the remaining steps are the same as those in Example 1, to obtain sterilized explants.

[0042] Comparative Example 2: A method for tissue culture and rapid propagation of Cucurbita pepo seedlings, which is different from Example 1 in that the explant washing solution in step S1.3 is replaced by a 3 g / L aqueous salicylic acid solution, and the remaining steps are the same as those in Example 1, to obtain sterilized explants.

[0043] The method for tissue culture and rapid propagation of Cynanchum Komarovii to seedlings is different from that of Example 1 in that the washing solution of explants in step S1.3 is replaced by a sterile aqueous solution of 3 g / L metformin hydrochloride, and the remaining steps are the same as those of Example 1 to obtain sterilized and disinfected explants.

[0044] The method for tissue culture and rapid propagation of Cynanchum Komarovii to seedlings is different from that of Example 1 in that the stem segments are not soaked with a 3,6-di-jasmine acetyl sucrose solution in step S3.1, and the remaining steps are the same as those of Example 1 to obtain rootless tissue culture seedlings.

[0045] The method for tissue culture and rapid propagation of Cynanchum Komarovii to seedlings is different from that of Example 1 in that the red and blue composite light in step S4.1 is replaced by white light with an illumination intensity of 2500 lx, and the remaining steps are the same as those of Example 1 to obtain rooted tissue culture seedlings.

[0046] Experiment I: 100 sterilized and disinfected explants obtained from Examples 1-3 and Comparative Examples 1-3 were taken to statistically determine the contamination rate and survival rate of the sterilized and disinfected explants. The contamination rate = (number of contaminated explants / total number of explants) x 100%, and the survival rate = (number of surviving explants / total number of explants). The statistical results are shown in Table 1.

[0047] Table 1: Contamination rate and survival rate of sterilized and disinfected explants Contamination rate / % Survival rate / % Example 1 4 92 Example 2 3 93 Example 3 3 94 Comparative Example 1 9 85 Comparative Example 2 8 86 Comparative Example 3 6 88 As can be seen from the data of Examples 1-3 in Table 1, the sterilized and disinfected explants of the present application have a low contamination rate and a high survival rate. However, as can be seen from the data of Comparative Example 1, the contamination rate increases and the survival rate decreases after the carbendazim solution is replaced by a sodium hypochlorite solution, indicating that the conventional disinfectant sodium hypochlorite is not as suitable for sterilization and disinfection of Cynanchum Komarovii as carbendazim. As can be seen from the data of Comparative Examples 2-3, the sterilized and disinfected explants washed with sterile water containing salicylic acid and metformin hydrochloride have a low contamination rate and a high survival rate, proving that both can effectively remove mercury and sterilize, thereby increasing the survival rate of the explants.

[0048] Experiment II: 100 uncontaminated and surviving sterilized and disinfected explants obtained from Examples 1-3 and Comparative Examples 2-3 were taken to obtain primary clustered shoots after culture according to step S2, and the induction rate and growth of the clustered shoots were statistically determined, as shown in Table 2.

[0049] Table 2: Induction rate and growth of primary clustered shoots Average induction rate / % Sprout growth Example 1 93 Sprout healthy, bright green Example 2 93 Sprout healthy, bright green Example 3 95 Sprout healthy, bright green Comparative Example 2 89 Sprout healthy, bright green Comparative Example 3 88 Sprout healthy, bright green As can be seen from the data of Table 2, the primary culture medium of the present application can well induce the cluster bud growth of explants, and after the explants are rinsed with sterile water containing salicylic acid and metformin hydrochloride, both of them can adhere to the surface of the explants, synergistically improve the absorption and utilization ability of the plant to the growth hormone in the primary culture medium, thereby improving the induction rate of the cluster buds.

[0050] Experiment three: 100 portions of the primary cluster buds obtained from Examples 1-3 and Comparative Example 4 were cultured according to the respective step S3 to obtain the rootless tissue culture seedlings, the proliferation coefficient and growth of the rootless tissue culture seedlings were counted, the proliferation coefficient = (total bud number of the rootless tissue culture seedlings after proliferation / total bud number of the primary cluster buds inoculated), as shown in Table 3.

[0051] Table 3: Proliferation coefficient and growth of rootless tissue culture seedlings Average multiplication factor Sprout growth Example 1 8.1 No callus, sprout healthy, leaves unfolded Example 2 8.4 No callus, sprout healthy, leaves unfolded Example 3 8.5 No callus, sprout healthy, leaves unfolded Comparative Example 4 7.2 A small amount of callus, sprout slightly shorter, leaves unfolded As can be seen from the data of Examples 1-3 in Table 3, the proliferation medium of the present application has a proliferation coefficient of 8.1-8.5 for the primary cluster bud induction, has good proliferation effect, and the growth of the buds is healthy. Moreover, as can be seen from the data of Comparative Example 4, when the stem segments are not treated with 3,6-dihydroxybenzyl sucrose solution, the proliferation coefficient decreases significantly, which can prove that 3,6-dihydroxybenzyl sucrose can adhere to the surface of the stem segments as a high-quality carbon source and play the role of a signal molecule to stimulate the growth of cluster buds, greatly promoting the proliferation of cluster buds of Cucubalus baccifer.

[0052] Experiment four: 100 portions of the rootless tissue culture seedlings obtained from Examples 1-3 and Comparative Example 5 were cultured according to the respective step S4 to obtain the rooting tissue culture seedlings, the rooting rate and average number of roots of the rooting tissue culture seedlings were counted, the average number of roots = (total number of roots of all rooting tissue culture seedlings / total number of rooting tissue culture seedlings), and the obtained rooting tissue culture seedlings were acclimatized and transplanted according to the respective step S5, the survival rate of transplantation was counted, the survival rate of transplantation = (the number of plants surviving after transplantation / the total number of plants at the time of transplantation) x 100%, and the statistical results are shown in Table 4.

[0053] Table 4: Rooting rate and average number of roots of rooting tissue culture seedlings Average rooting rate / % Average number of roots Transplanting survival rate / % Example 1 93 4.5 92 Example 2 93 4.7 94 Example 3 94 4.8 95 Comparative Example 5 87 3.8 90 As can be seen from the data of Examples 1-3 and Comparative Example 5 in Table 4, the rooting medium of the present application can well induce the rooting of tissue culture seedlings, and at the same time, the obtained rooting tissue culture seedlings have a high survival rate of transplantation, which can also prove that by adjusting the light during the rooting culture to 20 days of red and blue composite light + 20 days of white light, the rooting rate, the number of roots of the rooting tissue culture seedlings and the survival rate of transplantation of the rooting tissue culture seedlings can be further improved, and the production cost of the tissue culture seedlings can be reduced.

[0054] Experiment five: replace the formula of primary culture medium, proliferation culture medium and rooting culture medium respectively, and the rest of the steps are the same as example 1, test the growth index of melon seed at different stages, as shown in table 5, 6, 7.

[0055] Table 5: the influence of different primary culture medium formula on bud induction Primary culture medium formula Average induction rate / % MS+6-BA 1.5 mg / L+NAA 0.05 mg / L 77 MS+6-BA 0.5 mg / L+NAA 0.05 mg / L 71 MS+6-BA 1.0 mg / L+NAA 0.1 mg / L 68 MS+6-BA 1.0 mg / L+NAA 0.01 mg / L 61 MS+6-BA 1.0 mg / L+IAA 0.05 mg / L 58 MS+6-BA 1.0 mg / L+IBA 0.05 mg / L 55 Table 6: the influence of different proliferation culture medium formula on bud proliferation Multiplication medium formula Average multiplication factor MS+ZT 0.05 mg / L+6-BA 1 mg / L+NAA 0.01 mg / L 4.5 MS+ZT 0.15 mg / L+6-BA 1 mg / L+NAA 0.01 mg / L 4.6 MS+ZT 0.1 mg / L+6-BA 0.5 mg / L+NAA 0.02 mg / L 4.8 MS+ZT 0.1 mg / L+6-BA 2 mg / L+NAA 0.02 mg / L 5.1 MS+ZT 0.1 mg / L+6-BA 1.5 mg / L+NAA 0.005 mg / L 5.8 MS+ZT 0.1 mg / L+6-BA 1.5 mg / L+NAA 0.05 mg / L 5.5 Table 7: the influence of different rooting culture medium formula on root induction of rootless tissue culture seedling Rooting medium formula Average rooting rate / % Average number of roots Transplanting survival rate / % 1 / 2MS medium+0.5 mg / L of IBA+200 mg / L of activated carbon 75 3.4 82 1 / 2MS medium+0.5 mg / L of IBA+600 mg / L of activated carbon 68 2.5 75 1 / 2MS medium+0.1 mg / L of IBA+300 mg / L of activated carbon 76 2.9 78 1 / 2MS medium+1.0 mg / L of IBA+500 mg / L of activated carbon 73 2.7 76 Note: all the formula in table 5, 6 and 7 also need to add 30mg / L sucrose and 6g / L agar.

[0056] From the data in table 5, 6 and 7 and the data of example in table 2, 3 and 4, it can be known that the most suitable primary culture medium formula for melon seed in the process of tissue culture and rapid propagation is MS medium + 1mg / L 6-BA + 0.05mg / L NAA + 30mg / L sucrose + 6g / L agar, the most suitable proliferation culture medium formula is MS medium + 0.1mg / L ZT + 1-1.5mg / L 6-BA + 0.01-0.03mg / L NAA + 30mg / L sucrose + 6g / L agar, and the most suitable rooting culture medium formula is 1 / 2MS medium + 0.5mg / L IBA + 300-500mg / L activated carbon + 30mg / L sucrose + 6g / L agar, which are respectively used for cluster bud induction, cluster bud proliferation and root induction of explant material, and the total culture process only needs about 100 days, the propagation efficiency is high, and the induction rate of cluster bud, cluster bud proliferation coefficient and rooting rate all reach a high level.

[0057] Experiment 6: adopt the conventional seed propagation method of melon seed to cultivate seedlings, take 1kg melon seed, soak the seeds in 25% PEG water solution for 24h, mix the seeds with 3 times volume of fine sand after taking out, pile up for sand storage, then sow in early April, the row spacing is 25cm and the sowing width is 15cm, irrigate the prepared plot with water, then appropriate for soil loosening, raking, leveling and watering. When the seeds just germinate, record the number of seedlings, mark as N1, strengthen the management of seedlings, set up protection facilities, when the seedlings grow to about 2cm, remove the protection facilities, water to maintain soil moisture, and acclimate the seedlings. When the seedlings grow to about 4cm, loosen the soil and remove the weeds, carry out thinning and fixing seedlings, remove the weak, inferior and diseased seedlings, get healthy melon seed seedlings, continue to cultivate for 6 months, get melon seed plants, record the number of plants, mark as N2, and the survival rate of seedlings = (N1-N2) / N1x100%.

[0058] The 100 rooting tissue culture seedlings of each of the three groups of Example 1-3 were transplanted by the respective step S5, and then were further cultivated for 6 months to obtain the C. japonica plants. The number of plants was recorded as N3, and the survival rate of the seedlings was calculated as (100-N3) / 100*100%. The plant morphology of the C. japonica plants obtained by the conventional seed propagation method was compared, and the results are shown in Table 8.

[0059] Table 8: Survival rate of seedlings and plant morphology Seedling survival rate / % Plant morphology Example 1 93 92% of the plant leaves were full and not wilted, without yellow spots and edges, the top leaves were bright green, and the old leaves were uniform in color Example 2 94 91% of the plant leaves were full and not wilted, without yellow spots and edges, the top leaves were bright green, and the old leaves were uniform in color Example 3 92 93% of the plant leaves were full and not wilted, without yellow spots and edges, the top leaves were bright green, and the old leaves were uniform in color Conventional seed propagation method 71 78% of the plant leaves were full and not wilted, without yellow spots and edges, the top leaves were bright green, and the old leaves were uniform in color As can be seen from the survival rate of the seedlings and the plant morphology of Example 1-3, the C. japonica seedlings obtained by the method of the present application have good growth vigor, and most of the plant morphology is very healthy. The survival rate of the seedlings obtained by the conventional seed propagation method is only 71%, and only 78% of the plant morphology is very healthy. It can be proved that the method of cultivating C. japonica seedlings of the present application has a significant advantage compared with the conventional seed propagation method.

[0060] The above examples only illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. A method for rapid propagation of melon seedlings through tissue culture, characterized in that, Includes the following steps: Step S1: Sterilization and disinfection of explants. Cut the stem segments with buds from the Guazi Jin plant, clean them, and then sterilize and disinfect them to obtain sterilized and disinfected explants. Step S2: Primary culture of sterilized explants. Sterilized explants are inoculated into primary culture medium to induce axillary bud germination and obtain primary clustered buds. The primary culture medium consists of MS medium + 1 mg / L 6-BA + 0.05 mg / L NAA + 30 mg / L sucrose + 6 g / L agar. Step S3: Proliferation culture of primary cluster shoots. Primary cluster shoots are cut into 2-3 cm stem segments and soaked in 20-30 mg / L 3,6-disinyl sucrose solution for 8-10 minutes. Then they are inoculated into proliferation medium for culture to obtain rootless tissue culture seedlings. The proliferation medium consists of MS medium + 0.1 mg / L ZT + 1-1.5 mg / L 6-BA + 0.01-0.03 mg / L NAA + 30 mg / L sucrose + 6 g / L agar. Step S4: Rooting culture of rootless tissue culture seedlings. Rootless tissue culture seedlings are inoculated into rooting medium for in-bottle rooting culture to obtain rooted tissue culture seedlings. The rooting medium consists of 1 / 2 MS medium + 0.5 mg / L IBA + 300-500 mg / L activated carbon + 30 mg / L sucrose + 6 g / L agar. Step S5: Acclimatization and transplanting of rooted tissue culture seedlings. First, close the bottle to harden the seedlings, then open the bottle to harden them for 7 days. Transplant them into seedling trays and cultivate until they survive, and then transplant them to the field.

2. The method for rapid propagation of *Gynostemma pentaphyllum* seedlings by tissue culture according to claim 1, characterized in that, Step S1 specifically includes the following steps: S1.1: In spring, cut healthy, disease-free Guazi Jin plants into 5-6cm stem segments with buds, immerse them in soapy water and shake to clean for 25-30 minutes, rinse them with tap water, then immerse them in Tween-80 and shake for 5-8 minutes, and then rinse them under running water for 0.5-1 hour to obtain clean stem segments with buds. S1.2: Place salicylic acid and metformin hydrochloride in a container, add sterile water and stir until completely dissolved to obtain explant washing solution. The concentration of salicylic acid in the explant washing solution is 1-1.5 g / L and the concentration of metformin hydrochloride is 2-3 g / L. S1.3: Place the clean stem segments with buds in a clean bench. First, disinfect them with 70-75% ethanol for 40-45 seconds. Rinse them with sterile water 4-5 times, then immerse them in 1% carbendazim solution for 6-8 minutes for sterilization. Rinse them with sterile water 4-5 times, then place them in 0.05% mercuric chloride solution and shake for 4-5 minutes for sterilization. Finally, rinse them with explant cleaning solution 4-5 times to obtain sterilized explants.

3. The method for rapid propagation of *Gynostemma pentaphyllum* seedlings by tissue culture according to claim 2, characterized in that, Step S2 specifically includes the following steps: S2.1: Inoculate sterilized explants into primary culture medium and incubate in the dark for 7 days at 25-27℃; S2.2: Keep the temperature constant and carry out light-dark alternation culture for 25 days under the conditions of 10 hours of light per day and light intensity of 2500-3000 lx to obtain the first generation of clustered shoots.

4. The method for rapid propagation of *Gynostemma pentaphyllum* seedlings by tissue culture according to claim 3, characterized in that, Step S3 specifically includes the following steps: S3.1: Cut the primary clustered shoots into 2-3 cm stem segments, soak them in 20-30 mg / L 3,6-disinyl sucrose solution for 8-10 minutes, and then inoculate them into the proliferation medium; S3.2: Rootless tissue culture seedlings were obtained by alternating light and dark culture for 35 days under conditions of 25-27℃, 12 hours of light per day, and light intensity of 2500-3000 lx.

5. The method for rapid propagation of *Gynostemma pentaphyllum* seedlings by tissue culture according to claim 4, characterized in that... Step S4 specifically includes the following steps: S4.1: Inoculate the rootless tissue culture seedlings into culture bottles containing rooting medium and culture them under red-blue composite light at a temperature of 25-27℃ and a light intensity of 2000-2500 lx for 12 hours of light per day for 20 days. S4.2: Then switch to white light culture, keep the culture temperature constant, the light intensity is 2500-3000 lx, the light intensity is 10 hours per day, and the culture is carried out for 20 days to obtain rooted tissue culture seedlings.

6. The method for rapid propagation of *Gynostemma pentaphyllum* seedlings by tissue culture according to claim 5, characterized in that, Step S5 specifically includes the following steps: S5.1: Move the culture bottles containing rooted tissue culture seedlings to an outdoor shaded area and close the bottles for 7 days to harden off the seedlings. The shade level should be 60-70%. Then open the bottle mouth and leave it for another 7 days. During this time, rotate the bottle frequently to ensure even light exposure. S5.2: Then transplant them into seedling trays and cultivate them until they survive, and then transplant them into the field.

7. The method for rapid propagation of *Gynostemma pentaphyllum* seedlings by tissue culture according to claim 5, characterized in that, In step S4.1, the red light spectrum and blue light spectrum in the red-blue composite light are (1-1.5):1, where the red light wavelength is 680-700nm and the blue light wavelength is 400-420nm.

8. The method for rapid propagation of *Gynostemma pentaphyllum* seedlings by tissue culture according to claim 6, characterized in that, Step S5.2 The culture medium in the seedling trays consists of peat moss, vermiculite, and perlite in a mass ratio of 3:1:1.

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