A method for rapid propagation of wild persimmon through tissue culture

Abstract

The invention provides a method for rapid propagation of wild persimmon tissue culture, which belongs to the technical field of plant culture and comprises the following steps: primary culture; proliferation culture; rooting culture; acclimatization and transplantation. The invention firstly decomposes urea at high temperature, then places it in a sodium hydroxide solution for heating and reflux, then repeatedly disperses by ultrasound, centrifuges and filters with a microporous membrane to prepare a colloidal solution, then adds polyethyleneimine to the colloidal solution for modification, and finally adds an ascorbic acid solution, stirs and fully loads it, so that the ascorbic acid is loaded in a carrier material to form an anti-browning agent with a composite structure, and the anti-browning agent can slowly release the ascorbic acid due to the loading effect of the carrier material, thereby achieving the effect of continuously inhibiting the browning of tissue culture and avoiding the aggravation of the browning phenomenon due to the excessive one-time concentration of ascorbic acid.

Description

Technical Field

[0001] The invention relates to the technical field of plant cultivation, and in particular to a tissue culture rapid propagation method for wild persimmon. Background Art

[0002] Wild persimmon is an important economic crop and ornamental plant. Its fruit is not only delicious but also has high nutritional value. With the increasing demand for the quality and yield of wild persimmon, traditional propagation methods can no longer meet the requirements of modern planting industry. Tissue culture technology has been widely used in plant propagation due to its high efficiency, rapidity and scalability.

[0003] Browning usually occurs during plant tissue culture. Browning is manifested as the explant material secreting brown substances into the culture medium, which leads to poor growth or even death. It often occurs in woody plants. However, compared with general woody plants, wild persimmon contains more tannins, which makes the browning phenomenon of persimmon tissue culture more serious. Therefore, it is usually necessary to add anti-browning agents to limit the browning phenomenon. Common anti-browning agents include ascorbic acid and activated carbon. Although ascorbic acid has a good anti-browning effect, its sustainability is poor, and its concentration is usually difficult to control. Too high a concentration will aggravate the browning phenomenon, while too low a concentration will make it difficult to limit the browning phenomenon.

[0004] Therefore, we proposed a tissue culture rapid propagation method for wild persimmon that can continuously inhibit the browning phenomenon. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a tissue culture rapid propagation method for wild persimmon.

[0006] A tissue culture rapid propagation method for wild persimmon comprises the following steps:

[0007] S1: Primary Culture

[0008] In autumn, wild persimmon stem segments with buds are sterilized and disinfected, then inoculated into primary culture medium, and axillary buds are germinated for 25 days to obtain germinated axillary buds;

[0009] S2: Proliferation culture

[0010] Cut the above-mentioned axillary buds into stem segments of 1.5-2 cm, and inoculate them in a proliferation culture medium for proliferation culture. After 25 days, rootless tissue culture seedlings are obtained;

[0011] S3: Rooting culture

[0012] Placing the rootless tissue culture seedlings in a rooting medium and performing in-bottle rooting culture, and obtaining rooted tissue culture seedlings after 25 days;

[0013] S4: Acclimation and transplanting

[0014] Take the above rooted tissue culture seedlings, close the bottle for seedling hardening, then open the bottle for seedling hardening for 7 days, then transplant them to seedling trays for cultivation until they survive, and finally transplant them to the field.

[0015] Furthermore, the steps for preparing the primary culture medium are as follows:

[0016] T1.1: Add turmeric powder to 60% ethanol solution at a material-liquid ratio of 1g: (15-25)mL, stir and mix evenly, then heat at 50-60℃ for 40-50min, then ultrasonically treat at 180-220W for 30-40min, and finally continue heating and reflux extraction for 1-2h. After cooling, filter to obtain turmeric extract;

[0017] T1.2: Add Sophora flavescens powder to distilled water at a solid-liquid ratio of 1g: (20-30)mL, stir and mix evenly, then microwave at a microwave power of 100-200W for 10-20min, then add anhydrous ethanol, heat and reflux at 80-90℃ for 1-2h, cool and filter to obtain Sophora flavescens extract;

[0018] T1.3: Mix the turmeric extract and the sophora flavescens extract in a volume ratio of 1:(1-3), place in a concentrator, evaporate and concentrate, remove the solvent, and obtain a composite antibacterial agent;

[0019] T1.4: Add the above-mentioned composite antimicrobial agent, 6-benzyladenine, naphthylacetic acid, sucrose and agar to the MS medium, mix evenly, and obtain the primary culture medium.

[0020] Further, the preparation steps of the rooting medium are as follows:

[0021] T2.1: Place urea in a muffle furnace, heat it to 500-600℃ at 10-20℃ / min and keep it at that temperature for 3-4h. After cooling to room temperature, grind and wash it with water to obtain solid powder;

[0022] T2.2: Add the above solid powder into 0.1 mol / L sodium hydroxide solution at a solid-liquid ratio of 1 g: (40-50) mL, heat and reflux at 100-120 ° C for 10-12 h, then centrifuge at a speed of 3000-4000 r / min for 3-5 min, remove the supernatant, and obtain a precipitate;

[0023] T2.3: Ultrasonic disperse the above precipitate in distilled water at a solid-liquid ratio of 1g: (25-35)mL to form a suspension, and then continue to centrifuge at a rate of 3000-4000r / min for 3-5min, collect the supernatant, and continue to ultrasonically disperse the precipitate in distilled water, repeat several times until no precipitate appears, then combine the supernatant, filter with a microporous filter membrane, and finally re-disperse the precipitate on the microporous filter membrane with distilled water to obtain a colloidal solution;

[0024] T2.4: centrifuge the colloidal solution at a rate of 8000-9000 r / min for 20-30 min, collect the supernatant, add polyethyleneimine aqueous solution to the supernatant, adjust the pH to neutral, and then ultrasonicate for 20-30 min to obtain a mixed solution;

[0025] T2.5: adding ascorbic acid solution to the above mixed solution, stirring for 10-12 hours, uniformly mixing, and then centrifuging at a rate of 20000-30000 r / min for 30-40 minutes, removing the supernatant, and washing and vacuum drying the precipitate to obtain an anti-browning agent;

[0026] T2.6: Add the above anti-browning agent, indolebutyric acid, 6-benzyladenine, naphthylacetic acid, brassinolide, sucrose and agar to the MS medium, mix evenly, and obtain a rooting medium.

[0027] Furthermore, the primary culture medium consists of MS medium, 0.9 mg / L composite antimicrobial agent, 1.0 mg / L 6-benzyladenine, 0.05 mg / L naphthaleneacetic acid, 30 mg / L sucrose and 6 g / L agar.

[0028] Furthermore, the solid-liquid ratio of anhydrous ethanol to Sophora flavescens powder is 1 g: (10-12) mL.

[0029] Furthermore, the proliferation medium consists of MS medium, 0.9 mg / L composite antimicrobial agent, 0.5 mg / L zeatin, 1.0 mg / L 6-benzyladenine, 0.1 mg / L naphthaleneacetic acid, 30 mg / L sucrose and 6 g / L agar.

[0030] Furthermore, the rooting medium consists of MS medium, 1.0 mg / L indolebutyric acid, 0.5 mg / L 6-benzyladenine, 0.05 mg / L brassinolide, 0.01 mg / L naphthylacetic acid, 3 g / L anti-browning agent, 30 mg / L sucrose, and 6 g / L agar.

[0031] Furthermore, the pore size of the microporous filter membrane is 0.22 μm.

[0032] Furthermore, the concentration of the polyethyleneimine aqueous solution is 50-60%.

[0033] Furthermore, the volume ratio of the ascorbic acid solution to the mixed solution is (4-6):1, and the concentration of the ascorbic acid solution is 10-20 μmol / L.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] 1. The present invention first decomposes urea at high temperature, then places it in a sodium hydroxide solution for heating and reflux, and then repeatedly ultrasonically disperses, centrifuges and filters through a microporous membrane to prepare a colloidal solution, then adds polyethyleneimine to the colloidal solution for modification, and finally adds an ascorbic acid solution, stirs and fully loads it, so that the ascorbic acid is loaded in a carrier material to form an anti-browning agent with a composite structure. Due to the loading effect of the carrier material, the anti-browning agent can slowly release the ascorbic acid, thereby achieving the effect of continuously inhibiting tissue culture browning and avoiding the aggravation of browning due to excessive one-time concentration of ascorbic acid.

[0036] 2. The present invention prepares a turmeric extract by adding turmeric powder to an ethanol solution and performing ultrasonic-assisted heating reflux extraction, then adds sophora flavescens powder to distilled water, performs microwave treatment, then adds anhydrous ethanol, performs heating reflux extraction, and prepares a sophora flavescens extract; the turmeric extract and the sophora flavescens extract are mixed, concentrated, and the solvent is removed to prepare a composite antibacterial agent; the antibacterial agent is added to a primary culture medium and a proliferation culture medium, which can play an antibacterial effect, prevent bacterial contamination problems during tissue culture, and ensure the success rate of wild persimmon tissue culture; in addition, the turmeric extract and the sophora flavescens extract have a certain synergistic effect, and when the two are added at the same time, they have a better antibacterial effect and will not produce toxic side effects on wild persimmon cultures.

[0037] 3. The present invention adds indolebutyric acid to the MS culture medium and then adds a small amount of brassinolide to prepare a rooting culture medium. Since brassinolide has the effects of promoting cell division and growth, balancing plant vegetative growth and reproductive growth, after it is mixed with indolebutyric acid, the two can interact with each other and comprehensively promote plant growth and metabolic activities, thereby further promoting the rooting of rootless tissue culture seedlings and improving the success rate of wild persimmon tissue culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, further serve to explain the principles of the present disclosure and to enable those skilled in the relevant art to make and use the present disclosure.

[0039] Figure 1 The present invention is a flowchart of the method for rapid propagation of wild persimmon through tissue culture adopted in the embodiment of the present invention.

[0040] Figure 2 This is a diagram of the axillary buds that have germinated after primary culture in Example 1 of the present invention.

[0041] Figure 3 This is a diagram of the rootless tissue culture seedlings after proliferation and cultivation in Example 1 of the present invention.

[0042] Figure 4 This is a diagram of the rooted tissue culture seedlings after rooting culture in Example 1 of the present invention.

[0043] Figure 5 This is a diagram of surviving seedlings after domestication and transplantation in Example 1 of the present invention. DETAILED DESCRIPTION

[0044] The tissue culture rapid propagation method of wild persimmon provided by the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Embodiment 1: A method for rapid propagation of wild persimmon through tissue culture, such as Figure 1 As shown, the following steps are included:

[0046] (1) Preparation of primary culture medium:

[0047] Turmeric powder was added to 60% ethanol solution at a solid-liquid ratio of 1g:15mL, stirred and mixed evenly, then heated at 50°C for 40min, and then ultrasonically treated at an ultrasonic power of 180W for 30min, and finally continued to heat and reflux for extraction for 1h. After cooling, it was filtered to obtain turmeric extract for standby use. Subsequently, Sophora flavescens powder was added to distilled water at a solid-liquid ratio of 1g:20mL, stirred and mixed evenly, and then microwaved at a microwave power of 100W for 10min. Then, anhydrous ethanol was added, and heated and refluxed at 80°C for extraction for 1h. After cooling and filtering, Sophora flavescens extract was obtained, wherein anhydrous ethanol The solid-liquid ratio of alcohol to sophora flavescens powder is 1 g:10 mL, and then the turmeric extract and the sophora flavescens extract are stirred and mixed at a volume ratio of 1:1, and then placed in a concentrator for evaporation and concentration to remove the solvent to obtain a composite antibacterial agent, and finally the composite antibacterial agent, 6-benzyladenine, naphthylacetic acid, sucrose and agar are added to the MS medium, and after uniform mixing, a primary culture medium is obtained, wherein the primary culture medium consists of MS medium, 0.9 mg / L composite antibacterial agent, 1.0 mg / L 6-benzyladenine, 0.05 mg / L naphthylacetic acid, 30 mg / L sucrose and 6 g / L agar;

[0048] (2) Preparation of rooting medium:

[0049] The urea was placed in a muffle furnace, heated to 500°C at a rate of 10°C / min and kept warm for 3h. After cooling to room temperature with the furnace, it was ground and washed with water to obtain a solid powder. The solid powder was then added to a 0.1mol / L sodium hydroxide solution at a solid-liquid ratio of 1g:40mL, heated to reflux at 100°C for 10h, and then centrifuged at a rate of 3000r / min for 3min, the supernatant was removed to obtain a precipitate, and then the precipitate was ultrasonically dispersed in distilled water at a solid-liquid ratio of 1g:25mL to form a suspension, and then continued to centrifuge at a rate of 3000r / min for 3min, the supernatant was collected, and the precipitate was ultrasonically dispersed in distilled water, and repeated several times until no precipitate appeared, and then the supernatants were combined and filtered with a 0.22μm microporous filter membrane, and finally the precipitate on the microporous filter membrane was redispersed with distilled water to obtain a colloidal solution. Subsequently, the colloidal solution was centrifuged at a rate of 8000r / min for 20min, the supernatant was collected, and polyethyleneimine was added to the supernatant. The aqueous solution was prepared by ultrasonic treatment for 20 minutes to obtain a mixed solution, wherein the concentration of the polyethyleneimine aqueous solution was 50%, and then ascorbic acid solution was added to the mixed solution, stirred for 10 hours, uniformly mixed, and then centrifuged at a rate of 20000r / min for 30 minutes, the supernatant was removed, and the precipitate was washed with water and vacuum dried to obtain an anti-browning agent, wherein the volume ratio of the ascorbic acid solution to the mixed solution was 4:1, and the concentration of the ascorbic acid solution was 10 μmol / L, finally, adding anti-browning agent, indolebutyric acid, 6-benzyladenine, naphthylacetic acid, brassinolide, sucrose and agar to the MS medium, and uniformly mixing to obtain a rooting medium, wherein the rooting medium consists of MS medium, 1.0 mg / L indolebutyric acid, 0.5 mg / L 6-benzyladenine, 0.05 mg / L brassinolide, 0.01 mg / L naphthylacetic acid, 3 g / L anti-browning agent, 30 mg / L sucrose, and 6 g / L agar;

[0050] S1: Primary Culture

[0051] In autumn, wild persimmon stem segments with buds were sterilized and disinfected, and then inoculated into the primary culture medium for axillary bud germination. After 25 days, axillary buds were obtained. Figure 2 As shown;

[0052] S2: Proliferation culture

[0053] The above-mentioned axillary buds were cut into stem segments of 1.5 cm and inoculated into a proliferation medium for proliferation culture. After 25 days, rootless tissue culture seedlings were obtained. Figure 3 As shown, the proliferation medium consists of MS medium, 0.9 mg / L of the composite antibacterial agent prepared in step (1), 0.5 mg / L of zeatin, 1.0 mg / L of 6-benzyladenine, 0.1 mg / L of naphthaleneacetic acid, 30 mg / L of sucrose and 6 g / L of agar;

[0054] S3: Rooting culture

[0055] The rootless tissue culture seedlings are placed in a rooting medium and cultured in a bottle for rooting. After 25 days, rooted tissue culture seedlings are obtained. Figure 4 As shown;

[0056] S4: Acclimation and transplanting

[0057] Take the above rooted tissue culture seedlings, close the bottle and harden them for 7 days, then open the cover and harden them for 7 days, then transplant them to the seedling tray and culture them until they survive, and finally transplant them to the field. Figure 5 shown.

[0058] Embodiment 2: A method for rapid propagation of wild persimmon through tissue culture, such as Figure 1 As shown, the following steps are included:

[0059] (1) Preparation of primary culture medium:

[0060] Turmeric powder was added to 60% ethanol solution at a solid-liquid ratio of 1g:20mL, stirred and mixed evenly, then heated at 55°C for 45min, and then ultrasonically treated at an ultrasonic power of 200W for 35min, and finally continued to heat and reflux for extraction for 1.5h. After cooling, it was filtered to obtain a turmeric extract for standby use. Subsequently, Sophora flavescens powder was added to distilled water at a solid-liquid ratio of 1g:25mL, stirred and mixed evenly, and then microwaved at a microwave power of 150W for 15min. Then, anhydrous ethanol was added, and refluxed at 85°C for extraction for 1.5h. After cooling and filtering, a Sophora flavescens extract was obtained, wherein the Sophora flavescens extract was free of The solid-liquid ratio of water ethanol to sophora flavescens powder is 1 g:11 mL, and then the turmeric extract and the sophora flavescens extract are stirred and mixed at a volume ratio of 1:2, and then placed in a concentrator for evaporation and concentration to remove the solvent to obtain a composite antibacterial agent, and finally the composite antibacterial agent, 6-benzyladenine, naphthylacetic acid, sucrose and agar are added to the MS medium, and after uniform mixing, a primary culture medium is obtained, wherein the primary culture medium consists of MS medium, 0.9 mg / L composite antibacterial agent, 1.0 mg / L 6-benzyladenine, 0.05 mg / L naphthylacetic acid, 30 mg / L sucrose and 6 g / L agar;

[0061] (2) Preparation of rooting medium:

[0062] The urea was placed in a muffle furnace, heated to 550°C at 15°C / min and kept warm for 3.5h. After cooling to room temperature with the furnace, it was ground and washed with water to obtain a solid powder. The solid powder was then added to a 0.1mol / L sodium hydroxide solution at a solid-liquid ratio of 1g:45mL, heated to reflux at 110°C for 11h, and then centrifuged at a rate of 3500r / min for 4min, the supernatant was removed to obtain a precipitate, and then the precipitate was ultrasonically dispersed in distilled water at a solid-liquid ratio of 1g:30mL to form a suspension, and then continued to centrifuge at a rate of 3500r / min for 4min, the supernatant was collected, and the precipitate was continued to be ultrasonically dispersed in distilled water, and repeated several times until no precipitate appeared, and then the supernatants were combined and filtered with a 0.22μm microporous filter membrane, and finally the precipitate on the microporous filter membrane was re-dispersed with distilled water to obtain a colloidal solution. Subsequently, the colloidal solution was centrifuged at a rate of 8500r / min for 25min, the supernatant was collected, and polyethylene glycol was added to the supernatant. Amine aqueous solution, adjusting pH to neutral, and then ultrasonically treating for 25 minutes to obtain a mixed solution, wherein the concentration of polyethyleneimine aqueous solution is 55%, and then adding ascorbic acid solution to the mixed solution, stirring for 11 hours, uniformly mixing, and then centrifuging at a rate of 25000r / min for 35 minutes, removing the supernatant, and washing and vacuum drying the precipitate to obtain an anti-browning agent, wherein the volume ratio of ascorbic acid solution to the mixed solution is 5:1, and the concentration of ascorbic acid solution is 1 5 μmol / L, finally, adding anti-browning agent, indolebutyric acid, 6-benzyladenine, naphthylacetic acid, brassinolide, sucrose and agar to the MS medium, and uniformly mixing to obtain a rooting medium, wherein the rooting medium consists of MS medium, 1.0 mg / L indolebutyric acid, 0.5 mg / L 6-benzyladenine, 0.05 mg / L brassinolide, 0.01 mg / L naphthylacetic acid, 3 g / L anti-browning agent, 30 mg / L sucrose, and 6 g / L agar;

[0063] S1: Primary Culture

[0064] In autumn, wild persimmon stem segments with buds were sterilized and disinfected, and then inoculated into primary culture medium for axillary bud germination. After 25 days, axillary buds were obtained.

[0065] S2: Proliferation culture

[0066] Cut the above-mentioned axillary buds into stem segments of 1.75 cm, and inoculate them in a proliferation culture medium for proliferation culture. After 25 days, rootless tissue culture seedlings are obtained, wherein the proliferation culture medium consists of MS medium, 0.9 mg / L of the composite antibacterial agent prepared in step (1), 0.5 mg / L of zeatin, 1.0 mg / L of 6-benzyladenine, 0.1 mg / L of naphthaleneacetic acid, 30 mg / L of sucrose and 6 g / L of agar;

[0067] S3: Rooting culture

[0068] Placing the rootless tissue culture seedlings in a rooting medium and performing in-bottle rooting culture, and obtaining rooted tissue culture seedlings after 25 days;

[0069] S4: Acclimation and transplanting

[0070] Take the above rooted tissue culture seedlings, close the bottle for seedling hardening, then open the bottle for seedling hardening for 7 days, then transplant them to seedling trays for cultivation until they survive, and finally transplant them to the field.

[0071] Embodiment 3: A method for rapid propagation of wild persimmon through tissue culture, such as Figure 1 As shown, the following steps are included:

[0072] (1) Preparation of primary culture medium:

[0073] Turmeric powder was added to 60% ethanol solution at a solid-liquid ratio of 1g:25mL, stirred and mixed evenly, then heated at 60°C for 50min, and then ultrasonically treated at 220W of ultrasonic power for 40min, and finally continued to heat and reflux for 2h. After cooling, it was filtered to obtain turmeric extract for standby use. Subsequently, Sophora flavescens powder was added to distilled water at a solid-liquid ratio of 1g:30mL, stirred and mixed evenly, and then microwaved at 200W of microwave power for 20min, and then anhydrous ethanol was added, and heated and refluxed at 90°C for 2h, cooled and filtered to obtain Sophora flavescens extract, wherein anhydrous ethanol The solid-liquid ratio of alcohol to sophora flavescens powder is 1 g:12 mL, and then the turmeric extract and the sophora flavescens extract are stirred and mixed at a volume ratio of 1:3, and then placed in a concentrator for evaporation and concentration to remove the solvent to obtain a composite antibacterial agent, and finally the composite antibacterial agent, 6-benzyladenine, naphthylacetic acid, sucrose and agar are added to the MS medium, and after uniform mixing, a primary culture medium is obtained, wherein the primary culture medium consists of MS medium, 0.9 mg / L composite antibacterial agent, 1.0 mg / L 6-benzyladenine, 0.05 mg / L naphthylacetic acid, 30 mg / L sucrose and 6 g / L agar;

[0074] (2) Preparation of rooting medium:

[0075] The urea was placed in a muffle furnace, heated to 600°C at 20°C / min and kept warm for 4h. After cooling to room temperature with the furnace, it was ground and washed with water to obtain a solid powder. The solid powder was then added to a 0.1mol / L sodium hydroxide solution at a solid-liquid ratio of 1g:50mL, heated to reflux at 120°C for 12h, and then centrifuged at a rate of 4000r / min for 5min, the supernatant was removed to obtain a precipitate, and then the precipitate was ultrasonically dispersed in distilled water at a solid-liquid ratio of 1g:35mL to form a suspension, and then continued to centrifuge at a rate of 4000r / min for 5min, the supernatant was collected, and the precipitate was ultrasonically dispersed in distilled water, and repeated several times until no precipitate appeared, and then the supernatants were combined and filtered with a 0.22μm microporous filter membrane. Finally, the precipitate on the microporous filter membrane was redispersed with distilled water to obtain a colloidal solution. Subsequently, the colloidal solution was centrifuged at a rate of 9000r / min for 30min, the supernatant was collected, and polyethyleneimine was added to the supernatant. The aqueous solution was prepared by ultrasonic treatment for 30 minutes to obtain a mixed solution, wherein the concentration of the polyethyleneimine aqueous solution was 60%, and then the ascorbic acid solution was added to the mixed solution, stirred for 12 hours, uniformly mixed, and then centrifuged at a rate of 30000r / min for 40 minutes, the supernatant was removed, and the precipitate was washed with water and vacuum dried to obtain an anti-browning agent, wherein the volume ratio of the ascorbic acid solution to the mixed solution was 6:1, and the concentration of the ascorbic acid solution was 20 μmol / L, finally, adding anti-browning agent, indolebutyric acid, 6-benzyladenine, naphthylacetic acid, brassinolide, sucrose and agar to the MS medium, and uniformly mixing to obtain a rooting medium, wherein the rooting medium consists of MS medium, 1.0 mg / L indolebutyric acid, 0.5 mg / L 6-benzyladenine, 0.05 mg / L brassinolide, 0.01 mg / L naphthylacetic acid, 3 g / L anti-browning agent, 30 mg / L sucrose, and 6 g / L agar;

[0076] S1: Primary Culture

[0077] In autumn, wild persimmon stem segments with buds were sterilized and disinfected, and then inoculated into primary culture medium for axillary bud germination. After 25 days, axillary buds were obtained.

[0078] S2: Proliferation culture

[0079] Cut the above-mentioned axillary buds into stem segments of 2 cm, and inoculate them in a proliferation culture medium for proliferation culture. After 25 days, rootless tissue culture seedlings are obtained, wherein the proliferation culture medium consists of MS medium, 0.9 mg / L of the composite antibacterial agent prepared in step (1), 0.5 mg / L of zeatin, 1.0 mg / L of 6-benzyladenine, 0.1 mg / L of naphthaleneacetic acid, 30 mg / L of sucrose and 6 g / L of agar;

[0080] S3: Rooting culture

[0081] Placing the rootless tissue culture seedlings in a rooting medium and performing in-bottle rooting culture, and obtaining rooted tissue culture seedlings after 25 days;

[0082] S4: Acclimation and transplanting

[0083] Take the above rooted tissue culture seedlings, close the bottle for seedling hardening, then open the bottle for seedling hardening for 7 days, then transplant them to seedling trays for cultivation until they survive, and finally transplant them to the field.

[0084] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that the anti-browning agent in the rooting medium is replaced by ascorbic acid having the same mass as that of the ascorbic acid in the ascorbic acid solution in step (2) of Example 1.

[0085] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that the turmeric extract in the preparation process of the composite antibacterial agent is replaced by an equal amount of Sophora flavescens extract.

[0086] Comparative Example 3: The difference between Comparative Example 3 and Example 1 is that the Sophora flavescens extract in the preparation process of the composite antibacterial agent is replaced by an equal amount of Curcuma longa extract.

[0087] Comparative Example 4: The difference between Comparative Example 4 and Example 1 is that the brassinolide in the rooting medium is replaced by an equal amount of indolebutyric acid.

[0088] Experiment 1:

[0089] The browning conditions of the rootless tissue culture seedlings in Examples 1-3 and Comparative Example 1 during the rooting culture process in the rooting medium were counted and calculated, and the results are shown in Table 1 below.

[0090] Table 1: Browning rate of Examples 1-3 and Comparative Example 1

[0091] Browning rate (%) Example 1 1.3 Example 2 1.2 Example 3 1.4 Comparative Example 1 42.8

[0092] As can be seen from Table 1, when ascorbic acid is directly added to the rooting medium in Comparative Example 1, the browning rate is about 42.8%, which is much greater than that in Example 1, indicating that when ascorbic acid is directly added, the browning phenomenon will be aggravated due to the high concentration of ascorbic acid. It can be seen that by first thermally decomposing urea at high temperature, then placing it in a sodium hydroxide solution for heating and reflux, and then repeatedly ultrasonically dispersing, centrifuging and filtering with a microporous membrane to prepare a colloidal solution, and then adding polyethyleneimine to the colloidal solution for modification, and finally adding ascorbic acid solution, stirring for full loading, so that ascorbic acid is loaded in the carrier material to form an anti-browning agent with a composite structure, the anti-browning agent can slowly release ascorbic acid due to the loading effect of the carrier material, thereby achieving the effect of continuously inhibiting tissue culture browning and avoiding the aggravation of browning due to excessive one-time concentration of ascorbic acid.

[0093] Experiment 2:

[0094] 90 culture bottles were prepared and randomly divided into 6 groups, each with 15 bottles, namely, Example 1-3 group, Comparative Example 2-3 group and blank group, wherein Example 1-3 group and Comparative Example 2-3 group were respectively prepared into primary culture medium according to the formula of each primary culture medium in Example 1-3 and Comparative Example 2-3, and the difference between the blank group and Example 1 group was that no composite antibacterial agent was added, and then the culture bottles of each group were directly packaged without high-pressure sterilization, and then the primary culture method of step S3 in Example 1 was used for primary culture. After 25 days, the axillary bud induction rate was counted and calculated, and then the number of contaminated culture bottles in each group was counted, and the contamination rate was calculated. The results are shown in Table 2 below.

[0095] Table 2: Contamination of culture medium in Examples 1-3 and Comparative Examples 2-3

[0096] Number of contaminations (bottles) Pollution rate (%) Axillary bud induction rate (%) Example 1 Group 1 6.7 76.4 Example 2 Group 1 6.7 75.7 Example 3 Group 0 0 76.9 Comparative Example 2 4 26.7 52.6 Comparative Example 3 3 20 58.3 Blank group 15 100 23.4

[0097] As can be seen from Table 2, the antibacterial agent prepared by using a single Sophora flavescens extract in Comparative Example 2 and a single Curcuma longa extract in Comparative Example 3 was added to the culture medium, and the contamination rates were about 26.7% and 20%, respectively, both greater than those of Example 1. In the blank group, when no composite antibacterial agent was added, the contamination rate was 100%. It can be seen that by adding turmeric powder to an ethanol solution, performing ultrasonic-assisted heating reflux extraction to obtain a turmeric extract, then adding Sophora flavescens powder to distilled water, performing microwave treatment, and then adding anhydrous ethanol to perform heating reflux extraction to obtain a Sophora flavescens extract, and then adding Curcuma longa to an ethanol solution. After the extract and the Sophora flavescens extract were mixed and concentrated to remove the solvent, a composite antibacterial agent was prepared. The antibacterial agent was added to the primary culture medium and the proliferation culture medium, which could play an antibacterial effect, prevent bacterial contamination during tissue culture, and ensure the success rate of wild persimmon tissue culture. In addition, the turmeric extract and the Sophora flavescens extract had a certain synergistic effect, and had a better antibacterial effect when the two were added at the same time. It can be seen from Table 2 that the axillary bud induction rate of the primary culture medium in Examples 1-3 was higher than 75%, indicating that the addition of the turmeric extract and the Sophora flavescens extract would not produce toxic side effects on the wild persimmon culture.

[0098] Experiment 3:

[0099] The rooting rates after the rooting culture in the bottle in Examples 1-3 and Comparative Example 4 were counted and calculated, and the results are shown in Table 3.

[0100] Table 3: Rooting conditions of Examples 1-3 and Comparative Example 4

[0101] Rooting rate (%) Example 1 94.3 Example 2 93.7 Example 3 94.5 Comparative Example 4 86.2

[0102] As can be seen from Table 3, after replacing brassinolide with an equal amount of indolebutyric acid in Comparative Example 4, the rooting rate was about 86.2%, which was lower than that in Example 1. It can be seen that by adding indolebutyric acid to the MS medium and then adding a small amount of brassinolide to prepare a rooting medium, since brassinolide has the effects of promoting cell division and growth, balancing plant vegetative growth and reproductive growth, after mixing it with indolebutyric acid, the two can interact with each other and comprehensively promote plant growth and metabolic activities, thereby further promoting the rooting of rootless tissue culture seedlings and improving the success rate of wild persimmon tissue culture.

[0103] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A tissue culture rapid propagation method for wild persimmon, characterized in that: The steps include: S1: Primary Culture In autumn, wild persimmon stem segments with buds are sterilized and disinfected, then inoculated into primary culture medium, and axillary buds are germinated for 25 days to obtain germinated axillary buds; S2: Proliferation culture Cut the above-mentioned axillary buds into stem segments of 1.5-2 cm, and inoculate them in a proliferation culture medium for proliferation culture. After 25 days, rootless tissue culture seedlings are obtained; S3: Rooting culture Placing the rootless tissue culture seedlings in a rooting medium and performing in-bottle rooting culture, and obtaining rooted tissue culture seedlings after 25 days; S4: Acclimation and transplanting Take the rooted tissue culture seedlings, close the bottle and harden them for 7 days, then open the bottle and harden them for 7 days, then transplant them to seedling trays and culture them until they survive, and finally transplant them to the field; The steps for preparing the primary culture medium are as follows: T1.1: Add turmeric powder to 60% ethanol solution at a material-liquid ratio of 1g: (15-25)mL, stir and mix evenly, then heat at 50-60℃ for 40-50min, then ultrasonically treat at 180-220W for 30-40min, and finally continue heating and reflux extraction for 1-2h. After cooling, filter to obtain turmeric extract; T1.2: Add Sophora flavescens powder to distilled water at a solid-liquid ratio of 1g: (20-30)mL, stir and mix evenly, then microwave at a microwave power of 100-200W for 10-20min, then add anhydrous ethanol, heat and reflux at 80-90℃ for 1-2h, cool and filter to obtain Sophora flavescens extract; T1.3: Mix the turmeric extract and the sophora flavescens extract in a volume ratio of 1:(1-3), place in a concentrator, evaporate and concentrate, remove the solvent, and obtain a composite antibacterial agent; T1.4: Add the above-mentioned composite antimicrobial agent, 6-benzyladenine, naphthylacetic acid, sucrose and agar to the MS medium, mix evenly, and obtain the primary culture medium; The steps for preparing the rooting medium are as follows: T2.1: Place urea in a muffle furnace, heat it to 500-600℃ at 10-20℃ / min and keep it at that temperature for 3-4h. After cooling to room temperature, grind and wash it with water to obtain solid powder; T2.2: Add the above solid powder into 0.1 mol / L sodium hydroxide solution at a solid-liquid ratio of 1 g: (40-50) mL, heat and reflux at 100-120 ° C for 10-12 h, then centrifuge at a speed of 3000-4000 r / min for 3-5 min, remove the supernatant, and obtain a precipitate; T2.3: Ultrasonic disperse the above precipitate in distilled water at a solid-liquid ratio of 1g: (25-35)mL to form a suspension, and then continue to centrifuge at a rate of 3000-4000r / min for 3-5min, collect the supernatant, and continue to ultrasonically disperse the precipitate in distilled water, repeat several times until no precipitate appears, then combine the supernatant, filter with a microporous filter membrane, and finally re-disperse the precipitate on the microporous filter membrane with distilled water to obtain a colloidal solution; T2.4: centrifuge the colloidal solution at a rate of 8000-9000 r / min for 20-30 min, collect the supernatant, add polyethyleneimine aqueous solution to the supernatant, adjust the pH to neutral, and then ultrasonicate for 20-30 min to obtain a mixed solution; T2.5: adding ascorbic acid solution to the above mixed solution, stirring for 10-12 hours, uniformly mixing, and then centrifuging at a rate of 20000-30000 r / min for 30-40 minutes, removing the supernatant, and washing and vacuum drying the precipitate to obtain an anti-browning agent; T2.6: Add the above anti-browning agent, indolebutyric acid, 6-benzyladenine, naphthylacetic acid, brassinolide, sucrose and agar to the MS medium, mix evenly, and obtain a rooting medium.

2. The method for rapid propagation of wild persimmon through tissue culture according to claim 1, characterized in that: The primary culture medium consisted of MS medium, 0.9 mg / L composite antimicrobial agent, 1.0 mg / L 6-benzyladenine, 0.05 mg / L naphthaleneacetic acid, 30 mg / L sucrose and 6 g / L agar.

3. The method for rapid propagation of wild persimmon through tissue culture according to claim 1, characterized in that: The proliferation medium consists of MS medium, 0.9 mg / L composite antimicrobial agent, 0.5 mg / L zeatin, 1.0 mg / L 6-benzyladenine, 0.1 mg / L naphthaleneacetic acid, 30 mg / L sucrose and 6 g / L agar.

4. The method for rapid propagation of wild persimmon by tissue culture according to claim 1, characterized in that: The rooting medium consists of MS medium, 1.0 mg / L indolebutyric acid, 0.5 mg / L 6-benzyladenine, 0.05 mg / L brassinolide, 0.01 mg / L naphthaleneacetic acid, 3 g / L anti-browning agent, 30 mg / L sucrose, and 6 g / L agar.

5. The method for rapid propagation of wild persimmon through tissue culture according to claim 1, characterized in that: The solid-liquid ratio of anhydrous ethanol to Sophora flavescens powder is 1g:(10-12)mL.

6. The method for rapid propagation of wild persimmon through tissue culture according to claim 1, characterized in that: The pore size of the microporous membrane is 0.22 μm.

7. The method for rapid propagation of wild persimmon through tissue culture according to claim 1, characterized in that: The concentration of the polyethyleneimine aqueous solution is 50-60%.

8. The method for rapid propagation of wild persimmon through tissue culture according to claim 1, characterized in that: The volume ratio of the ascorbic acid solution to the mixed solution is (4-6):1, and the concentration of the ascorbic acid solution is 10-20 μmol / L.

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