Culture method for directly inducing clumpy buds by using gingko sterile stem segments

Abstract

The invention discloses a culture method for directly inducing clumpy buds by using gingko sterile stems, and relates to the technical field of gingko tissue culture. The culture method comprises the following steps: removing ginkgo fruit testa, washing, airing, storing, disinfecting, washing with sterile water, taking out ginkgo biloba embryos, inoculating the ginkgo biloba embryos into an initial culture medium for culture, cutting the cultured sterile seedlings into stem segments, inoculating into a proliferation culture medium for culture, and directly inducing clumpy buds to germinate. According to the method, the culture period is shortened, the cluster bud induction rate and the multiplication coefficient are improved, the genetic variation risk is avoided, and the problems of low multiplication efficiency, long culture period, high genetic variation risk and the like in the prior art are effectively solved.

Description

Technical Field

[0001] This invention relates to the field of Ginkgo tissue culture technology, specifically to a method for directly inducing cluster buds using aseptic stem segments of Ginkgo. Background Technology

[0002] ginkgo( Ginkgo biloba Ginkgo biloba, a relict plant from the Quaternary glacial period of the Cenozoic era, is a rare and precious tree species unique to my country, excelling in both leaves and fruit. Often referred to as a "living fossil" of gymnosperms, it possesses extremely high medicinal, edible, ornamental, economic, and ecological value. Its seeds contain various nutrients and active ingredients such as protein, fat, calcium, phosphorus, iron, carotene, and lactones, offering good therapeutic effects for cardiovascular and cerebrovascular diseases. The flavonoids and terpene lactones in ginkgo leaves have significant effects in eliminating free radicals, anti-tumor activity, anti-oxidation, and protecting the nervous system. Furthermore, ginkgo is easy to cultivate and manage, has a long lifespan, and excellent ornamental value, making it a major tree species for urban landscaping.

[0003] With the deepening development and utilization of ginkgo, the market demand for high-quality ginkgo seedlings, especially superior varieties, is increasing daily. Conventional ginkgo propagation methods include sowing, grafting, and cutting, but these methods suffer from low breeding efficiency, long cycles, and limited propagation coefficients, making it difficult to meet the needs of large-scale production. Plant tissue culture technology, as an effective means of rapidly propagating high-quality seedlings, can achieve efficient propagation of superior ginkgo varieties and is of great significance for promoting ginkgo clonal breeding and the industrial production of ginkgo secondary metabolites. However, ginkgo tissue culture not only faces technical challenges such as high contamination rates, difficulty in rooting, low proliferation coefficients, and long culture cycles, but also a key hidden danger: most existing technologies rely on callus-induced differentiation pathways to obtain clustered buds or adventitious buds. During callus formation, cells undergo dedifferentiation and redifferentiation, easily leading to genetic variations due to recombination or mutation of genetic material. This results in inconsistencies between the traits of regenerated plants and the parent plant, seriously affecting the stability of superior varieties. This is a core problem that urgently needs to be solved for large-scale ginkgo seedling production, which requires maintaining excellent genetic characteristics. Therefore, optimizing Ginkgo tissue culture technology, constructing a callus-free pathway for direct induction of shoot clusters from sterile stem segments, avoiding the risk of genetic variation, and simultaneously improving the shoot cluster induction rate and proliferation coefficient, shortening the culture cycle, and reducing the risk of contamination have become the key technical demands in the field of Ginkgo tissue culture.

[0004] In existing technologies, there are methods for culturing aseptic Ginkgo seedlings using Ginkgo nuclei and embryos as inoculation materials and adding different concentrations of Ginkgo endosperm extract. However, these methods only focus on the initial culture of aseptic seedlings and do not address related schemes for shoot induction and proliferation culture. This makes it impossible to achieve large-scale rapid propagation of seedlings. While the selected culture medium is suitable for the initial growth of aseptic seedlings, it is not optimized for the nutritional needs of shoot induction, and the culture cycle is long, failing to mitigate the risk of genetic variation. Other conventional Ginkgo tissue culture propagation methods have complex aseptic processes, culture medium components, and hormone combinations, limiting explant selection. They also suffer from limited proliferation efficiency, long proliferation cycles, and the risk of genetic variation. Summary of the Invention

[0005] To address the aforementioned shortcomings in existing technologies, this invention provides a method for directly inducing shoot clusters using aseptic stem segments of Ginkgo biloba, effectively solving the problems of low proliferation efficiency, long culture cycle, and high risk of genetic variation in existing technologies.

[0006] To achieve the above objectives, the technical solution adopted by this invention to solve its technical problem is: to provide a method for directly inducing cluster buds using aseptic stem segments of Ginkgo biloba, comprising the following steps: S1. Remove the outer seed coat of the ginkgo fruit, rinse it, then dry and store it. After disinfection, rinse it with sterile water, then take out the ginkgo embryo and inoculate it into the starting culture medium for culture. S2. Cut the cultured sterile seedlings into segments and then inoculate them into a proliferation medium for culture to directly induce the germination of shoot clusters.

[0007] Furthermore, in step S1, the ginkgo fruits are mature ginkgo fruits collected in the current year.

[0008] Furthermore, in step S1, the ginkgo nuts are placed in a nylon bag and rubbed repeatedly.

[0009] The beneficial effects of taking the above-mentioned further measures are: removing the outer seed coat through mechanical friction avoids the harmful substances produced by the rotting of the outer seed coat from affecting seed vigor.

[0010] Furthermore, in step S1, the sample is rinsed under running sterile water for 25-35 minutes.

[0011] Furthermore, in step S1, the sample is rinsed under running sterile water for 30 minutes.

[0012] The beneficial effects of taking the above-mentioned further measures are: to completely remove residual impurities and outer seed coat debris from the surface.

[0013] Furthermore, in step S1, air dry naturally in a well-ventilated, shady place, avoiding direct sunlight.

[0014] The beneficial effect of taking the above-mentioned further measures is to prevent seeds from losing water too quickly.

[0015] Furthermore, in step S1, the moisture content of the seeds after drying is controlled at 12-15%.

[0016] Furthermore, in step S1, the food is stored in a cool, well-ventilated indoor environment.

[0017] Furthermore, in step S1, the storage temperature is 20-25 ℃ and the relative humidity is 40-50%.

[0018] Furthermore, in step S1, the storage time is 2-3 months, during which the seeds are turned over every 15 days and the seed condition is checked regularly to ensure that the seed embryo activity is ≥90%.

[0019] The beneficial effect of taking the above-mentioned further measures is to prevent mold growth.

[0020] Further, in step S1, during disinfection, the sample is first placed in a 74-76 wt% ethanol solution for 25-35 seconds, and then placed in a sodium hypochlorite solution containing 0.02-0.04 wt% Tween-20 at a concentration of 0.4-0.6 wt% for 10-15 minutes.

[0021] Furthermore, in step S1, during disinfection, the sample is first placed in a 75 wt% ethanol solution for 30 seconds, and then in a sodium hypochlorite solution containing 0.03 wt% Tween-20 at a concentration of 0.5 wt% for 12 minutes.

[0022] Furthermore, in step S1, the container is gently shaken every 2-4 minutes during the disinfection process.

[0023] Furthermore, in step S1, the container is gently shaken every 3 minutes during the disinfection process.

[0024] The beneficial effect of taking the above-mentioned further measures is to ensure that the disinfectant solution is in full contact with the materials.

[0025] Furthermore, in step S1, rinse with sterile water for 1-2 minutes, repeating 3-5 times.

[0026] The beneficial effect of taking the above-mentioned further measures is that it completely removes the disinfectant residue from the surface.

[0027] Furthermore, in step S1, surface moisture is absorbed using sterile filter paper.

[0028] Further, in step S1, the ginkgo embryo is dissected under aseptic conditions and removed.

[0029] Further, in step S1, the starting culture medium is MS medium containing 0.4-0.6 mg / L 6-benzylaminopurine, 0.1-0.3 mg / L naphthaleneacetic acid, 25-35 g / L sucrose and 6-8 g / L agar, with a pH of 5.6-5.8.

[0030] Furthermore, in step S1, the starting culture medium is MS medium containing 0.5 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid, 30 g / L sucrose and 7 g / L agar, with a pH of 5.7.

[0031] Furthermore, in step S1, the pH of the culture medium is adjusted using hydrochloric acid or sodium hydroxide.

[0032] Furthermore, in step S1, the starting culture medium is dispensed into culture flasks, with each flask containing 20-30 mL.

[0033] Furthermore, in step S1, the initial culture medium is autoclaved at 121 °C and 0.1 MPa for 20 min.

[0034] Furthermore, in step S1, one embryo is inoculated into each bottle of starting culture medium, ensuring that the embryo's morphology remains intact and the cotyledons face upwards during the inoculation process.

[0035] Furthermore, in step S1, the culture temperature is 24-26 ℃, the light intensity is 2800-3000 lx, the photoperiod is 16h light / 8h dark, and the culture time is 20-30 days.

[0036] Furthermore, in step S2, the height of the sterile seedling is 2-3.5 cm.

[0037] Furthermore, in step S2, the internodes of the stem segment are plump and the buds are prominent.

[0038] Furthermore, in step S2, the sterile seedlings are cut into segments with a length of 0.45-0.55 cm, each segment having 1-2 internodes, and the cut is made at a 45° oblique angle.

[0039] The beneficial effects of taking the above-mentioned further measures are: increasing the nutrient absorption area and avoiding excessively slanted incisions or damage to buds.

[0040] Furthermore, in step S2, the proliferation medium is MS medium containing 0.5-0.7 mg / L cytokinin, 0.1-0.2 mg / L auxin, 15-25 mL / L sterile Ginkgo endosperm juice, 5-15 mL / L sterile Ginkgo cotyledon juice, 25-35 g / L sucrose and 6-8 g / L agar, with a pH of 5.6-5.8.

[0041] Furthermore, in step S2, the proliferation medium is MS medium containing 0.6 mg / L cytokinin, 0.1 mg / L auxin, 20 mL / L sterile Ginkgo endosperm juice, 10 mL / L sterile Ginkgo cotyledon juice, 30 g / L sucrose and 7 g / L agar, with a pH of 5.7.

[0042] Furthermore, in step S2, the cytokinin is 6-benzylaminopurine or kinetin.

[0043] Furthermore, in step S2, the auxin is indolebutyric acid or indoleacetic acid.

[0044] Furthermore, in step S2, the conductivity of the aseptic endosperm of Ginkgo biloba is 500-800 μs / cm.

[0045] Furthermore, in step S2, the conductivity of the sterile ginkgo cotyledon juice is 300-500 μs / cm.

[0046] Furthermore, in step S2, the aseptic endosperm juice of ginkgo is prepared by the following method: take the endosperm of ginkgo fruit, rinse and cut it into pieces, add ultrapure water and grind to obtain endosperm homogenate, then heat the endosperm homogenate, cool and filter, adjust the conductivity value and then sterilize to obtain aseptic endosperm juice of ginkgo.

[0047] Furthermore, in step S2, the aseptic endosperm juice of ginkgo is prepared by the following method: take the endosperm of ginkgo fruit, rinse it 2-3 times with sterile water, and then cut it into 0.4-0.6cm pieces. 3 Grind small pieces of endosperm with ultrapure water in a sterile mortar. The mass-to-volume ratio of endosperm pieces to ultrapure water is 1-2 g: 5-10 mL to obtain an endosperm homogenate. Then, pour the endosperm homogenate into a sterile beaker and place it on a constant temperature water bath. Heat to boiling and maintain a gentle boil for 25-35 minutes to cook the endosperm juice. Stir constantly during the process to prevent scorching. After cooling to room temperature, filter with sterile gauze to remove solid impurities. Measure the conductivity of the endosperm juice using a conductivity meter. Adjust the conductivity to 500-800 μs / cm by adding ultrapure water or by low-temperature drying and concentration. Autoclave at 121 ℃ and 0.1 MPa for 20 minutes. After sterilization, cool to room temperature and seal and store in a 4℃ refrigerator to obtain sterile Ginkgo endosperm juice.

[0048] Furthermore, in step S2, the storage time for aseptic Ginkgo endosperm juice is 0-7 days.

[0049] Further, in step S2, the aseptic cotyledon juice of ginkgo is prepared by the following method: cotyledons are cut from aseptic seedlings, rinsed, and then ground with ultrapure water to obtain cotyledon homogenate. The cotyledon homogenate is then heated, cooled, filtered, and the conductivity value is adjusted before sterilization to obtain aseptic cotyledon juice of ginkgo.

[0050] Further, in step S2, the sterile cotyledon juice of Ginkgo biloba is prepared by the following method: thick, green cotyledons are cut from sterile seedlings after 20-30 days of initial culture, rinsed 2-3 times with sterile water, and then placed in a sterile mortar. Ultrapure water is added and the mixture is ground. The mass-volume ratio of cotyledons to ultrapure water is 1-2 g: 4-8 mL to obtain a cotyledon homogenate. The cotyledon homogenate is poured into a sterile beaker, placed on a constant temperature water bath, heated to boiling, and then kept at a gentle boil for 15-25 min with constant stirring. After cooling to room temperature, the mixture is filtered to remove solid impurities. The conductivity value is adjusted to 300-500 μs / cm, and then autoclaved at 121℃ and 0.1MPa for 20 min. After sterilization, the mixture is cooled to room temperature, sealed, and stored in a refrigerator at 4℃ to obtain sterile cotyledon juice of Ginkgo biloba.

[0051] Furthermore, in step S2, the pH of the culture medium is adjusted using hydrochloric acid or sodium hydroxide.

[0052] Furthermore, in step S2, the proliferation medium is dispensed into culture flasks, with each flask containing 20-30 mL.

[0053] Furthermore, in step S2, the proliferation medium is autoclaved at 121 °C and 0.1 MPa for 20 min.

[0054] Furthermore, in step S2, 3-5 stem segments are inoculated into each bottle.

[0055] Furthermore, in step S2, during inoculation, ensure that the stem segment cut is in full contact with the culture medium, with the axillary bud facing upwards, to avoid suspension.

[0056] Furthermore, in step S2, the culture temperature is 24-26℃, the light intensity is 3000-3500 lx, the photoperiod is 16h light / 8h dark, and the culture time is 25-30 days.

[0057] In summary, the present invention has the following beneficial effects: 1. This invention is the first to clearly define the synergistic proliferation mechanism of "Ginkgo endosperm juice + cotyledon juice + 6-BA / IBA". By precisely controlling the EC value and addition ratio of the extract, it achieves direct induction of shoot clusters without callus, and simultaneously achieves the triple goals of "high-efficiency proliferation + no genetic variation + low pollution". It forms a unique three-in-one innovative system of "synergistic effect of natural extract + hormone regulation + callus-free pathway", which is different from the simple addition of components in the existing technology and has outstanding substantive characteristics and significant progress.

[0058] 2. Contamination and browning rates were reduced to extremely low levels, and culture stability was significantly improved. Using an optimized disinfection protocol, combined with aseptic operation and culture medium formulation, the contamination rate of all experimental groups was 0%, and the browning rate of the main experimental group was 0%, which is far superior to the level of ≥12% contamination rate and ≥5% browning rate in existing technologies. During the culture process, the shoots grew uniformly and vigorously, laying a good foundation for subsequent rooting and transplanting.

[0059] 3. The induction rate and proliferation coefficient of shoot clusters were significantly improved, and the synergistic effect was clear. The induction rate of shoot clusters in the main experimental group reached 68.9±1.9%, and the proliferation coefficient reached 5.23±0.29, which was 23.3% higher than that of the hormone-only group (control group 3) and 68.17% higher than that of the control group (p<0.01). This proves that the "endosperm juice + cotyledon juice + hormone" synergistic proliferation system constructed in this invention breaks through the proliferation efficiency bottleneck of the existing technology. It is not a simple superposition of components and has outstanding substantive characteristics.

[0060] 4. Constructing a direct callus-free induction pathway to completely avoid the risk of genetic variation. This invention achieves direct germination of clustered buds from axillary bud primordia of aseptic stem segments of Ginkgo biloba through the synergistic regulation of natural extracts and hormones, without callus formation throughout the entire process. This fundamentally avoids the recombination or mutation of genetic material during callus dedifferentiation / redifferentiation, resulting in regenerated plants with genetic stability ≥99%, perfectly maintaining the superior traits of the parent plant and solving the core hidden dangers of existing technologies.

[0061] 5. Simplified operation process, shortened cultivation cycle, and high value for large-scale promotion. The aseptic treatment and culture medium preparation process is simple, requiring no complex chemical additives or browning inhibitors; the initial culture is 20-30 days + proliferation culture is 30 days, and the entire cultivation cycle is only 50-60 days, which is 15-25 days shorter than the existing technology (≥75 days); the raw materials for natural extracts are widely available (ginkgo fruit, aseptic seedling cotyledons), and the preparation process is clear and quantifiable, reducing the difficulty of operation and production costs, and facilitating industrial-scale promotion.

[0062] 6. Adaptable to different production needs and highly practical. It can be flexibly selected according to the characteristics of ginkgo varieties and production cost requirements, which not only ensures the advanced nature of core technologies, but also enhances the practical application value of the technology, making it suitable for large-scale seedling cultivation in different scenarios. Attached Figure Description

[0063] Figure 1 A schematic diagram of the endosperm and embryo of a mature ginkgo seed; Figure 2 Figure showing the growth of Ginkgo embryos 7 days after inoculation into the initial culture medium; Figure 3 Figure showing the growth of Ginkgo embryos 25 days after inoculation into the initial culture medium; Figure 4This is a diagram showing the condition of aseptic Ginkgo stem segments after 25 days of transfer to the proliferation medium. Detailed Implementation

[0064] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0065] Example 1 A method for directly inducing clustered buds using aseptic stem segments of Ginkgo biloba includes the following steps: S1. Select mature ginkgo fruits collected in the current year, place them in a nylon bag and repeatedly rub them to remove the outer seed coat. Rinse under running sterile water for 30 minutes, then air dry naturally in a ventilated and cool place, avoiding direct sunlight. After drying, control the seed moisture content to 14%. Store in a cool and ventilated indoor environment at a temperature of 23 ℃ and a relative humidity of 45% for 2 months. During this period, turn the seeds over every 15 days and check their condition regularly to ensure that the seed embryo activity is ≥90%. For disinfection, first treat the seeds in a 75 wt% ethanol solution for 30 seconds, then treat them in a 0.5 wt% sodium hypochlorite solution containing 0.03 wt% Tween-20 for 12 minutes. During the disinfection process, gently shake the container every 3 minutes to ensure that the disinfection solution is in full contact with the material. After disinfection, rinse the seed embryos 4 times with sterile water, 1 liter each time. After thoroughly removing any residual disinfectant from the surface, blot the seed embryo dry with sterile filter paper. Then, in a laminar flow hood, use sterile forceps and a scalpel to remove the seed coat from the seed's medulla. Figure 1 As shown, intact ginkgo embryos are removed and inoculated into the initial culture medium for further culture. Figure 2 As shown, the starting medium was MS medium containing 0.5 mg / L 6-benzylaminopurine, 0.2 mg / L naphthaleneacetic acid, 30 g / L sucrose, and 7 g / L agar. The pH of the medium was adjusted to 5.7 with hydrochloric acid or sodium hydroxide. The starting medium was dispensed into culture flasks, 25 mL per flask. The starting medium was autoclaved at 121 ℃ and 0.1 MPa for 20 min. One embryo was inoculated into each flask, ensuring the embryo's morphology remained intact with cotyledons facing upwards. The culture temperature was 25 ℃, the light intensity was 2900 lx, and the photoperiod was 16 h light / 8 h dark. The culture period was 25 days. Figure 3 As shown; S2. When the aseptic seedlings reach a height of 3 cm, with plump internodes and prominent buds, in a clean bench, cut the initial cultured aseptic seedlings into 0.5 cm segments using sterile scissors. Each segment should have 1-2 internodes, and the cut should be made at a 45° angle. Then, inoculate the stem segments into a proliferation medium for further culture. Figure 4 As shown, 3-5 stem segments were inoculated into each bottle. The above-mentioned proliferation medium was MS medium containing 0.6 mg / L 6-benzylaminopurine, 0.1 mg / L indolebutyric acid, 20 mL / L sterile Ginkgo endosperm juice, 10 mL / L sterile Ginkgo cotyledon juice, 30 g / L sucrose, and 7 g / L agar. The pH was adjusted to 5.7 with hydrochloric acid or sodium hydroxide. The above-mentioned sterile Ginkgo endosperm juice was prepared by the following method: Ginkgo fruit endosperm was taken, rinsed twice with sterile water, and then cut into 0.5 cm pieces. 3 Small pieces of endosperm were ground in a sterile mortar with ultrapure water. The mass-to-volume ratio of endosperm pieces to ultrapure water was 1 g:5 mL to obtain an endosperm homogenate. The homogenate was then poured into a sterile beaker and placed in a constant temperature water bath. The mixture was heated to boiling and then kept at a gentle boil for 30 min to extract the endosperm juice. During this process, the mixture was stirred constantly to prevent scorching. After cooling to room temperature, the mixture was filtered through sterile gauze to remove solid impurities. The conductivity of the endosperm juice was measured using a conductivity meter. The conductivity was adjusted to 600 μs / cm by adding ultrapure water or by low-temperature drying and concentration. The mixture was then autoclaved at 121 ℃ and 0.1 MPa for 20 min. After sterilization, the mixture was cooled to room temperature, sealed, and stored in a 4℃ refrigerator to obtain sterile Ginkgo endosperm juice. The sterile Ginkgo endosperm juice can be stored for 0-7 days. The above sterile Ginkgo cotyledon juice was prepared by the following method: from the initial culture of 25... Thick, green cotyledons were cut from sterile seedlings after 3 days. After rinsing twice with sterile water, the cotyledons were placed in a sterile mortar and ground with ultrapure water. The mass-to-volume ratio of cotyledons to ultrapure water was 1 g:4 mL to obtain a cotyledon homogenate. The cotyledon homogenate was poured into a sterile beaker, placed in a constant temperature water bath, heated to boiling, and then kept at a gentle boil for 20 min with constant stirring. After cooling to room temperature, the mixture was filtered to remove solid impurities. The conductivity was adjusted to 400 μs / cm and then autoclaved at 121℃ and 0.1 MPa for 20 min. After sterilization, the mixture was cooled to room temperature, sealed, and stored in a 4℃ refrigerator to obtain sterile Ginkgo cotyledon juice. When inoculating, it was ensured that the stem segment cut was in full contact with the culture medium, with the axillary bud facing upwards to avoid suspension. The culture temperature was 25℃, the light intensity was 3200 lx, and the photoperiod was 16 h light / 8 h dark. After 30 days of culture, the bud clusters were directly induced to germinate.

[0066] Example 2 A method for directly inducing clustered buds using aseptic stem segments of Ginkgo biloba includes the following steps: S1. Select mature ginkgo fruits collected in the current year, place them in a nylon bag and repeatedly rub them to remove the outer seed coat. Rinse under running sterile water for 25 minutes, then air dry naturally in a ventilated and cool place, avoiding direct sunlight. After drying, control the seed moisture content to 12%. Store in a cool, ventilated indoor environment at a temperature of 20 ℃ and a relative humidity of 40% for 2 months. During this period, turn the seeds over every 15 days and check their condition regularly to ensure that the seed embryo activity is ≥90%. For disinfection, first treat the seeds in a 74 wt% ethanol solution for 25 seconds, then treat them in a 0.4 wt% sodium hypochlorite solution containing 0.02 wt% Tween-20 for 10 minutes. During disinfection, gently shake the container every 2 minutes to ensure that the disinfection solution is in full contact with the material. After disinfection, rinse the seed embryos 3 times with sterile water, 1 liter each time. After thoroughly removing any residual disinfectant from the surface, the seed embryos were dried with sterile filter paper. In a clean bench, the seed coat was removed from the bony core using sterile forceps and a scalpel, and the complete Ginkgo embryos were extracted. The embryos were then inoculated into the starting medium, which was MS medium containing 0.4 mg / L 6-benzylaminopurine, 0.1 mg / L naphthaleneacetic acid, 25 g / L sucrose, and 6 g / L agar. The pH was adjusted to 5.7 with hydrochloric acid or sodium hydroxide. The starting medium was dispensed into culture bottles, 25 mL per bottle. The starting medium was autoclaved at 121 ℃ and 0.1 MPa for 20 min. One embryo was inoculated into each bottle, ensuring the embryo remained morphologically intact with cotyledons facing upwards. The culture temperature was 25 ℃, the light intensity was 2900 lx, and the photoperiod was 16 h light / 8 h dark. The culture period was 25 days. S2. When the aseptic seedlings reach a height of 3 cm, with plump internodes and prominent buds, in a clean bench, cut the initial cultured aseptic seedlings into 0.45 cm segments using sterile scissors. Each segment should have 1-2 internodes, and the cut should be made at a 45° angle. Then, inoculate the stem segments into a proliferation medium, using 3-5 segments per bottle. The proliferation medium is MS medium containing 0.5 mg / L 6-benzylaminopurine, 0.1 mg / L indoleacetic acid, 15 mL / L aseptic endosperm of Ginkgo biloba, 5 mL / L aseptic cotyledon juice of Ginkgo biloba, 25 g / L sucrose, and 6 g / L agar. Adjust the pH to 5.6 with hydrochloric acid or sodium hydroxide. The aseptic endosperm of Ginkgo biloba is prepared as follows: Take the endosperm of Ginkgo biloba fruit, rinse twice with sterile water, and cut into 0.4 cm segments. 3Small pieces of endosperm were ground in a sterile mortar with ultrapure water. The mass-to-volume ratio of endosperm pieces to ultrapure water was 1 g: 5 mL to obtain an endosperm homogenate. The homogenate was then poured into a sterile beaker and placed in a constant temperature water bath. The mixture was heated to boiling and then kept at a gentle boil for 25 minutes to extract the endosperm juice. During this process, the mixture was stirred constantly to prevent scorching. After cooling to room temperature, the mixture was filtered through sterile gauze to remove solid impurities. The conductivity of the endosperm juice was measured using a conductivity meter. The conductivity was adjusted to 500 μs / cm by adding ultrapure water or by low-temperature drying and concentration. The mixture was then autoclaved at 121 ℃ and 0.1 MPa for 20 minutes. After sterilization, the mixture was cooled to room temperature, sealed, and stored in a 4℃ refrigerator to obtain sterile Ginkgo endosperm juice. The sterile Ginkgo endosperm juice can be stored for 0-7 days. The above sterile Ginkgo cotyledon juice was prepared by the following method: from the initial culture of 20... Thick, green cotyledons were cut from sterile seedlings after 3 days. After rinsing twice with sterile water, the cotyledons were placed in a sterile mortar and ground with ultrapure water. The mass-to-volume ratio of cotyledons to ultrapure water was 1 g:4 mL to obtain a cotyledon homogenate. The cotyledon homogenate was poured into a sterile beaker, placed in a constant temperature water bath, heated to boiling, and then kept at a gentle boil for 15 min with constant stirring. After cooling to room temperature, the mixture was filtered to remove solid impurities. The conductivity was adjusted to 300 μs / cm and then autoclaved at 121℃ and 0.1 MPa for 20 min. After sterilization, the mixture was cooled to room temperature, sealed, and stored in a 4℃ refrigerator to obtain sterile Ginkgo cotyledon juice. When inoculating, it was ensured that the stem segment cut was in full contact with the culture medium, with the axillary bud facing upwards to avoid suspension. The culture temperature was 24℃, the light intensity was 3000 lx, and the photoperiod was 16 h light / 8 h dark. After 25 days of culture, the bud clusters were directly induced to germinate.

[0067] Example 3 A method for directly inducing clustered buds using aseptic stem segments of Ginkgo biloba includes the following steps: S1. Select mature ginkgo fruits collected in the current year, place them in a nylon bag and repeatedly rub them to remove the outer seed coat. Rinse under running sterile water for 35 minutes, then air dry naturally in a ventilated and cool place, avoiding direct sunlight. After drying, control the seed moisture content to 15%. Store in a cool and ventilated indoor environment at a temperature of 25 ℃ and a relative humidity of 50% for 3 months. During this period, turn the seeds over every 15 days and check their condition regularly to ensure that the activity of the seed embryo is ≥90%. For disinfection, first treat the seeds in a 76 wt% ethanol solution for 35 seconds, then treat them in a 0.6 wt% sodium hypochlorite solution containing 0.04 wt% Tween-20 for 15 minutes. During the disinfection process, gently shake the container every 4 minutes to ensure that the disinfection solution is in full contact with the material. After disinfection, rinse the seed embryos 5 times with sterile water, 2 times each time. After thoroughly removing any residual disinfectant from the surface, the seed embryos were dried with sterile filter paper. In a clean bench, the seed coat was removed from the bony core using sterile forceps and a scalpel, and the complete Ginkgo embryos were extracted. The embryos were then inoculated into the starting medium, which was MS medium containing 0.6 mg / L 6-benzylaminopurine, 0.3 mg / L naphthaleneacetic acid, 35 g / L sucrose, and 8 g / L agar. The pH was adjusted to 5.8 with hydrochloric acid or sodium hydroxide. The starting medium was dispensed into culture bottles, 30 mL per bottle. The starting medium was autoclaved at 121 ℃ and 0.1 MPa for 20 min. One embryo was inoculated into each bottle, ensuring the embryo remained morphologically intact with cotyledons facing upwards. The culture temperature was 26 ℃, the light intensity was 3000 lx, and the photoperiod was 16 h light / 8 h dark. The culture period was 30 days. S2. When the aseptic seedlings reach a height of 3.5 cm, with plump internodes and prominent buds, in a clean bench, cut the initial cultured aseptic seedlings into 0.55 cm segments using sterile scissors. Each segment should have 1-2 internodes, and the cut should be made at a 45° angle. Then, inoculate the stem segments into a proliferation medium, using 3-5 segments per bottle. The proliferation medium is MS medium containing 0.7 mg / L kinetin, 0.2 mg / L indolebutyric acid, 25 mL / L aseptic endosperm of Ginkgo biloba, 15 mL / L aseptic cotyledon juice of Ginkgo biloba, 35 g / L sucrose, and 8 g / L agar. Adjust the pH to 5.8 with hydrochloric acid or sodium hydroxide. The aseptic endosperm of Ginkgo biloba is prepared as follows: Take the endosperm of Ginkgo biloba fruit, rinse it three times with sterile water, and cut it into 0.6 cm segments. 3Small pieces of endosperm were ground in a sterile mortar with ultrapure water. The mass-to-volume ratio of endosperm pieces to ultrapure water was 1 g: 5 mL to obtain an endosperm homogenate. The homogenate was then poured into a sterile beaker and placed in a constant temperature water bath. The mixture was heated to boiling and then kept at a gentle boil for 35 minutes to extract the endosperm juice. During this process, the mixture was stirred constantly to prevent scorching. After cooling to room temperature, the mixture was filtered through sterile gauze to remove solid impurities. The conductivity of the endosperm juice was measured using a conductivity meter. The conductivity was adjusted to 800 μs / cm by adding ultrapure water or by low-temperature drying and concentration. The mixture was then autoclaved at 121 ℃ and 0.1 MPa for 20 minutes. After sterilization, the mixture was cooled to room temperature, sealed, and stored in a 4℃ refrigerator to obtain sterile Ginkgo endosperm juice. The sterile Ginkgo endosperm juice can be stored for 0-7 days. The above sterile Ginkgo cotyledon juice was prepared by the following method: from the initial culture of 30... Thick, green cotyledons were cut from sterile seedlings after 3 days. After rinsing three times with sterile water, the cotyledons were placed in a sterile mortar and ground with ultrapure water. The mass-to-volume ratio of cotyledons to ultrapure water was 1 g:4 mL to obtain a cotyledon homogenate. The cotyledon homogenate was poured into a sterile beaker, placed in a constant temperature water bath, heated to boiling, and then kept at a gentle boil for 25 min with constant stirring. After cooling to room temperature, the mixture was filtered to remove solid impurities. The conductivity was adjusted to 500 μs / cm and then autoclaved at 121℃ and 0.1 MPa for 20 min. After sterilization, the mixture was cooled to room temperature, sealed, and stored in a 4℃ refrigerator to obtain sterile Ginkgo cotyledon juice. When inoculating, it was ensured that the stem segment cut was in full contact with the culture medium, with the axillary bud facing upwards to avoid suspension. The culture temperature was 26℃, the light intensity was 3500 lx, and the photoperiod was 16 h light / 8 h dark. After 28 days of culture, the bud clusters were directly induced to germinate.

[0068] Example 4 The difference between Example 4 and Example 1 is that the proliferation medium in Example 4 is MS medium containing 0.7 mg / L kinetin, 0.2 mg / L indoleacetic acid, 25 mL / L sterile Ginkgo endosperm juice, 15 mL / L sterile Ginkgo cotyledon juice, 35 g / L sucrose and 8 g / L agar.

[0069] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the proliferation culture medium of Comparative Example 1 does not contain sterile Ginkgo cotyledon juice.

[0070] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the proliferation culture medium of Comparative Example 2 does not contain sterile Ginkgo endosperm juice.

[0071] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the proliferation culture medium of Comparative Example 3 does not contain sterile cotyledon juice and sterile endosperm juice of Ginkgo biloba.

[0072] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the proliferation culture medium of Comparative Example 3 does not contain hormones.

[0073] Experimental Example 1 The shoot induction rate, proliferation coefficient, browning rate, contamination rate and callus condition of Example 1 and Example 4 were compared. Each group was repeated 3 times, with 30 explants each time, and cultured for 30 days. The results are shown in Table 1.

[0074] Table 1 Results of the validation experiment on the Ginkgo propagation alternative scheme

[0075] As shown in Table 1, the shoot induction rate of Examples 1 and 4 is ≥60%, the proliferation coefficient is ≥4.5, and no callus formation occurs, proving that different hormone regimens are feasible and can be flexibly selected according to production needs.

[0076] Experimental Example 2 To investigate the synergistic effect of natural nutrient extracts of Ginkgo biloba and hormones, the shoot induction rate, proliferation coefficient, browning rate, contamination rate and callus status of Example 1 and Comparative Examples 1-4 were compared. Each group was set up with 30 explants each time and cultured for 30 days. The results are shown in Table 2.

[0077] Table 2. Results of the synergistic effect test between Ginkgo biloba natural nutritional extract and hormones

[0078] The statistical analysis results (t-test) of the main experimental group and each control group are shown in Table 3. Table 3. Statistical analysis results of the main experimental group and each control group.

[0079] As shown in Tables 2 and 3, the synergistic effect of this invention is significant. The shoot induction rate and proliferation coefficient of the main experimental group were significantly higher than those of the single-component control groups. The differences were highly significant (p<0.01) according to the t-test, proving that the combination of endosperm juice + cotyledon juice + 6-BA / IBA is not a simple additive effect, but rather exhibits a clear synergistic proliferation effect. No callus tissue formed in the main experimental group or any of the natural extract addition groups, except for a small amount of callus tissue in Comparative Example 3. This demonstrates that the natural extract combination of this invention can effectively regulate the direct germination of axillary bud primordia and avoid the genetic variation risk associated with callus formation. Furthermore, the contamination rate was 0% in all groups of this invention, with a browning rate of 0% in the main experimental group and only slight browning in Comparative Example 2. This proves that the disinfection scheme and culture medium formulation of this invention can effectively reduce the risk of contamination and browning, and demonstrate strong culture stability.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for cultivating bud clusters by directly inducing them from aseptic stem segments of Ginkgo biloba, characterized in that, Includes the following steps: S1. Remove the outer seed coat of the ginkgo fruit, rinse it, then dry and store it. After disinfection, rinse it with sterile water, then take out the ginkgo embryo and inoculate it into the starting culture medium for culture. S2. Cut the cultured sterile seedlings into segments and then inoculate them into a proliferation medium for culture to directly induce shoot germination. The proliferation medium is MS medium containing 0.5-0.7 mg / L cytokinin, 0.1-0.2 mg / L auxin, 15-25 mL / L sterile Ginkgo endosperm juice, 5-15 mL / L sterile Ginkgo cotyledon juice, 25-35 g / L sucrose and 6-8 g / L agar, with a pH of 5.6-5.

8.

2. The cultivation method for directly inducing cluster buds using aseptic stem segments of Ginkgo biloba as described in claim 1, characterized in that, In step S1, the storage temperature is 20-25 ℃ and the relative humidity is 40-50%.

3. The method for cultivating bud clusters by directly inducing them from aseptic stem segments of Ginkgo biloba as described in claim 1, characterized in that, In step S1, during disinfection, the sample is first placed in a 74-76 wt% ethanol solution for 25-35 seconds, and then placed in a 0.02-0.04 wt% Tween-20 solution containing 0.4-0.6 wt% sodium hypochlorite for 10-15 minutes.

4. The method for directly inducing cluster buds using aseptic stem segments of Ginkgo biloba as described in claim 1, characterized in that, In step S1, the starting medium is MS medium containing 0.4-0.6 mg / L 6-benzylaminopurine, 0.1-0.3 mg / L naphthaleneacetic acid, 25-35 g / L sucrose and 6-8 g / L agar, with a pH of 5.6-5.

8.

5. The method for cultivating bud clusters by directly inducing them from aseptic stem segments of Ginkgo biloba as described in claim 1, characterized in that, In step S1, the culture temperature is 24-26 ℃, the light intensity is 2800-3000 lx, the photoperiod is 16 h light / 8 h dark, and the culture time is 20-30 days.

6. The method for directly inducing cluster buds using aseptic stem segments of Ginkgo biloba as described in claim 1, characterized in that, In step S2, the cytokinin is 6-benzylaminopurine or kinetin, and the auxin is indolebutyric acid or indoleacetic acid.

7. The method for cultivating bud clusters by directly inducing them from aseptic stem segments of Ginkgo biloba as described in claim 1, characterized in that, In step S2, aseptic endosperm juice of ginkgo is prepared by the following method: take ginkgo fruit endosperm, rinse and cut it into pieces, add ultrapure water and grind to obtain endosperm homogenate, then heat the endosperm homogenate, cool and filter, adjust the conductivity value and then sterilize to obtain aseptic endosperm juice of ginkgo.

8. The method for cultivating bud clusters by directly inducing them from aseptic stem segments of Ginkgo biloba as described in claim 1, characterized in that, In step S2, the sterile cotyledon juice of ginkgo is prepared by the following method: cotyledons are cut from sterile seedlings, rinsed, and then ground with ultrapure water to obtain cotyledon homogenate. The cotyledon homogenate is then heated, cooled, filtered, and the conductivity value is adjusted before sterilization to obtain sterile cotyledon juice of ginkgo.

9. The method for directly inducing cluster buds using aseptic stem segments of Ginkgo biloba as described in claim 1, characterized in that, In step S2, the electrical conductivity of the aseptic endosperm juice of Ginkgo biloba is 500-800 μs / cm, and the electrical conductivity of the aseptic cotyledon juice of Ginkgo biloba is 300-500 μs / cm.

10. The method for directly inducing cluster buds using aseptic stem segments of Ginkgo biloba as described in claim 1, characterized in that, In step S2, the culture temperature is 24-26℃, the light intensity is 3000-3500 lx, the photoperiod is 16h light / 8h dark, and the culture time is 25-30 days.

Images