Rooting induction and large-scale rapid propagation cultivation method for eucommia ulmoides tissue culture seedlings
By introducing modified culture medium, the rooting induction and large-scale rapid propagation method of Eucommia ulmoides tissue culture seedlings has solved the problems of long rooting induction cycle and low efficiency, and achieved efficient and stable rooting induction and high transplant survival rate, thus improving root quality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INNER MONGOLIA AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-02-26
- Publication Date
- 2026-05-01
AI Technical Summary
In existing technologies, the rooting induction cycle of Eucommia ulmoides tissue culture seedlings is long and inefficient, the rooting is uneven, the root quality of tissue culture seedlings is poor, the transplant survival rate is low, and the carbon source has no stress protection function.
A modified culture medium was used, which was modified MS medium by introducing Eucommia ulmoides leaf extract, chitosan and rare earth elements, combined with modified naphthaleneacetic acid, modified sucrose and fermentation broth of Bacillus subtilis endophyte of Eucommia ulmoides, to form a linkage system of intelligent hormones and protective carbon source, providing stable and continuous growth signals and stress resistance.
It improves the rooting rate, ensures robust root system and complete leaf function, enhances stress resistance, improves transplant survival rate and root quality, and shortens the seedling cycle.
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Abstract
Description
Methods for Rooting Induction and Large-Scale Rapid Propagation of Eucommia Tissue Culture Seedlings Technical Field
[0001] This invention relates to the field of plant cultivation technology, specifically to a method for inducing root growth in Eucommia ulmoides tissue culture seedlings and for large-scale rapid propagation. Background Technology
[0002] Eucommia ulmoides tissue culture seedling rooting induction is a key technology that promotes the growth of roots in seedlings by regulating the culture medium (such as using auxins NAA and IBA) under sterile tissue culture conditions. Its core function is to achieve efficient and rapid asexual reproduction of superior varieties (such as individuals with high gum production and high medicinal efficacy), obtaining a large number of complete plants with consistent genetic traits and robust root systems in a short period of time, thus shortening the seedling cycle and improving the transplant survival rate.
[0003] In existing technologies, conventional rooting media have a single function and lack bioactive signaling substances specific to Eucommia ulmoides, resulting in a long rooting induction cycle and low efficiency. The growth regulator naphthaleneacetic acid has poor stability and a crude mode of action, making it difficult to provide a continuous and gentle induction signal, resulting in uneven rooting and excessive callus growth. Moreover, the carbon source is only sucrose, which has no stress protection function, resulting in poor root quality of tissue culture seedlings and low transplant survival rate.
[0004] Based on this, the present invention provides a method for inducing root growth and large-scale rapid propagation of Eucommia ulmoides tissue culture seedlings. Summary of the Invention
[0005] The purpose of this invention is to provide a method for inducing root growth and large-scale rapid propagation of Eucommia ulmoides tissue culture seedlings. The rooting induction medium prepared by this invention has a high rooting rate, indicating that the medium formula has a high efficiency and stable rooting induction ability; the high transplant survival rate indicates that the cultured seedlings have strong root systems, complete leaf functions, and strong stress resistance; the high root quality index indicates that there are many roots and the roots are relatively long.
[0006] To achieve the above objectives, the present invention provides the following technical solution: Rooting induction of Eucommia ulmoides tissue culture seedlings, wherein the culture medium used for rooting induction comprises the following raw materials in parts by weight: 80-100 parts modified basic culture medium, 0.5-1.5 parts modified naphthaleneacetic acid, 20-30 parts modified sucrose, 0.5-1.5 parts inositol, 1-3 parts activated carbon, and 6-10 parts agar; the modified basic culture medium is prepared by modifying MS culture medium with Eucommia ulmoides leaf extract, chitosan, and rare earth elements; the modified naphthaleneacetic acid is prepared by naphthaleneacetic acid with β-cyclodextrin inclusion, nano-silica loading, and synergistic treatment with fermentation broth of Bacillus subtilis endophytic in Eucommia ulmoides; the modified sucrose is prepared by partially replacing trehalose, composite with magnetic iron oxide nanoparticles, and reinforced with Eucommia ulmoides gum.
[0007] Preferably, the modified basic culture medium is prepared as follows: Eucommia ulmoides leaves that have been pulverized and sieved are added to a 70% (v / v) ethanol solution and refluxed at 60-70°C for extraction. The filtrate is filtered and concentrated under reduced pressure to obtain an extract, which is the Eucommia ulmoides leaf extract. Chitosan is dissolved in a 1% (w / w) acetic acid solution to prepare a 2-3% (w / w) chitosan solution. Rare earth nitrate solution is added dropwise to the chitosan solution and stirred at 40-45°C to allow the rare earth ions to undergo a complexation reaction with the chitosan, resulting in a chitosan-rare earth composite solution. The Eucommia ulmoides leaf extract is added to the chitosan-rare earth composite solution and stirred at 35-40°C to form a composite modified solution. 4.3-4.8 g of MS culture medium powder is weighed, mixed with the composite modified solution, dissolved in water, and brought to a final volume of 1 L. The pH is then adjusted to 5.8-6.0 to obtain the modified basic culture medium.
[0008] Preferably, the pulverized and sieved Eucommia ulmoides leaves are mixed with a 70% ethanol solution at a material-to-liquid ratio of 1:10-15; the Eucommia ulmoides leaf extract and chitosan are mixed with the rare earth composite solution at a mass ratio of 1:5-8; the rare earth elements in the rare earth nitrate solution are lanthanum, cerium, and neodymium, and the rare earth nitrate solution is a mixture of nitrates of the three rare earth elements at a mass ratio of 1:1:1; the amount of rare earth nitrate solution added is 0.5-1% of the mass of the chitosan solution.
[0009] Preferably, the modified naphthaleneacetic acid is prepared as follows: β-cyclodextrin is mixed with deionized water, heated to 50-60℃ and stirred to dissolve, naphthaleneacetic acid powder is added at the same temperature and stirring is continued, and after cooling, a solid inclusion complex is obtained by freeze drying; nano-silica is dispersed in anhydrous ethanol and subjected to ultrasonic treatment, the solid inclusion complex is added, and stirred at 40-50℃ to allow the inclusion complex to be adsorbed and bonded to the high specific surface area of the nano-silica, the precipitate is collected by centrifugation and dried to obtain the loading material; the loading material is added to the fermentation broth of Bacillus subtilis endophytic in Eucommia ulmoides, and fermented by stirring at 28-30℃. After fermentation, the solid is collected by centrifugation, and the solid is washed, freeze-dried and sieved to obtain modified naphthaleneacetic acid.
[0010] Preferably, the β-cyclodextrin and deionized water are mixed at a mass ratio of 1:8-10; the amount of naphthaleneacetic acid powder added is 20-32% of the mass of β-cyclodextrin; the nano-silica and the solid inclusion complex are mixed at a mass ratio of 1:2-4; and the loading material and the fermentation broth of Bacillus subtilis endophyticus of Eucommia ulmoides are mixed at a solid-liquid ratio of 1:10-15.
[0011] Preferably, the modified sucrose is prepared by: mixing sucrose and trehalose, adding deionized water to prepare a mixed sugar solution with a mass fraction of 30-40%; adding magnetic iron oxide nanoparticles to the mixed sugar solution, first ultrasonically dispersing, then continuously stirring to uniformly disperse and composite the nanoparticles in the mixed sugar solution, forming a sugar and nanoparticle system; adding Eucommia ulmoides gum latex to the sugar and nanoparticle system, and continuing to stir to fully mix the Eucommia ulmoides gum and the sugar and nanoparticle system to form a mixed liquid; spray drying, pulverizing and sieving the mixed liquid to obtain powdered modified sucrose.
[0012] Preferably, the sucrose and trehalose are mixed at a mass ratio of 5:1-2; the amount of magnetic iron oxide nanoparticles added is 1-2% of the mass of the mixed sugar solution; and the amount of Eucommia ulmoides gum latex added is 0.5-1% of the mass of the sugar solution.
[0013] Preferably, the preparation method of the Eucommia ulmoides gum emulsion is as follows: Eucommia ulmoides gum fragments are added to a sodium hydroxide solution with a mass fraction of 0.5-1%, and the mixture is stirred at 70-80°C to carry out a hydrolysis reaction to obtain Eucommia ulmoides gum hydrolysate; the pH value of the Eucommia ulmoides gum hydrolysate is adjusted to 6.5-7.5, and emulsifiers Span-80 and Tween-80 are added to form a mixture; the mixture is sheared and emulsified at 60-70°C, and then cooled to room temperature to obtain an oil-in-water Eucommia ulmoides gum emulsion.
[0014] Preferably, the mass ratio of Eucommia ulmoides gum, Span-80 and Tween-80, based on dry glue content, is 5:0.5-1:0.5-1.
[0015] The method for large-scale rapid propagation of Eucommia ulmoides tissue culture seedlings includes the following steps: Explant preparation: Select healthy stem segments with axillary buds from Eucommia ulmoides branches as explants. After rinsing with running water, disinfect with a solution of 70% ethanol (v / v) and 0.1% mercuric chloride (w / w), and finally rinse with sterile water; Induction and proliferation of clustered buds: Inoculate the disinfected explants into a clustered bud induction medium, set the culture parameters, and culture for 25-35 days to induce the production of clustered buds; Divide the clustered buds into single buds or small clusters, and transfer them to the same clustered bud induction medium for subculture and proliferation culture. Subculture once every 25-35 days, for 3-5 consecutive subcultures, to establish a sterile propagation line; Rooting induction: Cut off the above-obtained healthy and tall... Rootless seedlings with a root length of 2-3 cm are immersed at the base in the liquid working solution of rooting induction medium, then removed and inserted into solidified rooting induction medium. Rooting culture is carried out using set culture parameters for 25-35 days. Hardening-off and transplanting: When the root system reaches a length of 2 cm or more, the culture bottle is moved to natural light for hardening-off for 3-5 days. The bottle cap is opened, and a small amount of tap water is added to continue hardening-off for 2-3 days. Subsequently, the tissue culture seedlings are removed, the root culture medium is washed off, and they are planted in a sterilized substrate composed of peat moss, perlite, and vermiculite in a volume ratio of 3:1:1. After culturing in the dark at 22-28℃ for 7-10 days, normal management is gradually resumed to obtain Eucommia ulmoides tissue culture seedlings suitable for field cultivation.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention, by modifying the traditional MS medium in combination, introduces Eucommia ulmoides leaf extract, chitosan and rare earth elements, endowing the medium with biological activity and signal initiation function; compared with the single-function inert nutrient matrix in the prior art, the modified basic medium can significantly promote the dedifferentiation of explant cells and callus formation, providing a stable and species-specific microenvironmental basis for the rooting process. It, together with modified naphthaleneacetic acid and modified sucrose, constitutes a linkage system of active substrate, intelligent hormone and protective carbon source, solving the problems of slow rooting induction and low efficiency in the prior art.
[0017] 2. This invention addresses the problems of poor stability and crude action of naphthaleneacetic acid (NAA) in existing technologies, which leads to uneven rooting. It employs a three-step synergistic modification process involving β-cyclodextrin inclusion, nano-silica loading, and endophytic fermentation to transform NAA into a stable, slow-release, and bioactive growth regulator. The inclusion and loading structures effectively extend the action period of NAA and avoid initial phytotoxicity, while endophytic fermentation introduces host-specific growth-promoting substances. This modified NAA, synergistically with a modified basic culture medium providing continuous trace elements and modified sucrose providing stable energy, achieves continuous, mild, and efficient induction of root primordia differentiation, improving root synchronicity and robustness.
[0018] 3. This invention, through partial substitution of trehalose, composite with nano-ferric oxide, and reinforcement with eucommia gum, enables sucrose to possess functions of stress resistance protection, mineral element supply, and physical support. Compared with ordinary sucrose in the prior art that only provides energy, this modified sucrose can effectively enhance the stress resistance of tissue culture seedlings during the rooting stage. Furthermore, by improving the physical structure of the culture medium through eucommia gum, it works synergistically with the modified basic culture medium that provides activity signals and the modified naphthaleneacetic acid that provides sustained hormone stimulation, comprehensively ensuring the quality of root development. The resulting tissue culture seedlings have well-developed roots and a high degree of lignification, solving the problem of low transplant survival rate of tissue culture seedlings in the prior art. Detailed Implementation
[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0020] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application are described clearly and completely.
[0021] Example 1: Rooting induction of Eucommia ulmoides tissue culture seedlings. The culture medium used for rooting induction included the following raw materials in parts by weight: 80 parts modified basic culture medium, 0.5 parts modified naphthaleneacetic acid, 20 parts modified sucrose, 0.5 parts inositol, 1 part activated carbon, and 6 parts agar. The modified basic culture medium was prepared by modifying MS medium with Eucommia ulmoides leaf extract, chitosan, and rare earth elements. The modified naphthaleneacetic acid was prepared by naphthaleneacetic acid being encapsulated with β-cyclodextrin, loaded with nano-silica, and synergistically treated with fermentation broth of Bacillus subtilis endophytic in Eucommia ulmoides. The modified sucrose was prepared by partially replacing trehalose, composite with magnetic iron oxide nanoparticles, and reinforced with Eucommia ulmoides gum.
[0022] The modified basic culture medium was prepared as follows: Fresh Eucommia ulmoides leaves were washed, dried, and pulverized, passed through an 80-mesh sieve, and added to a 70% (v / v) ethanol solution. The mixture was then refluxed at 60°C for 2 hours. After filtration, the filtrate was concentrated under reduced pressure to obtain an extract, which is the Eucommia ulmoides leaf extract. Chitosan was dissolved in a 1% (w / w) acetic acid solution to prepare a 2% (w / w) chitosan solution. Rare earth nitrate solution was added dropwise to the chitosan solution, and the mixture was stirred at 40°C and 200 rpm for 30 minutes. Rare earth ions were complexed with chitosan to obtain a chitosan-rare earth composite solution. Eucommia ulmoides leaf extract was added to the chitosan-rare earth composite solution and stirred at 35℃ and 200 rpm for 45 min to form a composite modified solution. 4.3 g of MS medium powder was weighed and mixed with the composite modified solution. An appropriate amount of water was added and stirred at 40℃ and 150 rpm for 30 min until completely dissolved. Water was added to make up to 1 L and the pH was adjusted to 5.8 to obtain the modified basic culture medium.
[0023] Crushed and sieved Eucommia ulmoides leaves were mixed with 70% ethanol solution at a material-to-liquid ratio of 1:10; Eucommia ulmoides leaf extract and chitosan were mixed with rare earth composite solution at a mass ratio of 1:5; the rare earth elements in the rare earth nitrate solution were lanthanum, cerium and neodymium, and the rare earth nitrate solution was a mixture of nitrates of the three rare earth elements at a mass ratio of 1:1:1; the amount of rare earth nitrate solution added was 0.5% of the mass of chitosan solution.
[0024] The preparation method of modified naphthaleneacetic acid is as follows: β-cyclodextrin is mixed with deionized water, heated to 50℃ and stirred to dissolve, and naphthaleneacetic acid powder is added at the same temperature and stirred for 2 hours to allow naphthaleneacetic acid to be encapsulated by β-cyclodextrin. After cooling, the solid inclusion complex is obtained by freeze-drying. Nano-silica (particle size 10nm, specific surface area 200m²) is used. 2 / g) was dispersed in anhydrous ethanol and sonicated for 20 min to ensure full dispersion. A solid inclusion complex was added and stirred at 40℃ and 300 rpm for 1 h to allow the inclusion complex to be adsorbed or bonded to the high specific surface area of nano-silica. The mixture was then dried at 60℃ to obtain the loaded material. The loaded material was added to the fermentation broth of Bacillus subtilis endophytic in Eucommia ulmoides and fermented at 28℃ and 120 rpm for 24 h. The solid was collected by centrifugation at 4℃ and 8000 rpm for 10 min. The solid was washed twice with sterile water, freeze-dried, and passed through a 200-mesh sieve to obtain modified naphthaleneacetic acid.
[0025] β-Cyclodextrin and deionized water were mixed at a mass ratio of 1:8; naphthaleneacetic acid powder was added at 20% of the mass of β-cyclodextrin; nano-silica and solid inclusion complex were mixed at a mass ratio of 1:2; the loading material and Bacillus subtilis fermentation broth were mixed at a solid-liquid ratio of 1:10.
[0026] The modified sucrose is prepared as follows: sucrose and trehalose are mixed, and deionized water is added to prepare a mixed sugar solution with a mass fraction of 30%. Magnetic iron oxide nanoparticles are added to the mixed sugar solution, first ultrasonically dispersed for 30 min, and then stirred at 50℃ and 200 rpm for 1 h to ensure that the nanoparticles are uniformly dispersed and composited in the sugar solution, forming a sugar-nanoparticle system. Eucommia ulmoides gum latex is added to the sugar-nanoparticle system, and stirring is continued at 50℃ and 300 rpm for 1 h to ensure that the eucommia ulmoides gum and sugar-nanoparticle system are fully mixed, thus obtaining liquid modified sucrose.
[0027] Sucrose and trehalose were mixed at a mass ratio of 5:1; the amount of magnetic iron oxide nanoparticles added was 1% of the mass of the mixed sugar solution; and the amount of Eucommia ulmoides gum latex added was 0.5% of the mass of the sugar solution.
[0028] The preparation method of Eucommia ulmoides gum emulsion is as follows: Eucommia ulmoides gum fragments are added to a 0.5% sodium hydroxide solution and stirred and hydrolyzed for 2 hours at 70℃ and 200 rpm to obtain Eucommia ulmoides gum hydrolysate; the pH value of Eucommia ulmoides gum hydrolysate is adjusted to 6.5, and emulsifiers Span-80 and Tween-80 are added to form a mixture; the mixture is emulsified by high-speed shearing at 60℃ and 8000 rpm for 10 minutes, and then cooled to room temperature to obtain an oil-in-water Eucommia ulmoides gum emulsion.
[0029] The mass ratio of Eucommia ulmoides gum, Span-80 and Tween-80, based on dry glue, is 5:0.5:0.5.
[0030] The method for large-scale rapid propagation of Eucommia ulmoides tissue culture seedlings includes the following steps: Explant preparation: Select healthy stem segments with axillary buds from Eucommia ulmoides branches as explants. After rinsing with running water, disinfect with 70% ethanol for 30 seconds, then soak in 0.1% mercuric chloride solution for 8 minutes, and finally rinse four times with sterile water; Induction and proliferation of clustered buds: Inoculate the disinfected explants into a clustered bud induction medium and culture for 25 days at 23℃, light intensity of 1500 Lux, and light duration of 12 h / d to induce the production of clustered buds; Divide the clustered buds into single buds or small clusters and transfer them to the same clustered bud induction medium for subculture and proliferation culture. Subculture once every 25 days for three consecutive subcultures to establish a sterile propagation line; Rooting induction: Cut off the healthy stem segments obtained above... Rootless seedlings with a height of 2cm were immersed at the base in the liquid working solution of rooting induction medium for 1 hour. Then, they were removed and inserted into solidified rooting induction medium and rooted for 25 days at 23℃, light intensity of 2000 Lux, and light duration of 14h / d. Hardening and transplanting: When the root length of the obtained tissue culture seedlings reached more than 2cm, the culture bottles were moved to natural light for hardening for 3 days. The bottle caps were opened, and a small amount of tap water was added to continue hardening for 2 days. Then, the tissue culture seedlings were removed, the root culture medium was washed off, and they were planted in a sterilized substrate made of peat moss, perlite, and vermiculite in a volume ratio of 3:1:1. The air humidity was maintained above 85%, the temperature was 22℃, and the seedlings were cultured in the dark for 7 days. After that, normal management was gradually restored to obtain Eucommia ulmoides tissue culture seedlings that could be used for field cultivation.
[0031] Example 2: Rooting induction of Eucommia ulmoides tissue culture seedlings. The culture medium used for rooting induction included the following raw materials in parts by weight: 90 parts modified basic culture medium, 1.0 part modified naphthaleneacetic acid, 25 parts modified sucrose, 1.0 part inositol, 2 parts activated carbon, and 8 parts agar. The modified basic culture medium was prepared by modifying MS medium with Eucommia ulmoides leaf extract, chitosan, and rare earth elements. The modified naphthaleneacetic acid was prepared by naphthaleneacetic acid being encapsulated with β-cyclodextrin, loaded with nano-silica, and synergistically treated with fermentation broth of Bacillus subtilis endophytic in Eucommia ulmoides. The modified sucrose was prepared by partially replacing trehalose, composite with magnetic iron oxide nanoparticles, and reinforced with Eucommia ulmoides gum.
[0032] The modified basic culture medium was prepared as follows: Fresh Eucommia ulmoides leaves were washed, dried, and pulverized, passed through a 90-mesh sieve, and added to a 70% (v / v) ethanol solution. The mixture was then refluxed at 65°C for 2.5 h. After filtration, the filtrate was concentrated under reduced pressure to obtain an extract, which is the Eucommia ulmoides leaf extract. Chitosan was dissolved in a 1% (w / w) acetic acid solution to prepare a 2.5% (w / w) chitosan solution. Rare earth nitrate solution was added dropwise to the chitosan solution, and the mixture was stirred at 42°C and 250 rpm for 30 minutes. In an induction process, rare earth ions are complexed with chitosan to obtain a chitosan-rare earth composite solution. Eucommia ulmoides leaf extract is added to the chitosan-rare earth composite solution and stirred at 37℃ and 250 rpm for 52 min to form a composite modified solution. 4.55 g of MS medium powder is weighed and mixed with the composite modified solution. An appropriate amount of water is added, and the mixture is stirred at 45℃ and 175 rpm for 35 min until completely dissolved. Water is added to bring the volume to 1 L, and the pH is adjusted to 5.9 to obtain the modified basic culture medium.
[0033] Crushed and sieved Eucommia ulmoides leaves were mixed with 70% ethanol solution at a material-to-liquid ratio of 1:12.5; Eucommia ulmoides leaf extract and chitosan were mixed with rare earth composite solution at a mass ratio of 1:6.5; the rare earth elements in the rare earth nitrate solution were lanthanum, cerium and neodymium, and the rare earth nitrate solution was a mixture of nitrates of the three rare earth elements at a mass ratio of 1:1:1; the amount of rare earth nitrate solution added was 0.75% of the mass of chitosan solution.
[0034] The preparation method of modified naphthaleneacetic acid is as follows: β-cyclodextrin is mixed with deionized water, heated to 55℃ and stirred to dissolve, and naphthaleneacetic acid powder is added at the same temperature and stirred for 2.5 h to allow naphthaleneacetic acid to be encapsulated by β-cyclodextrin. After cooling, the solid inclusion complex is obtained by freeze-drying. Nano-silica (particle size 15 nm, specific surface area 250 m²) is used. 2 / g) was dispersed in anhydrous ethanol and sonicated for 25 min to ensure full dispersion. The solid inclusion complex was added and stirred at 45℃ and 350 rpm for 1.5 h to allow the inclusion complex to be adsorbed or bonded to the high specific surface area of nano-silica. The mixture was dried at 65℃ to obtain the loaded material. The loaded material was added to the fermentation broth of Bacillus subtilis endophytic in Eucommia ulmoides and fermented at 29℃ and 135 rpm for 36 h. The solid was collected by centrifugation at 4℃ and 9000 rpm for 12 min. The solid was washed three times with sterile water, freeze-dried, and passed through a 250-mesh sieve to obtain modified naphthaleneacetic acid.
[0035] β-Cyclodextrin and deionized water were mixed at a mass ratio of 1:9; naphthaleneacetic acid powder was added at 26% of the mass of β-cyclodextrin; nano-silica and solid inclusion complex were mixed at a mass ratio of 1:3; the loading material and Bacillus subtilis fermentation broth were mixed at a solid-liquid ratio of 1:12.5.
[0036] The modified sucrose is prepared as follows: sucrose and trehalose are mixed, and deionized water is added to prepare a mixed sugar solution with a mass fraction of 35%. Magnetic iron oxide nanoparticles are added to the mixed sugar solution, first ultrasonically dispersed for 35 min, and then stirred at 55℃ and 250 rpm for 1.5 h to ensure that the nanoparticles are uniformly dispersed and composited in the sugar solution, forming a sugar-nanoparticle system. Eucommia ulmoides gum latex is added to the sugar-nanoparticle system, and stirring is continued at 55℃ and 350 rpm for 1.2 h to ensure that the eucommia ulmoides gum and sugar-nanoparticle system are fully mixed, thus obtaining liquid modified sucrose.
[0037] Sucrose and trehalose were mixed at a mass ratio of 5:1.5; the amount of magnetic iron oxide nanoparticles added was 1.5% of the mass of the mixed sugar solution; and the amount of Eucommia ulmoides gum latex added was 0.75% of the mass of the sugar solution.
[0038] The preparation method of Eucommia ulmoides gum emulsion is as follows: Eucommia ulmoides gum fragments are added to a 0.75% sodium hydroxide solution and stirred and hydrolyzed for 3 hours at 75℃ and 250 rpm to obtain Eucommia ulmoides gum hydrolysate; the pH value of Eucommia ulmoides gum hydrolysate is adjusted to 7.0, and emulsifiers Span-80 and Tween-80 are added to form a mixture; the mixture is emulsified by high-speed shearing at 65℃ and 10000 rpm for 15 minutes, and then cooled to room temperature to obtain an oil-in-water Eucommia ulmoides gum emulsion.
[0039] The mass ratio of Eucommia ulmoides gum, Span-80 and Tween-80, based on dry glue, is 5:0.75:0.75.
[0040] The method for large-scale rapid propagation of Eucommia ulmoides tissue culture seedlings includes the following steps: Explant preparation: Select healthy stem segments with axillary buds from Eucommia ulmoides branches as explants. After rinsing with running water, disinfect with 70% ethanol for 45 seconds, then soak in 0.1% mercuric chloride solution for 10 minutes, and finally rinse 5 times with sterile water; Induction and proliferation of clustered buds: Inoculate the disinfected explants into a clustered bud induction medium and culture them for 30 days at 25℃, light intensity of 1750 Lux, and light duration of 13 h / d to induce the production of clustered buds; Divide the clustered buds into single buds or small clusters and transfer them to the same clustered bud induction medium for subculture and proliferation culture. Subculture once every 30 days for 4 consecutive subcultures to establish a sterile propagation line; Rooting induction: Cut off the above-obtained healthy and tall roots... Rootless seedlings measuring 2.5cm in length were immersed at the base in the liquid working solution of rooting induction medium for 1.5 hours. They were then removed and inserted into solidified rooting induction medium for rooting culture at 25℃, light intensity of 2250 Lux, and light duration of 14 hours / day for 30 days. Hardening and transplanting: When the root length of the obtained tissue culture seedlings reached more than 2cm, the culture bottles were moved to natural light for hardening for 4 days. The bottle caps were opened, and a small amount of tap water was added to continue hardening for 2.5 days. Subsequently, the tissue culture seedlings were removed, the root culture medium was washed off, and they were planted in a sterilized substrate composed of peat moss, perlite, and vermiculite in a volume ratio of 3:1:1. The air humidity was maintained above 85%, the temperature at 25℃, and the seedlings were cultured in the dark for 8.5 days. Afterward, normal management was gradually resumed to obtain Eucommia ulmoides tissue culture seedlings suitable for field cultivation. Example 3: Rooting induction of Eucommia ulmoides tissue culture seedlings. The culture medium used for rooting induction included the following raw materials in parts by weight: 100 parts modified basic culture medium, 1.5 parts modified naphthaleneacetic acid, 30 parts modified sucrose, 1.5 parts inositol, 3 parts activated carbon, and 10 parts agar. The modified basic culture medium was prepared by modifying MS medium with Eucommia ulmoides leaf extract, chitosan, and rare earth elements. The modified naphthaleneacetic acid was prepared by naphthaleneacetic acid being encapsulated with β-cyclodextrin, loaded with nano-silica, and synergistically treated with fermentation broth of Bacillus subtilis endophytic in Eucommia ulmoides. The modified sucrose was prepared by partially replacing trehalose, composite with magnetic iron oxide nanoparticles, and reinforced with Eucommia ulmoides gum.
[0041] The modified basic culture medium was prepared as follows: Fresh Eucommia ulmoides leaves were washed, dried, and pulverized, passed through a 100-mesh sieve, and added to a 70% (v / v) ethanol solution. The mixture was then refluxed at 70°C for 3 hours. After filtration, the filtrate was concentrated under reduced pressure to obtain an extract, which is the Eucommia ulmoides leaf extract. Chitosan was dissolved in a 1% (w / w) acetic acid solution to prepare a 3% (w / w) chitosan solution. Rare earth nitrate solution was added dropwise to the chitosan solution, and the mixture was stirred at 45°C and 300 rpm for 30 minutes. n, to induce a complexation reaction between rare earth ions and chitosan, to obtain a chitosan-rare earth composite solution; add Eucommia ulmoides leaf extract to the chitosan-rare earth composite solution, and stir at 40℃ and 300rpm for 60min to form a composite modified solution; weigh 4.8g of MS culture medium powder, mix it with the composite modified solution, add an appropriate amount of water, and stir at 50℃ and 200rpm for 40min until completely dissolved, add water to make up to 1L and adjust the pH to 6.0 to obtain the modified basic culture medium.
[0042] Crushed and sieved Eucommia ulmoides leaves were mixed with 70% ethanol solution at a material-to-liquid ratio of 1:15; Eucommia ulmoides leaf extract and chitosan were mixed with rare earth composite solution at a mass ratio of 1:8; the rare earth elements in the rare earth nitrate solution were lanthanum, cerium and neodymium, and the rare earth nitrate solution was a mixture of nitrates of the three rare earth elements at a mass ratio of 1:1:1; the amount of rare earth nitrate solution added was 1% of the mass of chitosan solution.
[0043] The preparation method of modified naphthaleneacetic acid is as follows: β-cyclodextrin is mixed with deionized water, heated to 60℃ and stirred to dissolve, and naphthaleneacetic acid powder is added at the same temperature and stirred for 3 hours to allow naphthaleneacetic acid to be encapsulated by β-cyclodextrin. After cooling, the solid inclusion complex is obtained by freeze-drying. Nano-silica (particle size 20nm, specific surface area 300m²) is used. 2 / g) was dispersed in anhydrous ethanol and sonicated for 30 min to ensure full dispersion. A solid inclusion complex was added and stirred at 50℃ and 400 rpm for 2 h to allow the inclusion complex to be adsorbed or bonded to the high specific surface area of nano-silica. The mixture was then dried at 70℃ to obtain the loaded material. The loaded material was added to the fermentation broth of Bacillus subtilis endophytic in Eucommia ulmoides and fermented at 30℃ and 150 rpm for 48 h. The solid was collected by centrifugation at 4℃ and 10000 rpm for 15 min. The solid was washed four times with sterile water, freeze-dried, and passed through a 300-mesh sieve to obtain modified naphthaleneacetic acid.
[0044] β-Cyclodextrin and deionized water were mixed at a mass ratio of 1:10; naphthaleneacetic acid powder was added at 32% of the mass of β-cyclodextrin; nano-silica and solid inclusion complex were mixed at a mass ratio of 1:4; the loading material and Bacillus subtilis fermentation broth were mixed at a solid-liquid ratio of 1:15.
[0045] The modified sucrose is prepared as follows: sucrose and trehalose are mixed, and deionized water is added to prepare a mixed sugar solution with a mass fraction of 40%. Magnetic iron oxide nanoparticles are added to the mixed sugar solution, first ultrasonically dispersed for 40 min, and then stirred at 60℃ and 300 rpm for 2 h to ensure that the nanoparticles are uniformly dispersed and composited in the sugar solution, forming a sugar-nanoparticle system. Eucommia ulmoides gum latex is added to the sugar-nanoparticle system, and stirring is continued at 60℃ and 400 rpm for 1.5 h to ensure that the eucommia ulmoides gum and sugar-nanoparticle system are fully mixed, thus obtaining liquid modified sucrose.
[0046] Sucrose and trehalose were mixed at a mass ratio of 5:2; the amount of magnetic iron oxide nanoparticles added was 2% of the mass of the mixed sugar solution; and the amount of Eucommia ulmoides gum latex added was 1% of the mass of the sugar solution.
[0047] The preparation method of Eucommia ulmoides gum emulsion is as follows: Eucommia ulmoides gum fragments are added to a 1% sodium hydroxide solution and stirred and hydrolyzed for 4 hours at 80℃ and 300 rpm to obtain Eucommia ulmoides gum hydrolysate; the pH value of Eucommia ulmoides gum hydrolysate is adjusted to 7.5, and emulsifiers Span-80 and Tween-80 are added to form a mixture; the mixture is emulsified by high-speed shearing at 70℃ and 12000 rpm for 20 minutes, and then cooled to room temperature to obtain an oil-in-water Eucommia ulmoides gum emulsion.
[0048] The mass ratio of Eucommia ulmoides gum, Span-80 and Tween-80, based on dry glue content, is 5:1:1.
[0049] The method for large-scale rapid propagation of Eucommia ulmoides tissue culture seedlings includes the following steps: Explant preparation: Select healthy stem segments with axillary buds from Eucommia ulmoides branches as explants. After rinsing with running water, disinfect with 70% ethanol for 60 seconds, then soak in 0.1% mercuric chloride solution for 12 minutes, and finally rinse 6 times with sterile water; Induction and proliferation of clustered buds: Inoculate the disinfected explants into a clustered bud induction medium and culture them for 35 days at 27℃, light intensity of 2000 Lux, and light duration of 14 h / d to induce the production of clustered buds; Divide the clustered buds into single buds or small clusters and transfer them to the same clustered bud induction medium for subculture and proliferation culture. Subculture once every 35 days for 5 consecutive subcultures to establish a sterile propagation line; Rooting induction: Cut off the healthy branches obtained above... Rootless seedlings with a height of 3cm were immersed at the base in the liquid working solution of rooting induction medium for 2 hours. Then, they were removed and inserted into solidified rooting induction medium and rooted for 35 days at 27℃, light intensity of 2500 Lux, and light duration of 14h / d. Hardening and transplanting: When the root length of the obtained tissue culture seedlings reached more than 2cm, the culture bottles were moved to natural light for hardening for 5 days. The bottle caps were opened, and a small amount of tap water was added to continue hardening for 3 days. Then, the tissue culture seedlings were removed, the root culture medium was washed off, and they were planted in a sterilized substrate made of peat moss, perlite, and vermiculite in a volume ratio of 3:1:1. The air humidity was maintained above 85%, the temperature was 28℃, and the seedlings were cultured in the dark for 10 days. After that, normal management was gradually restored to obtain Eucommia ulmoides tissue culture seedlings that could be used for field cultivation.
[0050] Comparative Example 1: The difference between this comparative example and Example 1 is that this comparative example uses untreated commercially available MS culture medium.
[0051] Comparative Example 2 differs from Example 1 in that untreated commercially available naphthaleneacetic acid is used in this comparative example.
[0052] Comparative Example 3 differs from Example 1 in that it uses untreated commercially available sucrose.
[0053] Performance Testing: The rooting induction media prepared for Examples 1, 2, 3, Comparative Examples 1, 2, and 3 were subjected to performance testing: Rooting Rate Test: Rootless Eucommia ulmoides tissue culture seedlings with uniform growth were selected, randomly grouped, and inoculated into the solid rooting induction media of each example and comparative example. They were cultured for 30 days under the same conditions of constant temperature, constant light, and fixed photoperiod. After the culture period, the basal germination length ≥1 cm was counted for each seedling. The number of plants with mm root systems was counted, and the percentage of each plantlet relative to the total number of inoculated plants in the group was calculated; this was the rooting rate. Each treatment was tested in at least three independent replicates to ensure data reliability and reproducibility. The testing was conducted according to the "Technical Specifications for Plant Tissue Culture." Transplant survival rate testing: Healthy tissue culture seedlings after rooting were selected and subjected to a two-stage hardening-off process (first, acclimatization to natural light for 3-5 days, then 2-3 days of moisture retention with the cap open). The culture medium was then washed off the roots, and the seedlings were transplanted into a uniform sterile substrate. They were cultured for 30 days in a controlled environment (temperature 22-28℃, initial humidity ≥85% gradually decreasing, and moderate shading). The final survival rate was statistically analyzed. The percentage of plants with vigorous growth and no wilting or rot was determined by conducting at least three independent replicate experiments for each treatment to ensure the reliability of the results. The test was conducted in accordance with the "Technical Regulations for Transplanting and Acclimatization of Tissue Culture Seedlings of Forestry Plants". Root quality index test: Ten representative, robust rooted seedlings were randomly selected from each treatment group. After washing and gently separating their roots, the number of primary adventitious roots of each seedling was counted and the length of the longest root was measured. The average number of roots and the average root length of each treatment group were calculated, and the root quality index was obtained by multiplying the two. At least three independent replicate sampling and measurement were conducted for each treatment. The test was conducted in accordance with the "Methods for Plant Root Research".
[0054] The obtained test data are recorded in Table 1 below:
[0055] By comparing and analyzing the relevant data in Table 1, it can be seen that the rooting induction medium prepared by the method of Eucommia ulmoides tissue culture seedling rooting induction and large-scale rapid propagation of the present invention has a high rooting rate, indicating that the medium formula has a high efficiency and stable rooting induction ability; the high transplant survival rate indicates that the cultured seedlings have robust root systems, complete leaf functions, and strong stress resistance; the high root quality index indicates a large number of roots and relatively long roots. Therefore, the method of Eucommia ulmoides tissue culture seedling rooting induction and large-scale rapid propagation of the present invention has a broader market prospect and is more suitable for promotion.
[0056] In the description of this specification, references to terms such as "an embodiment," "example," and "specific example" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.
[0057] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims
1. Rooting induction of Eucommia ulmoides tissue culture seedlings, characterized in that, The rooting induction culture medium comprises the following raw materials in parts by weight: 80-100 parts modified basic culture medium, 0.5-1.5 parts modified naphthaleneacetic acid, 20-30 parts modified sucrose, 0.5-1.5 parts inositol, 1-3 parts activated carbon, and 6-10 parts agar; the modified basic culture medium is prepared by modifying MS culture medium with Eucommia ulmoides leaf extract, chitosan, and rare earth elements. The modified naphthaleneacetic acid was prepared by naphthaleneacetic acid through β-cyclodextrin inclusion, nano-silica loading, and synergistic treatment with fermentation broth of Bacillus subtilis endophyticus from Eucommia ulmoides; the modified sucrose was prepared by partial substitution of trehalose, composite with magnetic iron oxide nanoparticles, and reinforcement with Eucommia ulmoides gum.
2. The method for inducing rooting of Eucommia ulmoides tissue culture seedlings according to claim 1, characterized in that, The modified basic culture medium is prepared as follows: Crushed and sieved Eucommia ulmoides leaves are added to a 70% (v / v) ethanol solution and extracted by reflux at 60-70°C. The filtrate is filtered and concentrated under reduced pressure to obtain an extract, which is the Eucommia ulmoides leaf extract. Chitosan is dissolved in a 1% (w / w) acetic acid solution to prepare a 2-3% (w / w) chitosan solution. Rare earth nitrate solution is added dropwise to the chitosan solution and stirred at 40-45°C to allow the rare earth ions to complex with the chitosan, resulting in a chitosan-rare earth composite solution. The Eucommia ulmoides leaf extract is added to the chitosan-rare earth composite solution and stirred at 35-40°C to form a composite modified solution. 4.3-4.8 g of MS culture medium powder is weighed, mixed with the composite modified solution, dissolved in water, and brought to a final volume of 1 L. The pH is then adjusted to 5.8-6.0 to obtain the modified basic culture medium.
3. The method for inducing rooting of Eucommia ulmoides tissue culture seedlings according to claim 2, characterized in that: The pulverized and sieved Eucommia ulmoides leaves are mixed with a 70% ethanol solution at a material-to-liquid ratio of 1:10-15; the Eucommia ulmoides leaf extract and chitosan are mixed with a rare earth composite solution at a mass ratio of 1:5-8; the rare earth elements in the rare earth nitrate solution are lanthanum, cerium, and neodymium, and the rare earth nitrate solution is a mixture of nitrates of the three rare earth elements at a mass ratio of 1:1:1; the amount of rare earth nitrate solution added is 0.5-1% of the mass of the chitosan solution.
4. The method for inducing rooting of Eucommia ulmoides tissue culture seedlings according to claim 1, characterized in that, The modified naphthaleneacetic acid is prepared as follows: β-cyclodextrin is mixed with deionized water, heated to 50-60℃ and stirred to dissolve. Naphthaleneacetic acid powder is added at the same temperature and stirring is continued. After cooling, a solid inclusion complex is obtained by freeze-drying. Nano-silica is dispersed in anhydrous ethanol and subjected to ultrasonic treatment. The solid inclusion complex is added and stirred at 40-50℃ to allow the inclusion complex to be adsorbed and bonded to the high specific surface area of the nano-silica. The precipitate is collected by centrifugation and dried to obtain the loading. The loading is added to the fermentation broth of Bacillus subtilis endophytic in Eucommia ulmoides and fermented by stirring at 28-30℃. After fermentation, the solid is collected by centrifugation. The solid is washed, freeze-dried, and sieved to obtain modified naphthaleneacetic acid.
5. The rooting induction method for Eucommia ulmoides tissue culture seedlings according to claim 4, characterized in that: The β-cyclodextrin and deionized water are mixed at a mass ratio of 1:8-10; the amount of naphthaleneacetic acid powder added is 20-32% of the mass of β-cyclodextrin; the nano-silica and the solid inclusion complex are mixed at a mass ratio of 1:2-4; the loading material and the fermentation broth of Bacillus subtilis endophyticus of Eucommia ulmoides are mixed at a solid-liquid ratio of 1:10-15.
6. The method for inducing rooting of Eucommia ulmoides tissue culture seedlings according to claim 1, characterized in that, The modified sucrose is prepared as follows: sucrose and trehalose are mixed, and deionized water is added to prepare a mixed sugar solution with a mass fraction of 30-40%; magnetic iron oxide nanoparticles are added to the mixed sugar solution, first ultrasonically dispersed, and then continuously stirred to ensure uniform dispersion and aggregation of the nanoparticles in the mixed sugar solution, forming a sugar-nanoparticle system; Eucommia ulmoides gum emulsion is added to the sugar-nanoparticle system, and stirring is continued to ensure thorough mixing of the Eucommia ulmoides gum and the sugar-nanoparticle system, forming a mixed liquid; the mixed liquid is spray-dried, pulverized, and sieved to obtain powdered modified sucrose.
7. The method for inducing rooting of Eucommia ulmoides tissue culture seedlings according to claim 6, characterized in that: The sucrose and trehalose are mixed at a mass ratio of 5:1-2; the amount of magnetic iron oxide nanoparticles added is 1-2% of the mass of the mixed sugar solution; the amount of Eucommia ulmoides gum latex added is 0.5-1% of the mass of the sugar solution.
8. The rooting induction method for Eucommia ulmoides tissue culture seedlings according to claim 7, characterized in that, The preparation method of the Eucommia ulmoides gum emulsion is as follows: Eucommia ulmoides gum fragments are added to a sodium hydroxide solution with a mass fraction of 0.5-1% and stirred at 70-80℃ to carry out a hydrolysis reaction to obtain Eucommia ulmoides gum hydrolysate; the pH value of the Eucommia ulmoides gum hydrolysate is adjusted to 6.5-7.5, and emulsifiers Span-80 and Tween-80 are added to form a mixture; the mixture is sheared and emulsified at 60-70℃, and then cooled to room temperature to obtain an oil-in-water Eucommia ulmoides gum emulsion.
9. The method for inducing rooting and large-scale rapid propagation of Eucommia ulmoides tissue culture seedlings according to claim 8, characterized in that: The mass ratio of Eucommia ulmoides gum, Span-80 and Tween-80, based on dry glue content, is 5:0.5-1:0.5-1.
10. A method for large-scale rapid propagation and cultivation of Eucommia ulmoides tissue culture seedlings, comprising the rooting induction method for Eucommia ulmoides tissue culture seedlings according to any one of claims 1-9, characterized in that, The process includes the following steps: Explant preparation: Select healthy stem segments with axillary buds from Eucommia ulmoides branches as explants. After rinsing with running water, disinfect with a solution of 70% ethanol (v / v) and 0.1% mercuric chloride (w / w), and finally rinse with sterile water; Bud induction and proliferation: Inoculate the disinfected explants into a bud induction medium and culture for 25-35 days to induce bud clusters; Divide the bud clusters into single buds or small clusters and transfer them to the same bud induction medium for subculture and proliferation. Subculture every 25-35 days for 3-5 consecutive subcultures to establish a sterile propagation line; Rooting induction: Cut off healthy stem segments with a height of 2-3 cm obtained above. Rootless seedlings are immersed at the base in the liquid working solution of rooting induction medium, then removed and inserted into solidified rooting induction medium. Rooting culture is carried out using set parameters for 25-35 days. Hardening and transplanting: When the root system reaches a length of 2cm or more, the culture bottle is moved to natural light for hardening for 3-5 days. The bottle cap is opened, and a small amount of tap water is added to continue hardening for 2-3 days. Subsequently, the tissue culture seedlings are removed, the root culture medium is washed off, and they are planted in a sterilized substrate composed of peat moss, perlite, and vermiculite in a volume ratio of 3:1:
1. After 7-10 days of shading at 22-28℃, normal management is gradually resumed to obtain Eucommia ulmoides tissue culture seedlings suitable for field cultivation.