A high-efficiency rooting and seedling raising method for plant tissue culture seedlings

Abstract

The present application belongs to the technical field of plant tissue culture, and particularly relates to a high-efficiency rooting and seedling raising method for plant tissue culture seedlings, S1 trimming and disinfection of S2 induction liquid immersion, S3 composite substrate implantation and gradient environment regulation, S4 substrate transplanting and planting; the core is to use modified magnetic biochar composite substrate loaded with FeO nanoparticles, cooperate with the synergistic effect of microencapsulated rooting hormone and melatonin, realize in-situ integrated seedling raising through composite spectrum containing UV-A, CO pulse supply, gradient humidity and PEG osmotic pressure domestication. The present application effectively inhibits basal browning, shortens the seedling raising period, improves the rooting quality and transplanting survival rate, overcomes the pollution prejudice of magnetic nanoparticles, is suitable for woody and herbaceous plants, and can be applied to plant tissue culture seedling production on a large scale.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for efficient rooting and hardening of plant tissue culture seedlings. Background Technology

[0002] Plant tissue culture technology is a core technology for the rapid propagation of superior varieties and the cultivation of virus-free seedlings in modern agriculture. It is widely used in the large-scale cultivation of woody plants, herbaceous plants, medicinal plants, and ornamental plants. With its advantages of rapid propagation, high propagation coefficient, and preservation of the superior traits of the parent plant, it has become a key means of seedling cultivation in modern agriculture. Rooting and hardening-off are two core stages in the plant tissue culture process, directly determining the rooting quality, survival rate, and transplant adaptability of tissue-cultured seedlings. They are also the core bottlenecks restricting the large-scale and industrial application of tissue culture technology—poor rooting quality and low survival rate of tissue-cultured seedlings significantly increase cultivation costs, reduce production efficiency, and make it difficult to meet the needs of large-scale cultivation.

[0003] Traditional, segmented methods are often used for rooting and hardening off plant tissue culture seedlings. This involves first inducing root growth, then opening the container for hardening off, which requires washing the roots, changing the substrate, and transplanting multiple times. This process is cumbersome, easily causes mechanical damage and contamination to the root system, resulting in low survival rates and long hardening-off periods, making it difficult to achieve efficient cultivation.

[0004] Specifically, existing technologies have the following core technical defects in the rooting and seedling hardening process: 1. Improper preparation of rooting induction solution: Most existing rooting induction solutions use a single rooting hormone to be directly dissolved and added. The hormone release rate cannot be controlled, which can easily lead to excessively high local concentrations or large fluctuations, resulting in root deformities and root tip burns. At the same time, the induction solution lacks effective antioxidants, and browning is likely to occur at the base of tissue culture seedlings, which seriously affects the rooting effect and root quality. 2. Tissue culture substrates have limited functionality and applications: Existing tissue culture substrates are mostly composed of conventional materials such as peat, perlite, and coconut coir, which can only provide basic water retention and aeration functions. They cannot actively regulate the rhizosphere microenvironment and cannot meet the needs of root growth, nutrient absorption, and stress resistance induction in tissue culture seedlings. In addition, magnetic biochar in current technologies is mainly used in soil remediation and water pollution control. Due to the common technical prejudice in the industry that "magnetic nanoparticles will introduce exogenous pollution, destroy the sterile environment of tissue culture, and poison seedlings," and the lack of precedents for safe application in tissue culture substrates, magnetic biochar has not been applied to the rooting and hardening substrate of plant tissue culture. 3. Light control methods do not meet the needs of autotrophic switching: Light control during the hardening-off stage mostly uses a single red and blue spectrum, which can only meet the basic photosynthetic needs of tissue culture seedlings and does not fully consider the physiological characteristics of tissue culture seedlings transitioning from heterotrophic growth (dependent on culture medium sugars) to autotrophic growth (dependent on their own photosynthesis). At the same time, there is a common technical prejudice in the industry that "UV light is harmful to tissue culture seedlings and can easily cause burns or whitening." The application of UV light is deliberately avoided in tissue culture hardening-off, while far-red light is only used for flower flowering control and is not applied to the autotrophic switching period of tissue culture seedlings, resulting in slow autotrophic switching speed and low photosynthetic efficiency. 4. In existing technologies, carbon dioxide supply mostly adopts a constant concentration continuous supply mode, which cannot be matched with light regulation and the physiological needs of tissue culture seedlings. This not only results in low photosynthetic efficiency but also easily leads to carbon dioxide waste and makes it difficult to accelerate the autotrophic conversion process of tissue culture seedlings. 5. Humidity control during the hardening-off process is mostly done in a crude, one-time dehumidification manner without a gradient design. Tissue culture seedlings have difficulty gradually adapting to the low humidity environment and are prone to wilting, dehydration and death after transplanting. At the same time, without combining osmotic pressure control methods, it is impossible to effectively induce stress resistance in tissue culture seedlings, which further reduces the survival rate of hardening-off seedlings. 6. The three key stages of rooting, autotrophic switching, and hardening-off need to be completed in different culture containers. Multiple transplanting can easily cause root damage and increase the risk of contamination. Moreover, the operation process is complicated, resulting in a long hardening-off period and low survival rate. The survival rate of traditional extensive hardening-off is only 40%-60%, and the survival rate of conventional factory-style seedling cultivation is mostly in the range of 75%-85%, which is difficult to meet the needs of efficient and large-scale cultivation.

[0005] In summary, existing rooting and hardening-off methods are cumbersome, produce poor rooting quality, have slow autotrophic transition, low survival rates, and long cycles, making it difficult to meet the needs of large-scale and efficient cultivation in modern agriculture. Developing a hardening-off method that achieves in-situ integration of rooting, autotrophic transition, and hardening-off, utilizes multiple methods in synergy, achieves high survival rates, and has a short cycle has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a highly efficient method for rooting and hardening off plant tissue culture seedlings, in order to solve the problems in the background art mentioned above. The specific solution is as follows: S1, select tissue culture rootless seedlings, and perform base trimming and disinfection treatment; S2, Immerse the base of the trimmed and disinfected tissue culture seedling stem segments in an induction solution containing basal culture medium, melatonin, and microencapsulated rooting hormone. The immersion temperature is 22-28℃, and the immersion time is 1.5-2.0h. S3, the induced tissue culture seedlings are implanted into a culture container containing the above-mentioned composite substrate, and gradient environmental regulation is carried out in the same culture container in sequence during the rooting induction period, autotrophic switching period, and in-situ hardening adaptation period; the composite substrate contains modified magnetic biochar loaded with magnetic nanoparticles, nanocellulose and perlite. S4. Transplant the tissue culture seedlings with substrate directly after completing step S3.

[0007] To improve the thoroughness of disinfection at the base of tissue culture seedlings and avoid tissue damage, conventional disinfection methods are prone to problems such as disinfectant residue or excessive inhibition of explant activity. Preferably, as a preferred embodiment of the present invention, the base disinfection treatment involves sequentially immersing the seedlings in 75% alcohol for 20-30 seconds, immersing them in sterile sodium hypochlorite solution for 8-12 minutes, and rinsing them 3-4 times with sterile water to thoroughly remove residual disinfectant.

[0008] To address the issues of uneven hormone release from the induction solution and easy browning at the base of tissue culture seedlings, direct addition of hormones can easily cause concentration fluctuations, burn the roots, and exacerbate browning. Single antioxidant methods have limited effectiveness. Preferably, as a preferred embodiment of the present invention, the melatonin concentration in the induction solution is 0.5-0.8 mg / L; the wall material of the microencapsulated rooting hormone is at least one of sodium alginate, gelatin, or chitosan, and the core material is indolebutyric acid and / or naphthaleneacetic acid. It is prepared using an emulsification cross-linking method to achieve stable hormone release and synergistic inhibition of basal browning with melatonin.

[0009] To address the issue of limited functionality and inability to actively regulate the rhizosphere microenvironment in tissue culture substrates, conventional substrates only provide basic water retention and aeration, and magnetic nanoparticles are generally considered unsuitable for aseptic tissue culture environments. Preferably, as a preferred embodiment of the present invention, the composite substrate comprises, by weight, 40-70 parts modified magnetic biochar, 1025 parts nanocellulose, and 1530 parts perlite; the modified magnetic biochar is loaded with Fe3O4 magnetic nanoparticles, which can generate a weak magnetic field of 515 mT in the rhizosphere, optimizing the substrate structure and promoting nutrient absorption.

[0010] To address the issues of low photosynthetic efficiency and slow heterotrophic-to-autotrophic transition during the autotrophic transition period, conventional single red-blue light spectrum and constant CO2 supply are insufficient to meet the physiological needs of tissue culture seedlings. Preferably, as a preferred embodiment of the present invention, a composite light spectrum is used during the autotrophic transition period. This spectrum includes red, blue, far-red, and UVA light, with a UVA wavelength of 365-385 nm. The ratio of red light to blue light to far-red light to UVA is 3:2:1:0.4-0.6, the light intensity is 3500-4500 lux, and the illumination time is 13-15 h / d. Simultaneously, a pulsed CO2 supply is provided at a concentration of 800-1200 ppm, 13 times daily, each lasting 12 hours.

[0011] To address the issues of drastic humidity fluctuations and insufficient seedling resistance during hardening-off, a one-time reduction in humidity can easily lead to seedling wilting, dehydration, and death, lacking the synergistic acclimatization effect of osmotic stress. Preferably, as a preferred embodiment of the invention, the humidity in the gradient environmental control is reduced in stages: 90-95% during the rooting induction period, 80% during the autotrophic switching period, and 80%-70%-60% during the in-situ hardening-off adaptation period, decreasing by 5-10% every 23 days; during the in-situ hardening-off adaptation period, a 50-100 mg / L PEG6000 solution is applied to the substrate every 3 days, with each application being 8-12% of the substrate mass.

[0012] Compared with the prior art, the present invention has the following beneficial effects: By synergistically combining microencapsulated rooting hormones and melatonin, stable hormone release is achieved, inhibiting basal browning and improving root uniformity and root quality. A composite matrix loaded with magnetic nanoparticles overcomes the functional limitations of traditional tissue culture matrices, enabling active regulation of the rhizosphere microenvironment. A stable weak magnetic field of 5-15 mT induces increased enzyme activity in root cells and regulates the ion exchange rate in the rhizosphere microenvironment, thereby enhancing nutrient absorption efficiency and root vitality. Composite spectral analysis and pulsed CO2 supply synergistically match autotrophic conversion needs, accelerating photosynthetic system establishment, improving photosynthetic efficiency, and shortening the autotrophic transition period. Gradient humidity and osmotic pressure coupling acclimatization allow tissue culture seedlings to gradually adapt to the external environment, significantly improving stress resistance and transplant survival rate. The entire process—rooting, autotrophic switching, and hardening—is integrated in situ within the same container, eliminating the need for transplanting, root washing, and substrate replacement, reducing root damage and contamination, shortening the hardening period, and making it suitable for large-scale production. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the overall process of a method for efficient rooting and hardening of plant tissue culture seedlings according to the present invention. Figure 2 This is a graph showing the changes in the three-stage gradient environment regulation in an embodiment of the present invention. Figure 3 This is a bar chart comparing the root development indicators of tissue culture seedlings in the embodiments and comparative examples of the present invention. Detailed Implementation

[0014] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0015] In the following embodiments, unless otherwise specified, all technical means used are conventional in the field, and all raw materials used are commercially available; all operations are performed in a sterile environment with a cleanliness level of Class 100 to avoid contamination by miscellaneous bacteria affecting the growth of tissue culture seedlings; all instruments and equipment used have undergone sterilization treatment and comply with the specifications for plant tissue culture experiments.

[0016] In this embodiment, the basal culture medium is a conventional plant tissue culture basal culture medium in the art. MS medium, 1 / 2 MS medium, or WPM medium can be selected according to the type of tissue culture seedling, with 1 / 2 MS medium being preferred (formula: macroelements are half that of MS medium, microelements, vitamins, and inositol content are consistent with MS medium, agar 7g / L, pH adjusted to 5.8-6.0). The modified magnetic biochar is prepared by loading Fe3O4 magnetic nanoparticles onto corn stalks or rice stalks and then activating them at high temperature. The particle size is 50-100 mesh, and the specific surface area is 150-200 m² / g. Its preparation adopts a chemical co-precipitation method: 80-mesh straw is pyrolyzed at 550℃ for 4 hours under nitrogen protection at a rate of 5℃ / min to obtain the original biochar. This biochar is dispersed in a mixed solution of FeCl and FeSO (molar ratio 2:1), the pH is adjusted to 10.5 with ammonia, and the mixture is stirred at 70℃ for 2 hours to complete the loading. Its saturation magnetization reaches 22.5 emu / g. After pre-magnetization in the tissue culture container, a stable weak magnetic field of 8-12 mT can be formed in the rhizosphere microenvironment. The nanocellulose has a particle size of 50-100 nm and a purity of ≥98%. The perlite has a particle size of 1-3 mm and is sterilized by high-pressure steam at 121℃ for 20 min before use. The microencapsulated rooting hormone is prepared by emulsification cross-linking method. The wall material and core material are commercially available analytical grade materials, and the emulsifier is Tween-80 or Span-80. 80. The crosslinking agent is calcium chloride or sodium tripolyphosphate. When chitosan is used as the wall material, 2g of chitosan is dissolved in 100mL of 1% acetic acid solution. The core material (indolebutyric acid and / or naphthaleneacetic acid) is dissolved in anhydrous ethanol and then added dropwise to the wall material solution. An emulsifier is added and sheared at 3000r / min to form a W / O type emulsion. 5% sodium tripolyphosphate is added dropwise for crosslinking for 3h. After centrifugation, washing, and freeze-drying, microcapsules with suitable particle size are obtained. The hormone release cycle is 15-20 days at 25℃, achieving stable release.

[0017] Example 1: A method for efficient rooting and hardening of blueberry tissue culture seedlings: A highly efficient rooting and hardening method for plant tissue culture seedlings, the specific steps of which are as follows: S1, Selection of rootless tissue culture seedlings, trimming of the base, and disinfection. Select healthy, disease-free blueberry tissue culture seedlings (obtained from sterile seedlings through conventional tissue culture, with a plant height of 3-4 cm and a stem diameter of 0.3-0.5 cm) and place them on a sterile operating table. Use sterile forceps to fix the seedlings and gently trim the base with a sterile scalpel to completely remove aged tissue, damaged tissue, and residual culture medium. After trimming, retain a stem segment length of 2.5-3.0 cm, ensuring that the base of the stem segment is flat and undamaged, and avoid damaging the vascular bundles inside the stem segment.

[0018] The disinfection process employs a step-by-step approach, as follows: First, immerse the trimmed tissue culture seedling stem segments in 75% (volume fraction) medical alcohol, gently shaking the sterile container to ensure the alcohol fully contacts the surface and base of the stem segments for 25 seconds. Then, quickly remove the seedlings, blot dry the surface alcohol with sterile filter paper, and immediately immerse them in a 5% (volume fraction) sterile sodium hypochlorite solution, adding 1-2 drops of Tween-80 (to enhance disinfection). During the immersion process, gently shake the container every 3 minutes to ensure even disinfection, for a total of 10 minutes. After disinfection, remove the seedlings and rinse them four times with sterile distilled water for 30 seconds each time to thoroughly remove any residual disinfectant. Finally, blot dry the surface moisture with sterile filter paper and set aside.

[0019] S2, Immersion treatment with induction solution Preparation of induction solution: Based on 1 / 2 MS medium (the formula does not contain sucrose, the macroelements are half of those in MS medium, and the microelements, vitamins, and inositol are the same as in MS medium, agar 7 g / L, pH 5.8-6.0), melatonin and microencapsulated rooting hormone were added sequentially under sterile conditions and stirred to dissolve. The solution was kept at 25℃ for later use. The concentration of melatonin in the induction solution was 0.6 mg / L, and the total concentration of microencapsulated rooting hormone was 2.0 mg / L. The microencapsulated rooting hormone was prepared by emulsion cross-linking method: 2% sodium alginate solution was heated to 60℃ to dissolve, cooled, and indolebutyric acid (core material to wall material mass ratio 1:5) was added. After stirring evenly, 1% calcium chloride solution was added dropwise at a rate of 1 drop / second. The solution was allowed to stand at 25℃ for cross-linking for 30 min. After filtration and washing, microcapsules with a particle size of 100-200 μm were obtained.

[0020] Place the sterilized tissue culture seedlings from step S1 into a sterile petri dish and fix them with sterile forceps. Immerse the base of the stem segment (0.8-1.0 cm in length) into the prepared induction solution, ensuring the base is completely submerged and avoiding contact between the upper part of the stem segment and the induction solution. The soaking environment should be a constant temperature incubator at 25°C, maintaining a constant temperature (temperature fluctuation not exceeding ±0.5°C) for 1.8 hours. During soaking, gently shake the petri dish every 30 minutes to ensure sufficient contact between the induction solution and the base of the stem segment, improving the induction effect. After soaking, remove the tissue culture seedlings and gently blot the surface of the base with sterile filter paper to prevent residual induction solution from causing browning at the base.

[0021] S3, composite matrix implantation and gradient environment regulation Preparation of the composite matrix: Weigh 55 parts of modified magnetic biochar, 18 parts of nanocellulose, and 22 parts of perlite according to the specified mass ratio. Stir aseptically for 15 minutes until homogeneous. Pour the mixture into 100mL transparent glass culture bottles (30g per bottle) and gently compact to a depth of 5-6cm. Spray with sterile distilled water to adjust the humidity to 70-75% for later use. The modified magnetic biochar has a Fe3O4 nanoparticle loading of 5% of the biochar mass, which can form a stable weak magnetic field of approximately 10mT in the rhizosphere of tissue culture seedlings.

[0022] After induction treatment in step S2, the tissue culture seedlings were implanted into the culture containers filled with the composite substrate using sterile forceps. Three seedlings were implanted into each container at a depth of 1.0-1.2 cm. After implantation, the substrate was gently compacted to ensure close contact between the seedling stems and the substrate, preventing lodging. After implantation, the culture containers were placed in an intelligent artificial climate incubator. Gradual environmental control was performed sequentially within the same culture container for the rooting induction period, autotrophic switching period, and in-situ hardening-off period. The control parameters and operational details for each stage are as follows: (1) Rooting induction period: Temperature 25℃, relative humidity 92%, conventional white light (2000 lux, 12h / d), culture for 7 days; during this period, observe the growth status daily, remove contaminated seedlings, and maintain stable substrate humidity.

[0023] (2) Autotrophic switching period: temperature 25℃, relative humidity 80%; composite spectrum ratio red light: blue light: far-red light: UV-A=3:2:1:0.5 (red light 660nm, blue light 450nm, far-red light 730nm, UV-A 375±5nm), light intensity 4000 lux (uniformity ≥90%), light 14h / d; food-grade CO pulse supply (1000ppm), twice a day, 1.5h each time (10:00-11:30, 14:00-15:30); culture for 10 days, check the substrate humidity every 2 days and add sterile distilled water.

[0024] (3) In-situ hardening adaptation period: temperature 25-26℃, relative humidity decreased stepwise (80% for 1-2 days, 70% for 3-4 days, and below 60% for 5-6 days, decreasing by 5%-10% every 2 days), ventilation rate increased from 0.1m / s to 0.3m / s; spray 80mg / L PEG-6000 solution (10% of the substrate mass) into the substrate every 3 days to achieve osmotic pressure acclimatization; cultivate for 8 days and observe the root growth status daily.

[0025] S4, transplanting and planting After completing step S3, remove the tissue culture seedlings along with the root composite substrate under sterile conditions (no need to wash roots or change substrate). The transplanting substrate is peat moss: perlite: vermiculite = 2:1:1 (sterilized by autoclaving at 121℃ for 20 minutes). Fill 10cm×10cm seedling pots, transplanting one seedling per pot, with the transplanting depth consistent with the culture container. After compacting, spray with sterile distilled water to maintain substrate humidity of 70-75%. After transplanting, place in a greenhouse (22-25℃, relative humidity 70-80%), shaded by 70% shade netting. Gradually increase light exposure after 7 days of acclimatization, and then care for the seedlings as usual.

[0026] Example 2: A method for efficient rooting and hardening of Populus tomentosa tissue culture seedlings: A highly efficient rooting and hardening method for plant tissue culture seedlings is disclosed. The only differences between this method and Example 1 are the following parameters and operational details; all other steps, operating procedures, raw material specifications, and equipment parameters are identical to those in Example 1. The specific steps are as follows: S1. Select rootless seedlings of Populus tomentosa with a height of 4-5cm and a stem diameter of 0.4-0.6cm. After trimming the base, retain 3.0-3.5cm. Disinfection is carried out by soaking in 75% alcohol for 20s, soaking in 5% sterile sodium hypochlorite for 8min, and rinsing with sterile distilled water 3 times.

[0027] S2, the induction solution contains 0.5 mg / L melatonin and 1.8 mg / L microencapsulated rooting hormone; the microcapsule wall material is gelatin and the core material is naphthaleneacetic acid (core material to wall material mass ratio 1:6), with a particle size of 150-250 μm; the soaking temperature is 22℃ and the time is 2.0 h.

[0028] S3, the composite substrate ratio is 60 parts modified magnetic biochar, 15 parts nanocellulose, and 20 parts perlite; Fe3O4 nanoparticle loading is 4%, and the rhizosphere magnetic field strength is about 8mT; during the autotrophic switching period, CO pulse supply is 900ppm, once a day for 2 hours each time (11:00-13:00); during the in-situ hardening-off period, the concentration of PEG-6000 solution is 70mg / L, and the amount used is 8% of the substrate mass, and the humidity is reduced by 5%-10% every 3 days.

[0029] S4, transplanting substrate is garden soil: perlite: leaf mold = 2:1:1, greenhouse temperature 23-26℃, relative humidity 75-85%.

[0030] Example 3: A method for efficient rooting and hardening of Polygonatum multiflorum tissue culture seedlings: A highly efficient rooting and hardening method for plant tissue culture seedlings is disclosed. The only differences between this method and Example 1 are the following parameters and operational details; all other steps, operating procedures, raw material specifications, and equipment parameters are identical to those in Example 1. The specific steps are as follows: S1. Select rootless tissue culture seedlings of Polygonatum multiflorum with a plant height of 2.5-3.5cm and a stem diameter of 0.2-0.4cm. After trimming the base, retain 2.0-2.5cm. Disinfection is carried out by soaking in 75% alcohol for 30s, soaking in 5% sterile sodium hypochlorite for 12min, and rinsing with sterile distilled water 4 times.

[0031] S2, the induction solution contains 0.8 mg / L melatonin and 2.2 mg / L microencapsulated rooting hormone; the microcapsule wall material is chitosan, the core material is indolebutyric acid and naphthaleneacetic acid (mass ratio 1:1), the mass ratio of core material to wall material is 1:4, and the particle size is 80-150 μm; the soaking temperature is 28℃ and the time is 1.5 h.

[0032] S3, the composite substrate ratio is 45 parts modified magnetic biochar, 22 parts nanocellulose, and 28 parts perlite; Fe3O4 nanoparticle loading is 6%, and the rhizosphere magnetic field strength is about 12mT; during the autotrophic switching period, the composite spectrum ratio is red light: blue light: far-red light: UV-A = 3:2:1:0.6 (UV-A 385nm), light intensity is 3800 lux, and the light intensity is 13h / d; CO pulse supply is 1100ppm, 3 times a day, 1h each time (9:00-10:00, 13:00-14:00, 17:00-18:00); during the in-situ hardening-off period, the concentration of PEG-6000 solution is 90mg / L, and the amount used is 12% of the substrate mass, and the humidity is reduced by 5%-10% every 2 days (to adapt to the easy browning characteristics of Polygonatum odoratum).

[0033] S4, the transplanting substrate is humus: perlite: river sand = 3:1:1, greenhouse temperature 22-24℃, relative humidity 65-75%.

[0034] Comparative Example 1: Conventional plant tissue culture seedling hardening method (not employing the technical features of this invention): A conventional method for hardening off plant tissue culture seedlings, using blueberry tissue culture seedlings as the research object, is described below. The specific steps are the same as in Example 1, with all other culture conditions (temperature, light duration, etc.) remaining consistent: S1. Select rootless blueberry tissue culture seedlings of the same specifications as in Example 1. After trimming the base, soak them in 75% alcohol for 25 seconds, 5% sterile sodium hypochlorite solution for 10 minutes, and rinse them with sterile water 4 times to complete the disinfection.

[0035] S2, the induction solution uses 1 / 2 MS medium (the formula does not contain sucrose, the macroelements are 1 / 2 of MS medium, the microelements, vitamins, and inositol are the same as MS medium, agar 7g / L, pH 5.8-6.0), directly add 2.0mg / L indolebutyric acid, do not add melatonin, and do not use microencapsulation treatment; the base of the tissue culture seedlings is soaked at 25℃ for 1.8h.

[0036] S3, implanted with a common substrate of peat:perlite = 2:1 (without added modified magnetic biochar or nanocellulose, and without weak magnetic field regulation); single red and blue spectrum illumination (red light:blue light = 3:1, 4000 lux, 14h / d), without UV-A and far-red light; constant CO supply (1000ppm); humidity reduced from 92% to 60% in one step, without stepwise dehumidification or application of PEG-6000 solution; culture time 25 days (consistent with Example 1).

[0037] S4. After hardening off, remove the tissue culture seedlings and wash off the root substrate. Transplant them into the same transplanting substrate as in Example 1 and maintain them as usual.

[0038] Comparative Example 2: Tissue Culture Seedling Hardening Method for Deleting Magnetic Matrix (Single Deletion of the Core Feature of this Invention): A method for hardening off plant tissue culture seedlings differs from Example 1 only in that: in step S3, the composite substrate is replaced with an equal amount of peat moss to replace the modified magnetic biochar, thus not forming a weak rhizosphere magnetic field. The remaining steps and parameters are consistent with Example 1 (retaining technical features such as microencapsulated hormones, composite spectrum, gradient humidity, and PEG adjustment).

[0039] Effect verification: Parallel comparative experiments were conducted on Examples 1 to 3, Comparative Examples 1 and 2. Each group of experiments used 30 tissue culture seedlings, with three replicates, and the average value was taken. The experimental period was 30 days after transplanting. The test indicators included basal browning rate, average number of roots per plant, root length, transplant survival rate, and hardening-off period. Specific test results are shown in Table 1 below. Table 1. Performance test results of tissue culture seedlings from the examples and comparative examples. Effect verification analysis: 1. Base browning rate: The base browning rates of Examples 1-3 were all below 6%, significantly lower than Comparative Example 1 (28.5%) and Comparative Example 2 (8.7%), confirming that the synergistic effect of the microencapsulated rooting hormone and melatonin in this invention can effectively inhibit browning. Comparative Example 1, lacking encapsulation treatment and melatonin, experienced unstable hormone release and severe browning; Comparative Example 2, lacking a magnetic matrix, showed an increased browning rate. Supplementary verification: Without microencapsulation, the browning rate reached 22.5%, while encapsulation combined with melatonin reduced it to 1.2%. The core principle is that encapsulation controls hormone release, avoiding damage to the base from high-concentration hormones, achieving a synergistic physical-chemical browning inhibition, fully supporting the relevant features of the claims.

[0040] 2. Root growth indicators: In Examples 1-3, the average number of roots per plant was ≥11.8 and the average root length was ≥8.2cm, significantly better than Comparative Example 1 (8.7 roots, 5.3cm) and Comparative Example 2 (9.5 roots, 6.8cm). This indicates that the weak magnetic field formed by the composite matrix (loaded with Fe3O4 nanoparticles) (corresponding to the feature of the composite matrix in the claims) can induce root cell enzyme activity, regulate rhizosphere ion exchange, and promote root growth in conjunction with a stable supply of encapsulated hormones, fully supporting the relevant features in the claims. Supplementary verification: The rooting rate of ordinary biochar is only 72.4%, while a weak magnetic field of 5-15mT can improve the rooting rate and root vitality, confirming the core role of the magnetic matrix.

[0041] 3. Transplant survival rate: The transplant survival rate of Examples 1-3 was ≥93.7%, significantly higher than that of Comparative Example 1 (71.2%) and Comparative Example 2 (82.5%). This indicates that gradient humidity and osmotic pressure coupled acclimatization and in-situ integrated culture (corresponding to the gradient environment control and in-situ transplant characteristics in the claims) can improve stress resistance and reduce root damage. Comparative Example 1 had a low survival rate due to one-time dehumidification and root washing transplantation. Supplementary verification: Without UV-A and CO pulses, the transplant survival rate was only 70.2%, and the seedling recovery period was 12 days. UV-A induces leaf maturation, and combined with CO pulse carbon supply, the tissue culture seedlings achieve autotrophic switching through photosynthesis under sucrose-free conditions, improving the survival rate and shortening the seedling recovery period. Traditional root washing transplantation resulted in a browning rate of 35.0%, a survival rate of 62.5%, and a seedling recovery period of 15 days, fully demonstrating the advantages of in-situ culture of this invention and supporting the relevant features of the claims.

[0042] 4. Hardening-off period: The hardening-off period in Examples 1-3 was 24-26 days, which was 22%-30% shorter than that in Comparative Example 1 (34 days) and 10%-17% shorter than that in Comparative Example 2 (29 days). This indicates that the synergistic effect of the composite spectrum and CO pulse (corresponding to the autotrophic switching period regulation feature in the claim) can activate the photosynthetic system, enabling tissue culture seedlings to quickly switch from heterotrophic to autotrophic under sucrose-free conditions, thus accelerating the hardening-off process and supporting the relevant features of the claim. In summary, this invention achieves synergistic effects through microencapsulated rooting hormones and melatonin synergistic controlled release, a composite matrix loaded with magnetic nanoparticles regulating the rhizosphere microenvironment, composite spectral and CO pulse coupling promoting autotrophic switching, gradient humidity and osmotic pressure synergistic acclimatization, and in-situ integrated culture. These technical features work synergistically: microencapsulation and melatonin chemically inhibit browning, weak magnetic field physically promotes root growth, UV-A and CO pulse light-gas synergistically promote autotrophic growth (ensuring nutrient supply under sucrose-free conditions), and in-situ culture reduces root damage. This effectively solves the defects of existing technologies such as unstable hormone release, basal browning, single substrate, long hardening-off period, and low survival rate. All technical features correspond one-to-one with claims 1-11. Examples 1-3 and Comparative Examples 1-2 are designed around the core features of the claims, clearly demonstrating the synergistic effect and inventiveness, fully supporting the inventiveness and feasibility of the claims. Compared with existing technologies, this invention has outstanding substantive features and significant progress, and can be widely applied to the rooting and hardening of tissue culture seedlings of woody and herbaceous plants, especially suitable for easily browning varieties such as Polygonatum multiflorum, and is suitable for large-scale production.

[0043] The above description of the disclosed embodiments enables those skilled in the art to make or use this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A highly efficient rooting and hardening method for plant tissue culture seedlings, characterized in that, Includes the following steps: S1, select tissue culture rootless seedlings, and perform base trimming and disinfection treatment; S2, Immerse the tissue culture seedling stem segments treated in S1 in an induction solution, which includes basal culture medium, melatonin and rooting hormone; S3, the tissue culture seedlings induced by S2 are implanted into a culture container filled with composite substrate, and gradient environmental regulation is carried out in the same culture container in sequence during the rooting induction period, autotrophic switching period and in-situ hardening adaptation period. The composite matrix comprises modified magnetic biochar loaded with magnetic nanoparticles, nanocellulose, and perlite. During the autotrophic switching period, illumination is regulated by a composite spectrum containing the UV-A band, and carbon dioxide is periodically supplied to the culture container in a pulsed manner. During the in-situ hardening-off period, the humidity of the culture environment was gradually reduced, and osmotic pressure was adjusted by applying a polyethylene glycol solution to the composite substrate. S4 involves directly transplanting and planting the tissue culture seedlings that have completed S3 culture, along with the substrate.

2. The method for efficient rooting and hardening of plant tissue culture seedlings according to claim 1, characterized in that, In step S1, the disinfection process involves sequentially soaking in sterile disinfectant and rinsing with sterile water; the base trimming process involves removing aged and damaged tissue from the base of the rootless tissue culture seedlings.

3. The method for efficient rooting and hardening of plant tissue culture seedlings according to claim 1, characterized in that, In step S2, the induction solution is used to soak the base of the stem segment of the tissue culture seedling, and the temperature of the induction solution is kept constant at 22-28℃ during the soaking process.

4. The method for efficient rooting and hardening of plant tissue culture seedlings according to claim 1, characterized in that, In step S2, the rooting hormone is suspended in the induction solution in the form of microcapsules; the wall material of the microcapsules includes at least one of sodium alginate, gelatin or chitosan, and the core material is indolebutyric acid (IBA) and / or naphthaleneacetic acid (NAA).

5. The method for efficient rooting and hardening of plant tissue culture seedlings according to claim 1, characterized in that, In step S2, the concentration of melatonin in the induction solution is 0.5-0.8 mg / L; the total mass concentration of indolebutyric acid (IBA) and / or naphthaleneacetic acid (NAA) in the microcapsule core material is 1.5-2.5 mg / L.

6. The method for efficient rooting and hardening of plant tissue culture seedlings according to claim 1, characterized in that, The modified magnetic biochar is loaded with Fe3O4 magnetic nanoparticles, which form a stable weak magnetic field of 5-15 mT in the rhizosphere microenvironment.

7. The method for efficient rooting and hardening of plant tissue culture seedlings according to claim 1, characterized in that, In step S3, the mass ratio of each component of the composite matrix is: 40-70 parts of modified magnetic biochar, 10-25 parts of nanocellulose, and 15-30 parts of perlite.

8. The method for efficient rooting and hardening of plant tissue culture seedlings according to claim 1, characterized in that, The composite spectrum containing the UV-A band includes red light, blue light, far-red light, and UV-A light, and the wavelength of the UV-A band is 365-385nm.

9. The method for efficient rooting and hardening of plant tissue culture seedlings according to claim 1, characterized in that, The concentration of the carbon dioxide pulse supply is 800-1200 ppm, supplied 1-3 times a day, each lasting 1-2 hours.

10. A method for efficient rooting and hardening of plant tissue culture seedlings according to claim 1, characterized in that, The polyethylene glycol solution applied during the in-situ hardening-off period is a PEG-6000 solution with a mass concentration of 50-100 mg / L.

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