A method for cultivating rootless and leafless ginger tissue culture seedlings

Abstract

The application provides a cultivation method of rootless and leafless ginger tissue culture seedlings, relates to the field of seed seedling cultivation, and comprises the following steps: material preparation, mixed substrate preparation and tissue culture seedling planting, wherein the tissue culture seedling planting is specifically as follows: the rootless and leafless tissue culture seedlings are vertically implanted into the planting holes of the mixed substrate, the root neck part is completely buried into the substrate hole, the stem section is vertically arranged, the substrate around the seedlings is gently compacted, and the seedlings are fixed without lodging; finally, four stage cultivation is sequentially carried out according to time, and the transplanting tissue culture seedlings are obtained. The method is transplanting the rootless and leafless tissue culture seedlings and reconstructing the root system, so that the ginger plant groups with consistent tillering and balanced structure are cultivated, and the adaptability and survival rate after transplanting are improved.

Description

Technical Field

[0001] This invention relates to the field of seed and seedling cultivation technology, specifically to a method for cultivating rootless and leafless ginger tissue culture seedlings. Background Technology

[0002] In recent years, ginger detoxification technology has become increasingly mature and improved, and ginger tissue culture seedlings have been gradually applied to production. However, compared with the traditional tuber propagation method, ginger tissue culture seedlings still have the disadvantage of a more complex domestication and transplanting process.

[0003] Specifically, the problems are as follows: 1. Weak physiological foundation and poor adaptability: The root system of ginger tissue culture seedlings is tender and has weak regeneration ability. Most of them are adventitious roots, and their ability to absorb water and nutrients is not perfect. They are prone to wilting after transplanting due to water loss. At the same time, the guard cells of the stomata of ginger tissue culture seedlings have low potassium content and cannot synthesize enough nutrients on their own, relying on external supply, resulting in slow growth. In addition, tissue culture seedlings are susceptible to infection and death in the early stage and are extremely sensitive to environmental fluctuations (temperature, humidity, light). 2. The physiological state of tissue culture seedlings induced in bottles differs from that of plants in the field: The bottle provides a stable environment with constant temperature and humidity, sterility, and low light. After transplanting, they need to adapt to the fluctuations in temperature and humidity, microbial competition, and changes in light intensity in the field, making physiological regulation difficult. 3. Complex operation process and high cost: It requires many steps, such as opening the bottle to harden the seedlings, cleaning the culture medium, disinfection, transplanting, and seedling management. These steps are numerous, rely on manual labor, and have a long acclimatization period, resulting in a long cultivation cycle and high human influence. 4. Limited survival rate and growth performance: The overall survival rate is usually 60% to 80%, which is much lower than that of traditional divided seedlings. Furthermore, the survival rate of seedlings is slow, the early growth is weak, and the growth period is delayed, which affects the enlargement of ginger rhizomes and the final yield. After transplanting, problems such as trait separation and quality decline are also likely to occur.

[0004] Furthermore, tissue-cultured ginger seedlings present a contradiction between root development and yield: while the root system of tissue-cultured ginger seedlings can be 2.21 times that of traditionally propagated ginger rhizomes, the imbalance in endogenous hormone ratios (e.g., an abnormally high IAA / ZR ratio) inhibits the normal expansion of underground ginger rhizomes, resulting in "exceptionally developed roots, but small rhizomes and low yield." Moreover, tissue-cultured seedlings face severe environmental stress when transplanted to soil, leading to a long recovery period and unstable survival rates, further increasing the production risks and costs associated with tissue-cultured seedlings. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a cultivation method for rootless and leafless ginger tissue culture seedlings. This method involves transplanting rootless and leafless tissue culture seedlings and rebuilding the root system to cultivate a ginger plant population with uniform tillering and balanced structure, thereby improving the adaptability and survival rate after transplanting.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for cultivating rootless and leafless ginger tissue culture seedlings includes:

[0008] Step S1, Material Preparation: Remove all original roots and callus tissue at the base of the ginger virus-free tissue culture seedlings, leaving only the root neck connecting the above-ground part and the root system; at the same time, cut off the excessively long stems and all leaves of the above-ground part, leaving only a 2-3cm healthy stem segment, with a clean cut without tearing.

[0009] Step S2, Mixed matrix preparation: Prepare the mixed matrix in advance, and after non-destructive sterilization, put the mixed matrix into a container to make the surface of the mixed matrix smooth;

[0010] Step S3, Transplanting of Tissue Culture Seedlings: Select a seedling tray with drainage holes at the bottom and a matching tray as the transplanting container. Evenly fill each hole of the seedling tray with the mixed substrate, filling it to a height of 1cm above the top edge of the hole. Gently scrape it level and lightly compact it. Then, vertically punch a hole in the center of the substrate in each hole, with a depth of 0.5-1cm and a diameter matching the stem segment of the tissue culture seedling. Next, vertically insert the rootless and leafless tissue culture seedlings obtained in Step S1 into the hole, ensuring that the root neck is completely buried in the substrate hole and the stem segment is upright. Gently compact the substrate around the seedling to fix it and prevent it from falling over. Finally, carry out the four stages of cultivation in sequence according to the time, and the transplanted tissue culture seedlings are obtained.

[0011] Based on further optimization of the above scheme, in step S1, the ginger virus-free tissue culture seedlings are selected from those that have grown for 30-45 days, are 5-8cm tall, have 3-5 fully expanded leaves, have thick stems, are vigorous, and are free from vitrification and obvious pests and diseases. After the ginger virus-free tissue culture seedlings are taken out of the culture bottle, they are first rinsed with sterile water 3-4 times to wash away the culture medium attached to the roots. At the same time, they are handled gently throughout the process to avoid damaging the stems, leaves and root neck.

[0012] Based on further optimization of the above scheme, in step S2, the mixed matrix includes hydrophobically modified expanded perlite microspheres (30-50 mesh), aminated silica aerogel microparticles (100-200 mesh), bacterial humic acid-grafted vermiculite composite carrier (200 mesh), and enzymatically hydrolyzed peat fiber (0.5-1 mm short fiber), with their mass percentages being 29.5-30.5%, 8.5-9.5%, 37.5-38.5%, and 22.5-23.5%, respectively.

[0013] Based on further optimization of the above scheme, the preparation method of the hydrophobically modified expanded perlite microspheres is as follows: 30-50 mesh perlite is washed, dried, and then immersed in a 1.8-2.2% KH570-anhydrous ethanol solution. The mass-to-volume ratio of perlite to solution is 0.9-1.1 g: 4.5-5.5 mL. The mixture is first shaken at 38-42℃ and 120-150 rpm for 3.8-4.2 h, then filtered, washed, and dried at 102-108℃ for 2-3 h to obtain the microspheres.

[0014] Based on further optimization of the above scheme, the preparation method of the aminated silica aerogel particles is as follows: 100-200 mesh silica aerogel is vacuum dried and then immersed in a 2.8-3.2% KH550-anhydrous ethanol solution, with a mass-to-volume ratio of silica aerogel to solution of 0.9-1.1 g: 3.6-4.4 mL. The mixture is first shaken at 100-130 rpm for 5.8-6.2 h under light-protected conditions at 48-52℃. After filtration and washing, it is vacuum dried at 58-62℃ and -0.08--0.09 MPa for 4-6 h to obtain the final product.

[0015] Based on further optimization of the above scheme, the preparation method of the bacterial humic acid-grafted vermiculite composite carrier is as follows: 200-mesh vermiculite is added to a 1 mol / L hydrochloric acid solution, with a mass-to-volume ratio of vermiculite to hydrochloric acid solution of 0.9–1.1 g: 4.5–5.5 mL. The mixture is first stirred at 78–82℃ and 140–160 rpm for 3.8–4.2 h, then filtered and washed until neutral, and dried at 102–108℃ for 2–3 h. Afterward, the activated vermiculite and humic acid are mixed at a mass ratio of 4.75–5.2. Mix 5:1.9–2.1, and add a mixed crosslinking agent of EDC and NHS at a molar ratio of 1:1. The mixed crosslinking agent accounts for 0.48–0.52% of the total mass of vermiculite and humic acid. Stir at 180–220 rpm for 5.8–6.2 h in a constant temperature water bath at 48–52 °C. After filtration and washing, vacuum dry at 58–62 °C and -0.08–-0.09 MPa for 3–5 h to obtain the carrier. Finally, place the sterilized carrier into a sterile vacuum adsorption bottle and add bacterial solution with a concentration of 1×10⁻⁶. 9 A CFU / mL suspension of *Candida langiocarpa* was prepared with a carrier-to-suspension solid-liquid ratio of 0.9–1.1 g: 2.7–3.3 mL. The suspension was maintained under a vacuum of -0.08–0.09 MPa for 15–20 min, followed by slow introduction of sterile air. The bacterial suspension permeated into the pores of the carrier under the pressure difference. This process was maintained for 10–12 min, and the suspension was then air-dried at 27–29 °C until the moisture content reached 30%.

[0016] Based on further optimization of the above scheme, the specific preparation method of the enzymatically hydrolyzed peat fiber is as follows: Peat is pulverized and added to a buffer solution composed of a pH 4.8 acetic acid solution and a sodium acetate solution, with a peat to buffer solution mass-to-volume ratio of 0.9–1.1 g: 9–11 mL. The mixture is stirred at 180–220 rpm to form a peat suspension. Then, cellulase is added to the peat suspension at an amount of 1.8–2.2% of the peat mass, and the mixture is stirred at 48–52°C and 140–160 rpm for 3.8–4.2 h. Afterward, the enzymatically hydrolyzed suspension is placed in a constant-temperature wastewater bath at 98–102°C and kept at that temperature for 8–10 min. The peat fiber is repeatedly washed with deionized water until the pH of the filtrate stabilizes at 5.5–6.0. ​​Finally, the fiber filter cake is collected by filtration, air-dried to semi-dry, sieved, and graded. The graded fibers are then dried at 58–62°C to constant weight to obtain the final product.

[0017] In the mixed substrate, silica aerogel with amino-active groups on its surface is used to electrostatically adsorb cations (such as potassium ions, magnesium ions, and trace element ions) in the nutrient solution, achieving slow release of mineral nutrients and preventing rapid nutrient loss. Hydrophobically modified perlite prevents the substrate from hardening after absorbing water, maintaining the permeability of its large pores. Simultaneously, it forms a hydrophobic-hydrophilic balance with hydrophilic vermiculite and peat fiber, regulating the substrate's water-holding capacity. Furthermore, the combination of perlite and silica aerogel provides stable physical support for seedlings, preventing rootless seedlings from lodging. The use of a microbially grafted vermiculite composite carrier with humic acid and enzymatically hydrolyzed peat fiber jointly regulates and stabilizes the substrate's pH value, buffering pH fluctuations in the nutrient solution and ensuring a suitable pH environment (5.5–6.5) for tissue culture seedlings. Moreover, the increased surface porosity of vermiculite after hydrochloric acid activation, combined with humic acid grafting modification, further enhances its adsorption capacity for organic substances such as amino acids and hormones, achieving slow release of organic nutrients.

[0018] Based on further optimization of the above scheme, step S3 includes four stages: root primordia differentiation and cultivation stage, adventitious root construction and cultivation stage, root system stress-resistant tillering cultivation stage, and robust seedling cultivation stage. The root primordia differentiation and cultivation stage lasts for 1 to 7 days after transplanting, the adventitious root construction and cultivation stage lasts for 8 to 20 days after transplanting, the root system stress-resistant tillering cultivation stage lasts for 21 to 35 days after transplanting, and the robust seedling cultivation stage lasts for 36 to 40 days after transplanting.

[0019] Based on further optimization of the above scheme, the root primordia differentiation cultivation stage is carried out in an environment with a temperature of 24-26℃, an air humidity of not less than 80%RH, a light intensity of 2400-2600Lux, and a light duration of 12h / d. Root primordia differentiation nutrient solution is used for irrigation: the first irrigation after transplanting involves thoroughly watering the substrate with the root primordia differentiation nutrient solution and then emptying any excess liquid from the tray; when the humidity of the mixed substrate is lower than 55%RH, supplementary irrigation is carried out by evenly spraying the nutrient solution along the substrate surface using a fine-nozzle sprayer, directly pouring the nutrient solution into the substrate;

[0020] The root primordium differentiation nutrient solution includes 1 / 10 MS medium, pH-sensitive chitosan-coated amino acid nitrogen, N-(1-naphthyl)-o-carbamoylbenzoic acid, 24-epibrassinolide, 1,4-butanediamine, tea tree oil nanoemulsion, urea peroxide, γ-polyglutamic acid (molecular weight 100,000–700,000 Da), and potassium humate; among which, pH-sensitive chitosan-coated amino acid nitrogen, N-(1-naphthyl)-o-carbamoylbenzoic acid, 24-epibrassinolide, 1,4-butanediamine, tea tree oil nanoemulsion, urea peroxide, γ-polyglutamic acid (molecular weight 100,000–700,000 Da), and potassium humate; The concentrations of 4-butanediamine, tea tree oil nanoemulsion, urea peroxide, γ-polyglutamic acid, and potassium humate were 0.78–0.82 g / L, 0.09–0.11 μmol / L, 0.18–0.22 μmol / L, 0.9–1.1 μmol / L, 0.09–0.11 g / L, 4.8–5.2 mg / L, 0.28–0.32 g / L, and 0.18–0.22 g / L, respectively.

[0021] During the root primordium differentiation and cultivation stage, N-(1-naphthyl)-o-carbamoylbenzoic acid is used to promote auxin accumulation, 24-epibrassinolide is used to regulate endogenous hormones and promote cell division, and 1,4-butanediamine is used to promote root cell differentiation. The three work synergistically to increase auxin and cytokinin levels at the root collar, thereby precisely inducing root primordium formation and solving the problem of rootless seedlings lacking meristems. Tea tree oil nanoemulsion is used for broad-spectrum antibacterial activity to prevent root collar infection, and pH-sensitive chitosan-coated amino acid nitrogen possesses antibacterial properties, thus achieving dual antibacterial effects and constructing a... The sterile root primordium differentiation environment, coupled with the slow release of amino acid nitrogen from the pH-sensitive chitosan-coated rhizosphere microenvironment (supplying root primordium differentiation), works synergistically with γ-polyglutamic acid (for water and fertilizer retention, adsorption of mineral nutrients and hormones) and potassium humate (for regulating rhizosphere pH and improving nutrient absorption efficiency) to achieve a slow and continuous supply of nutrients, effectively matching the slow differentiation rhythm of root primordium. The slow release of oxygen by urea peroxide improves the aeration environment at the root collar, and in conjunction with the macroporous permeability of the substrate, further enhances the differentiation efficiency of root primordium.

[0022] Based on further optimization of the above scheme, the specific preparation method of the pH-sensitive chitosan-coated amino acid nitrogen is as follows: First, chitosan is added to a 2.5% aqueous solution of glacial acetic acid, with a mass-to-volume ratio of chitosan to glacial acetic acid aqueous solution of 3g:80mL. The solution is stirred at 280-320rpm at room temperature until dissolved to obtain a transparent solution. Then, the solution is ultrasonically treated at a power of 190-210W and a frequency of 18-22kHz for 14-16min. Finally, the pH is adjusted to 4.6-4 using a 0.1mol / L NaOH solution. 8; Then, the complex amino acids, EDC, and NHS were added to deionized water in a mass-to-volume ratio of 8 g: 3.5 g: 2.1 g: 200 mL. The mixture was stirred at 480–520 rpm for 28–32 min at room temperature to obtain an activated amino acid solution. The complex amino acid solution consisted of L-lysine hydrochloride, L-arginine, and L-glutamic acid in a mass ratio of 7:2:1. Subsequently, under light-protected conditions at room temperature, the activated amino acid solution was added dropwise to the chitosan solution at a rate of 1 drop / s. The mixture was stirred at 780–820 rpm for 0.8–1.2 h, then at 180–220 rpm for 11.5–12.5 h. Sodium tripolyphosphate was then added, with a mass ratio of sodium tripolyphosphate to complex amino acids of 0.3:2. The mixture was stirred continuously at 180–220 rpm for 1.8–2.2 h to obtain the coupling reaction solution. The coupling reaction solution was then diluted with deionized water to a solid content of 10% (w / v), and sonicated for 8–12 min to ensure homogeneity. Spray drying was performed, the dried powder was collected, and vacuum dried at 58–62℃ and -0.08–-0.09 MPa for 3.8–4.2 h to obtain pH-sensitive chitosan-amino acid composite powder. Finally, the spray-dried powder was soaked in 95% ethanol aqueous solution for 2–2.5 h, then the pH was adjusted to 5.9–6.1 with 0.1 mol / L NaOH, and vacuum dried at 58–62℃ and -0.08–-0.09 MPa to constant weight to obtain the final product.

[0023] Based on further optimization of the above scheme, the adventitious root construction and cultivation stage is carried out in an environment with a temperature of 24-26℃, an air humidity of 75%-80%RH, a light intensity of 3400-3600Lux, and a light duration of 14h / d. Adventitious root construction nutrient solution is used for irrigation: the first irrigation is performed on the 8th day after transplanting, thoroughly soaking the substrate with the nutrient solution, and then emptying any excess liquid from the tray; thereafter, supplementary irrigation is performed every 5 days, with each irrigation amount being 60% of the substrate's water holding capacity.

[0024] The nutrient solution for adventitious root construction included 1 / 10 MS medium, N6-isopentenyl adenosine, L-tryptophan, strigolactone analogue, sodium borate, zinc sulfate, salicin, humic acid chelated trace elements, γ-polyglutamic acid, chitosan oligosaccharide, and Bacillus subtilis; wherein the concentrations of N6-isopentenyl adenosine, L-tryptophan, strigolactone analogue, sodium borate, zinc sulfate, salicin, humic acid chelated trace elements, γ-polyglutamic acid, and chitosan oligosaccharide were 0.5%. 8–0.62 μmol / L, 0.08–0.12 μmol / L, 0.28–0.32 μmol / L, 1.9–2.1 μmol / L, 0.08–0.12 μmol / L, 0.48–0.52 μmol / L, 0.95–1.05 mL / L, 0.28–0.32 g / L, 0.33–0.37 g / L; the concentration of Bacillus subtilis was 2 × 10⁻⁶. 8 CFU / mL.

[0025] During the adventitious root formation and cultivation stage, N6 - Isoprene adenosine promotes adventitious root cell division, L-tryptophan promotes adventitious root elongation, and strigolactone analogues regulate root architecture and promote lateral root development. These three synergistically regulate the IAA / ZR ratio to achieve adventitious root elongation and lateral root germination, thereby constructing a balanced root architecture and preventing excessive root development. Simultaneously, sodium borate and zinc sulfate promote root vascular bundle formation and enhance root activity. Humic acid chelates complex trace elements for a stable trace element supply and prevent ion precipitation. γ-polyglutamic acid adsorbs trace elements and improves absorption efficiency. These three synergistically solve the problem of easy precipitation of ordinary trace elements and ensure a continuous supply of trace elements required for adventitious root growth. In addition, salicin protects young adventitious roots and enhances stress resistance, chitosan oligosaccharides promote root growth and induce root stress resistance, and Bacillus subtilis inhibits bacteria and secretes root-promoting substances. These three synergistically construct a stress-resistant protection system for young roots, preventing adventitious roots from being damaged by pathogens or environmental fluctuations.

[0026] Based on further optimization of the above scheme, the root-resistant tillering cultivation stage is carried out in an environment with a daytime temperature of 25-27℃ and a diurnal temperature range of 4℃, an air humidity of 60%-70%RH, a light intensity of 3900-4100Lux, and a light duration of 16h / d. Root-resistant tillering nutrient solution is used for irrigation: the first irrigation is performed on the 21st day after transplanting, thoroughly irrigating the substrate with the root-resistant tillering nutrient solution, and then emptying any excess liquid from the tray; thereafter, supplementary irrigation is performed every 4 days, with each irrigation amount being 50% of the substrate's water holding capacity.

[0027] The root stress-resistant tillering nutrient solution includes 1 / 10 MS medium, methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex, 6-benzyl adenosine, potassium dihydrogen phosphate, nano silica (30-50 nm) solution, γ-polyglutamic acid, and potassium humate; wherein the concentrations of methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex, 6-benzyl adenosine, potassium dihydrogen phosphate, nano silica, γ-polyglutamic acid, and potassium humate are 0.48-0.52 μmol / L, 1.18-1.22 μmol / L, 0.78-0.82 g / L, 0.28-0.32 g / L, 0.18-0.22 g / L, and 0.15-0.2 g / L, respectively.

[0028] During the root system stress-resistance and tillering cultivation stage, methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex is used to induce the expression of root stress-resistance genes. Combined with potassium humate, it gradually enhances root stress resistance while simultaneously inducing systemic stress resistance in the plant. 6-Benzyladenosine promotes lateral bud germination and synchronizes tillering. Combined with the tillering regulation effect of methyl jasmonate, it regulates apical dominance and lateral bud germination, avoiding uneven tillering in tissue culture seedlings. Potassium dihydrogen phosphate, combined with nano-silica, achieves dual enhancement of root physiological and physical stress resistance, providing a robust root foundation for transplanting. Furthermore, potassium dihydrogen phosphate, combined with γ-polyglutamic acid, prevents the precipitation of phosphorus and potassium ions by binding with cations in the matrix, ensuring an effective supply of phosphorus and potassium and providing sufficient nutrition for root lignification and tillering.

[0029] Based on further optimization of the above scheme, the specific preparation method of the methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex is as follows: First, 0.1 mmol of methyl jasmonate is dissolved in 1 mL of anhydrous ethanol to obtain a stock solution; then, 1 mmol of hydroxypropyl-β-cyclodextrin is dissolved in 80 mL of sterile water and stirred at 280–320 rpm at room temperature until completely dissolved to obtain a mother liquor; then, under a constant temperature water bath environment of 38–42℃, the stock solution is added dropwise to the mother liquor at a rate of 1–2 mL / min while stirring at 380–420 rpm. After the addition is completed, the mixture is stirred for 3.8–4.2 h, and then sonicated at room temperature for 14–16 min; finally, the mixture is filtered through a 0.22 μm filter membrane to obtain a 1 mmol / L methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex mother liquor.

[0030] Based on further optimization of the above scheme, the seedling cultivation stage is carried out in an environment with a daytime temperature of 26-30℃ and a diurnal temperature range of 8℃, an air humidity of 60%-70%RH, a light intensity of 4900-5100Lux, and a light duration of 14h / d. Seedling nutrient solution is used for irrigation: the first irrigation is performed on the 36th day after transplanting, using the seedling nutrient solution to moisten the substrate but not to saturate it; thereafter, supplementary irrigation is performed every 3 days, with each irrigation amount being 18-22% of the substrate's water holding capacity.

[0031] The seedling nutrient solution includes 1 / 10 MS medium, trehalose, potassium dihydrogen phosphate, potassium nitrate, betaine, L-proline, potassium humate, Bacillus subtilis, and Bacillus megaterium. The concentrations of trehalose, potassium dihydrogen phosphate, potassium nitrate, *Glomus mosy* spore suspension, betaine, L-proline, and potassium humate are 1.9–2.1 g / L, 0.9–1.1 g / L, 0.48–0.52 g / L, 0.08–0.12 g / L, 0.08–0.12 g / L, and 0.28–0.32 g / L, respectively. The concentration of Bacillus subtilis is 2.5 × 10⁻⁶ g / L. 7 The internal activity of Bacillus megaterium is 2.5 × 10⁻⁶ CFU / mL. 7 CFU / mL.

[0032] During the seedling cultivation stage, the synergistic effect of trehalose, betaine, and L-proline enhances the plant's osmotic regulation capacity, enabling the plant to adapt to environmental stresses such as temperature and humidity fluctuations and drought in the field in advance, achieving pre-adaptation to the field without additional hardening-off. The combination of potassium dihydrogen phosphate, potassium nitrate, and potassium humate provides sufficient phosphorus, potassium, and nitrogen supply, promotes the synthesis and accumulation of photosynthetic products, and achieves strong seedlings, solving the problem of weak growth in the early stage of tissue culture seedlings. The addition of compound microbial agents (i.e., Bacillus subtilis and Bacillus megaterium) achieves the synergistic function of promoting root resistance and efficient phosphorus solubilization, avoiding excessive distribution of photosynthetic products to the root system, and solving the yield contradiction of "well-developed root system but small ginger rhizomes".

[0033] The following are the technical effects of this invention:

[0034] This invention addresses the root causes of problems in ginger tissue culture seedling cultivation and transplantation, such as weak physiological foundation, poor adaptability, differences in physiological state between in-bottle induced tissue culture seedlings and field plants, complex operation procedures, high costs, and limited survival rate and growth performance. It also resolves the contradiction between root development and yield, thereby achieving high survival rate, uniform tillering, no need for hardening-off seedlings, low cost, and excellent yield.

[0035] This invention constructs a balanced and robust adventitious root system by removing the original root system with imbalanced endogenous hormones and precisely regulating endogenous hormones using a four-stage nutrient solution. This solves problems such as imperfect root absorption function and wilting due to water loss. A dual antibacterial system of mixed substrate and nutrient solution prevents pathogen infection in the early stages of tissue culture seedlings and alleviates environmental stress. Hormone balance regulation achieves a balanced, rather than overdeveloped, root structure, while preventing excessive translocation of photosynthetic products to the roots, thus solving the problem of "well-developed roots but small ginger rhizomes and low yield" in tissue culture seedlings. Furthermore, the environmental parameters of the four-stage cultivation process are adjusted progressively, and the regulation of the nutrient solution enables a synchronous transition between endogenous hormones and the rhizosphere microenvironment. This avoids the problem of large differences in physiological state between the tissue culture bottle and the field, and the difficulty of physiological regulation, significantly improving the adaptability of plants after transplanting and ensuring the survival rate of transplanted plants. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of rootless and leafless ginger tissue culture seedlings being transplanted into a mixed substrate in an embodiment of the present invention.

[0037] Figure 2 This is a schematic diagram of the roots of rootless and leafless ginger tissue culture seedlings during the root system stress-resistant tillering cultivation stage (approximately 30 days) in an embodiment of the present invention. Detailed Implementation

[0038] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0039] Example 1:

[0040] A method for cultivating rootless and leafless ginger tissue culture seedlings includes:

[0041] Step S1, Material Preparation: Select ginger virus-free tissue culture seedlings that have grown for 37 days, are 6.5cm tall, have 4 fully expanded leaves, have thick stems, are vigorous, and are free from vitrification and obvious pests and diseases. After removing the ginger virus-free tissue culture seedlings from the culture bottle, rinse them 3 times with sterile water to wash away the culture medium attached to the roots. Handle them gently throughout the process to avoid damaging the stems, leaves, and root neck.

[0042] Remove all original roots and callus tissue from the ginger tissue culture seedlings, leaving only the root neck connecting the above-ground part and the root system; at the same time, cut off the excessively long stems and all leaves from the above-ground part, leaving only a 2.5cm healthy stem segment with a clean cut and no tears (after completion, place the rootless individual plant on sterile damp gauze to keep it moist and prevent it from wilting due to water loss).

[0043] Step S2, Mixed Matrix Preparation: Prepare the mixed matrix in advance, and after non-destructive sterilization, put the mixed matrix into a container to make the surface of the mixed matrix smooth.

[0044] The mixed matrix includes hydrophobically modified expanded perlite microspheres (30-50 mesh), aminated silica aerogel microparticles (100-200 mesh), bacterial humic acid-grafted vermiculite composite carrier (200 mesh), and enzymatically hydrolyzed peat fiber (0.5-1 mm short fiber), with their mass percentages being 29.5%, 9.5%, 37.5%, and 23.5%, respectively. The specific preparation method of hydrophobically modified expanded perlite microspheres is as follows: 30-50 mesh perlite is washed and dried (temperature 102℃, drying time 2h), then immersed in a 1.8% concentration KH570 (3-(methacryloyloxy)propyltrimethoxysilane, CAS: 2530-85-0)-anhydrous ethanol solution, with a perlite to solution mass-volume ratio of 0.9g:4.5mL. The mixture is first shaken at 38℃ and 120rpm (rotary shaker) for 4.2h, then filtered, washed, and dried at 102℃ for 3h to obtain the microspheres. The specific preparation method of aminated silica aerogel particles is as follows: 100-200 mesh silica aerogel is vacuum dried (temperature 118℃, vacuum degree -0.08MPa, drying time 2.2h), and then immersed in a 2.8% KH550 (3-aminopropyltriethoxysilane, CAS: 919-30-2)-anhydrous ethanol solution. The mass-volume ratio of silica aerogel to solution is 0.9g:3.6mL. The mixture is first shaken at 100rpm (rotary shaker) for 6.2h under light-protected conditions at 48℃, then filtered and washed, and finally vacuum dried at 58℃ and -0.08MPa for 6h to obtain the desired product. The preparation method of the humic acid-grafted vermiculite composite carrier is as follows: 200-mesh vermiculite is added to a 1 mol / L hydrochloric acid solution, with a mass-to-volume ratio of vermiculite to hydrochloric acid solution of 0.9 g: 4.5 mL. The mixture is first stirred at 78℃ and 140 rpm for 4.2 h, then filtered and washed until neutral (pH 6.5), and dried at 102℃ for 3 h. Afterward, the activated vermiculite is mixed with humic acid at a mass ratio of 4.75: 1.9, and EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide salt) is added. A mixed crosslinking agent of vermiculite (CAS: 25952-53-8) and NHS (N-hydroxysuccinimide, CAS: 6066-82-6), with a molar ratio of EDS to NHS of 1:1, and the mixed crosslinking agent accounting for 0.48% of the total mass of vermiculite and humic acid, was stirred at 180 rpm for 6.2 h in a constant temperature water bath at 48 °C. After filtration and washing, the mixture was vacuum dried at 58 °C and -0.08 MPa for 5 h to obtain the carrier. Finally, the sterilized carrier was placed in a sterile vacuum adsorption bottle, and a bacterial solution concentration of 1×10⁻⁶ was added. 9A CFU / mL suspension of *Candida langiocarpa* (purchased from the China General Microbiological Culture Collection Center, accession number: CGMCC 2.1763), with a carrier-to-suspension solid-liquid ratio of 0.9 g: 2.7 mL, was maintained under a vacuum of -0.08 MPa for 20 min, followed by slow introduction of sterile air. The bacterial suspension permeated into the carrier pores under the pressure difference, and this process was maintained for 12 min. After removal, the suspension was air-dried at 27°C until the moisture content reached 30%. The preparation method for enzymatically hydrolyzed peat fiber is as follows: Peat is pulverized and added to a buffer solution composed of a pH 4.8 acetic acid solution and a sodium acetate solution, with a peat-to-buffer volume ratio of 0.9 g: 9 mL. The mixture is stirred at 180 rpm to form a peat suspension. Then, cellulase (enzyme activity not less than 1000 U / g) is added to the peat suspension at an amount of 1.8% of the peat mass, and the mixture is then heated at 48°C. Stir at 140 rpm for 4.2 hours; then, place the enzymatically hydrolyzed suspension in a 98°C constant temperature wastewater bath and keep it at that temperature for 10 minutes. Wash the peat fibers repeatedly with deionized water until the pH of the filtrate stabilizes at 5.5. Finally, filter and collect the fiber filter cake and air-dry it to semi-dry (moisture content of about 40%). Then, sieve and classify the fibers (first pass them through a 1 mm standard sieve to remove long fibers and impurities; then use a 0.5 mm standard sieve to remove fine powder and collect short fibers of 0.5–1 m). Dry the classified fibers at 58°C to constant weight (moisture content not greater than 10%) to obtain the final product.

[0045] The specific preparation method of the mixed matrix is ​​as follows: The four components are poured into a horizontal mixer according to the mass ratio and dry-mixed at 30 rpm for 16 minutes to obtain the mixed matrix; then, the mixed matrix is ​​subjected to a 500 mg / m³... 3 The gaseous sterilization process involves chlorine dioxide, followed by a 6-hour sealed sterilization at room temperature and a 2-hour ventilation period to remove any residue.

[0046] Step S3, Transplanting of Tissue Culture Seedlings: Select a seedling tray with water absorption holes at the bottom and a matching tray as the transplanting container. Evenly fill each hole of the seedling tray with the mixed substrate, filling it to a height of 1cm above the top edge of the hole. Gently scrape it flat and lightly compact it. Then, vertically punch a hole in the center of the substrate in each hole, with a depth of 0.7cm and a diameter matching the stem segment of the tissue culture seedling. After that, vertically insert the rootless and leafless tissue culture seedlings obtained in Step S1 into the holes, ensuring that the root neck is completely buried in the substrate hole and the stem segment is upright. Gently compact the substrate around the seedling to fix the seedling and prevent it from falling over.

[0047] Finally, the cultivation is carried out in four stages according to time: root primordia differentiation cultivation stage, adventitious root construction cultivation stage, root system stress-resistant tillering cultivation stage, and strong seedling cultivation stage. The root primordia differentiation cultivation stage is 1 to 7 days after transplanting, the adventitious root construction cultivation stage is 8 to 20 days after transplanting, the root system stress-resistant tillering cultivation stage is 21 to 35 days after transplanting, and the strong seedling cultivation stage is 36 to 40 days after transplanting.

[0048] The root primordia differentiation cultivation stage is carried out in an environment with a temperature of 24℃, an air humidity of not less than 80%RH, a light intensity of 2400Lux, and a light duration of 12h / d (using cool white LED lights for supplemental lighting, avoiding direct strong light, and a light quality of red to blue light ratio of 7:3). Root primordia differentiation nutrient solution is used for irrigation: the first irrigation after transplanting involves thoroughly watering the substrate with the root primordia differentiation nutrient solution (until just enough nutrient solution seeps out from the bottom tray of the seedling tray), and then emptying any excess liquid from the tray; when the humidity of the mixed substrate is lower than 55%RH, supplemental irrigation is carried out by evenly spraying the nutrient solution along the surface of the substrate using a fine-nozzle sprayer, directly pouring the nutrient solution into the substrate;

[0049] The root primordium differentiation nutrient solution includes 1 / 10 MS medium (i.e., the concentration is diluted to 1 / 10 of the original MS medium concentration), pH-sensitive chitosan-coated bound amino acid nitrogen, N-(1-naphthyl)-o-carbamoylbenzoic acid (CAS: 132-66-1), 24-epibrassinolide (CAS: 78821-43-9), 1,4-butanediamine (CAS: 110-60-1), tea tree oil nanoemulsion, urea peroxide (CAS: 124-43-6), and γ-polyglutamic acid (molecular weight 100,000–700,000 Da, CAS: 25513-46-6), potassium humate; wherein, the concentrations of pH-sensitive chitosan-coated bound amino acid nitrogen, N-(1-naphthyl)-o-carbamoylbenzoic acid, 24-epibrassinolide, 1,4-butanediamine, tea tree oil nanoemulsion, urea peroxide, γ-polyglutamic acid, and potassium humate are 0.78 g / L, 0.09 μmol / L, 0.18 μmol / L, 0.9 μmol / L, 0.09 g / L, 4.8 mg / L, 0.28 g / L, and 0.18 g / L, respectively.The specific preparation method of pH-sensitive chitosan-coated amino acid nitrogen is as follows: First, chitosan is added to a 2.5% glacial acetic acid aqueous solution, with a mass-to-volume ratio of 3g:80mL. The solution is stirred at 280rpm at room temperature (25±1℃) until dissolved to obtain a transparent solution. This solution is then sonicated at 190W power and 18kHz frequency for 16min. The pH is then adjusted to 4.6 using a 0.1mol / L NaOH solution. Finally, the composite amino acids, EDC, and NHS are added to the solution... In deionized water, the mass-to-volume ratio of the amino acids was 8 g: 3.5 g: 2.1 g: 200 mL. The mixture was stirred at 480 rpm for 32 min at room temperature to obtain an activated amino acid solution. The complex amino acid solution consisted of L-lysine hydrochloride (CAS: 657-27-2), L-arginine (CAS: 74-79-3), and L-glutamic acid (CAS: 56-86-0) in a mass ratio of 7:2:1. Subsequently, under light-protected conditions at room temperature, the activated amino acid solution was added dropwise to a chitosan solution at a rate of 1 drop / s. The mixture was stirred at 780 rpm for 1.2 h and then at 180 rpm for 12.5 h. Sodium tripolyphosphate (CAS; 7758-29-4) was then added, with a mass ratio of sodium tripolyphosphate to complex amino acids of 0.3:2. The mixture was stirred continuously at 180 rpm for 2.2 h to obtain the coupling reaction solution. The coupling reaction solution was then diluted with deionized water to a solid content of 10% (w / v), ultrasonicated for 8 min to ensure homogeneity, and then spray-dried (inlet temperature 120℃, outlet temperature 55℃). The powder was collected at ℃, feed rate 5 mL / min, atomization pressure 0.25 MPa, and fan speed 2800 rpm. The dried powder was then vacuum-dried at 58℃ and -0.08 MPa for 4.2 h to obtain pH-sensitive chitosan-amino acid composite powder. Finally, the spray-dried powder was soaked in a 95% ethanol aqueous solution for 2 h, then the pH was adjusted to 5.9 with 0.1 mol / L NaOH, and vacuum-dried at 58℃ and -0.08 MPa to constant weight (moisture content less than 5%) to obtain the final product.The specific preparation method of tea tree oil nanoemulsion is as follows: First, Tween-80 and anhydrous ethanol are mixed at a mass ratio of 3:1 and stirred evenly at 280 rpm at room temperature to form a transparent emulsion system. Then, tea tree oil is added to the transparent emulsion system at a mass ratio of 1:3 to Tween-80, and stirred at 280 rpm for 15 min to obtain a transparent oil-emulsion mixture. Next, the oil-emulsion mixture is placed in a high-speed shear mill and sheared at 8000 rpm while adding deionized water dropwise at a rate of 1 g / min for 10 min to obtain a pre-emulsion. Then, the pre-emulsion is transferred to an ultrasonic disruptor and disrupted at a power of 160 W and a temperature below 25℃ for 10 min to obtain a nanoemulsion. Finally, the nanoemulsion is filtered through a 0.45 μm filter membrane and then sterilized through a 0.22 μm filter membrane to obtain the final product.

[0050] The adventitious root establishment and cultivation stage was carried out in an environment with a temperature of 24℃, an air humidity of 75%RH, a light intensity of 3400Lux, and a light duration of 14h / d (using cool white LED lights for supplemental lighting, avoiding direct strong light, and a light quality of red to blue light ratio of 7:3). Adventitious root establishment nutrient solution was used for irrigation: the first irrigation was performed on the 8th day after transplanting, thoroughly soaking the substrate with the nutrient solution (until just enough nutrient solution seeped out from the bottom tray of the seedling tray), and then emptying the excess liquid from the tray; thereafter, supplemental irrigation was performed once every 5 days, with each irrigation amount being 60% of the substrate's water holding capacity;

[0051] The nutrient solution for adventitious root construction includes 1 / 10 MS medium (i.e., the concentration is diluted to 1 / 10 of the original MS medium concentration), N6-isopentenyl adenosine (CAS: 7724-76-7), L-tryptophan (CAS: 73-22-3), strigolactone analogue (GR24, CAS: 76974-79-3), sodium borate, zinc sulfate, salicin (CAS: 138-52-3), humic acid chelated complex trace elements, γ-polyglutamic acid, chitosan oligosaccharide, and Bacillus subtilis (purchased from China General Microbiological Culture Collection Center, accession number: CGMCC). 1.1086); wherein, the concentrations of N6-isopentenyl adenosine, L-tryptophan, strigolactone analog, sodium borate, zinc sulfate, salicin, humic acid chelated trace elements, γ-polyglutamic acid, and chitosan oligosaccharide (low degree of polymerization, such as degree of polymerization 2-10) were 0.58 μmol / L, 0.08 μmol / L, 0.28 μmol / L, 1.9 μmol / L, 0.08 μmol / L, 0.48 μmol / L, 0.95 mL / L, 0.28 g / L, and 0.33 g / L, respectively; the concentration of Bacillus subtilis was 2 × 10⁻⁶. 8CFU / mL. The specific preparation method of humic acid chelated complex trace elements is as follows: First, add 5g of sodium humate to 800mL of deionized water, adjust the pH to 8 with 1mol / L NaOH solution, and stir until completely dissolved; then add 2.49g of FeSO4 sequentially. 7H2O, 0.88g ZnSO4 7H2O, 0.67g MnSO4 4H₂O, 0.2g CuSO₄ 5H2O and 0.57g of H3BO3 were stirred at 380rpm for 2.2h in a constant temperature water bath at 58℃. After the reaction was completed, the pH of the solution was lowered to 5.5 and the volume was adjusted to 1L to obtain the final product.

[0052] During the root-resistant tillering cultivation stage, the plants were cultivated in an environment with a daytime temperature of 25℃ and a diurnal temperature range of 4℃, an air humidity of 60%RH, a light intensity of 3900Lux, and a light duration of 16h / d. Root-resistant tillering nutrient solution was used for irrigation: the first irrigation was performed on the 21st day after transplanting, thoroughly soaking the substrate with the root-resistant tillering nutrient solution (until just enough nutrient solution seeped out from the bottom tray of the seedling tray), and then emptying any excess liquid from the tray. Subsequent irrigations were performed every 4 days, with each irrigation amount being 50% of the substrate's water holding capacity.

[0053] The root stress-resistant tillering nutrient solution includes 1 / 10 MS medium (i.e., the concentration is diluted to 1 / 10 of the original MS medium concentration), methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex, 6-benzyladenine (CAS: 4294-16-0), potassium dihydrogen phosphate, nano silica (30-50nm) solution (i.e., hydrophilic nano-fumed silica is added to sterile water, ultrasonically dispersed, and then sterilized through a 0.22μm filter membrane to obtain a solution of the corresponding concentration), γ-polyglutamic acid, and potassium humate; wherein the concentrations of methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex, 6-benzyladenine, potassium dihydrogen phosphate, nano silica, γ-polyglutamic acid, and potassium humate are 0.48μmol / L, 1.18μmol / L, 0.78g / L, 0.28g / L, 0.18g / L, and 0.15g / L, respectively. The specific preparation method of methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex is as follows: First, 0.1 mmol of methyl jasmonate (CAS: 1211-29-6) was dissolved in 1 mL of anhydrous ethanol to obtain a stock solution; then, 1 mmol of hydroxypropyl-β-cyclodextrin (CAS: 128446-35-5) was dissolved in 80 mL of sterile water and stirred at 280 rpm at room temperature until completely dissolved to obtain a mother liquor; then, under a constant temperature water bath at 38℃, the stock solution was added dropwise to the mother liquor at a rate of 1 mL / min while stirring at 380 rpm. After the addition was completed, the mixture was stirred for 4.2 h, and then ultrasonicated (power 200 W, frequency 20 kHz) for 14 min at room temperature; finally, the mixture was filtered through a 0.22 μm filter membrane to obtain a 1 mmol / L methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex mother liquor.

[0054] During the seedling cultivation stage, seedlings were grown in an environment with a daytime temperature of 26℃ and a diurnal temperature range of 8℃, an air humidity of 60%RH, a light intensity of 4900Lux, and a light duration of 14h / d. Seedling nutrient solution was used for irrigation: the first irrigation was performed on the 36th day after transplanting, using the seedling nutrient solution to moisten the substrate but not to saturate it; thereafter, supplementary irrigation was performed every 3 days, with each irrigation amount being 18% of the substrate's water holding capacity.

[0055] The seedling nutrient solution consisted of 1 / 10 MS medium (i.e., diluted to 1 / 10 of the original MS medium concentration), trehalose, potassium dihydrogen phosphate, potassium nitrate, betaine, L-proline, potassium humate, Bacillus subtilis (purchased from the China General Microbiological Culture Collection Center, accession number: CGMCC 1.1086), and Bacillus megaterium (purchased from the China General Microbiological Culture Collection Center, accession number: CGMCC 1.6721). The concentrations of trehalose, potassium dihydrogen phosphate, potassium nitrate, *Glomus mosy* spore suspension, betaine, L-proline, and potassium humate were 1.9 g / L, 0.9 g / L, 0.48 g / L, 0.08 g / L, 0.08 g / L, and 0.28 g / L, respectively; the concentration of Bacillus subtilis was 2.5 × 10⁻⁶. 7 The internal activity of Bacillus megaterium is 2.5 × 10⁻⁶ CFU / mL. 7 CFU / mL.

[0056] Example 2:

[0057] A method for cultivating rootless and leafless ginger tissue culture seedlings includes:

[0058] Step S1, Material Preparation: Select ginger virus-free tissue culture seedlings that have grown for 37 days, are 6.5cm tall, have 4 fully expanded leaves, have thick stems, are vigorous, and are free from vitrification and obvious pests and diseases. After removing the ginger virus-free tissue culture seedlings from the culture bottle, rinse them 3 times with sterile water to wash away the culture medium attached to the roots. Handle them gently throughout the process to avoid damaging the stems, leaves, and root neck.

[0059] Remove all original roots and callus tissue from the ginger tissue culture seedlings, leaving only the root neck connecting the above-ground part and the root system; at the same time, cut off the excessively long stems and all leaves from the above-ground part, leaving only a 2.5cm healthy stem segment with a clean cut and no tears (after completion, place the rootless individual plant on sterile damp gauze to keep it moist and prevent it from wilting due to water loss).

[0060] Step S2, Mixed Matrix Preparation: Prepare the mixed matrix in advance, and after non-destructive sterilization, put the mixed matrix into a container to make the surface of the mixed matrix smooth.

[0061] The mixed matrix comprises hydrophobically modified expanded perlite microspheres (30-50 mesh), aminated silica aerogel microparticles (100-200 mesh), a bacterial humic acid-grafted vermiculite composite carrier (200 mesh), and enzymatically hydrolyzed peat fiber (0.5-1 mm short fiber), with mass percentages of 30%, 9%, 38%, and 23%, respectively. The preparation method of the hydrophobically modified expanded perlite microspheres is as follows: 30-50 mesh perlite is washed and dried (temperature 105℃, drying time 1.5h), then immersed in a 2% concentration KH570 (3-(methacryloyloxy)propyltrimethoxysilane, CAS: 2530-85-0)-anhydrous ethanol solution, with a perlite-to-solution mass-to-volume ratio of 1g:5mL. The mixture is first shaken at 40℃ and 135rpm (rotary shaker) for 4h, then filtered, washed, and dried at 105℃ for 2.5h to obtain the final product. The specific preparation method of aminated silica aerogel particles is as follows: 100-200 mesh silica aerogel is vacuum dried (temperature 120℃, vacuum degree -0.085MPa, drying time 2h), and then immersed in a 3% KH550 (3-aminopropyltriethoxysilane, CAS: 919-30-2)-anhydrous ethanol solution. The mass-volume ratio of silica aerogel to solution is 1g:4mL. The mixture is first shaken at 115rpm (rotary shaker) for 6h under light-protected conditions at 50℃, then filtered and washed, and finally vacuum dried at 60℃ and -0.085MPa for 5h to obtain the desired product. The preparation method of the humic acid-grafted vermiculite composite carrier is as follows: 200-mesh vermiculite is added to a 1 mol / L hydrochloric acid solution, with a mass-to-volume ratio of vermiculite to hydrochloric acid solution of 1 g: 5 mL. The mixture is first stirred at 80℃ and 150 rpm for 4 h, then filtered and washed until neutral (pH 6.7), and dried at 105℃ for 2.5 h. Afterward, the activated vermiculite is mixed with fulvic acid at a mass ratio of 5:2, and EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) is added. A mixed crosslinking agent consisting of vermiculite (CAS: 25952-53-8) and NHS (N-hydroxysuccinimide, CAS: 6066-82-6), with a molar ratio of EDS to NHS of 1:1, and the mixed crosslinking agent accounting for 0.5% of the total mass of vermiculite and humic acid, was stirred at 200 rpm for 6 hours in a constant temperature water bath at 50℃. After filtration and washing, the mixture was vacuum dried at 60℃ and -0.085 MPa for 4 hours to obtain the carrier. Finally, the sterilized carrier was placed in a sterile vacuum adsorption bottle, and a bacterial solution concentration of 1×10⁻⁶ was added. 9A CFU / mL suspension of *Candida langiocarpa* (purchased from the China General Microbiological Culture Collection Center, accession number: CGMCC 2.1763), with a carrier-to-suspension solid-liquid ratio of 1 g:3 mL, was maintained under a vacuum of -0.085 MPa for 17 min, followed by slow introduction of sterile air. The bacterial suspension permeated into the carrier pores under the pressure difference, and this process was maintained for 11 min. After removal, the suspension was air-dried at 28°C until the moisture content reached 30%. The preparation method for enzymatically hydrolyzed peat fiber is as follows: Peat is pulverized and added to a buffer solution composed of a pH 4.8 acetic acid solution and a sodium acetate solution, with a peat-to-buffer volume ratio of 1 g:10 mL. The mixture is stirred at 200 rpm to form a peat suspension. Then, cellulase (enzyme activity not less than 1000 U / g) is added to the peat suspension at 2% of the peat mass, and the mixture is heated at 50°C. Stir at 150 rpm for 4 hours; then, place the enzymatically hydrolyzed suspension in a 100℃ constant temperature wastewater bath and keep it at that temperature for 9 minutes. Wash the peat fibers repeatedly with deionized water until the pH of the filtrate stabilizes at 5.7. Finally, filter and collect the fiber filter cake and air dry it to semi-dry (moisture content of about 40%). Then, sieve and classify the fibers (first pass them through a 1 mm standard sieve to remove long fibers and impurities; then use a 0.5 mm standard sieve to remove fine powder and collect short fibers of 0.5-1 m). Dry the classified fibers at 60℃ to constant weight (moisture content not greater than 10%) to obtain the final product.

[0062] The specific preparation method of the mixed matrix is ​​as follows: The four components are poured into a horizontal mixer according to the mass ratio and dry-mixed at 35 rpm for 15 minutes to obtain the mixed matrix; then, the mixed matrix is ​​subjected to a 500 mg / m³... 3 The gaseous sterilization process involves chlorine dioxide, followed by a 6-hour sealed sterilization at room temperature and a 2-hour ventilation period to remove any residue.

[0063] Step S3, Transplanting of Tissue Culture Seedlings: Select a seedling tray with water absorption holes at the bottom and a matching tray as the transplanting container. Evenly fill each hole of the seedling tray with the mixed substrate, filling it to a height of 1cm above the top edge of the hole. Gently scrape it flat and lightly compact it. Then, vertically punch a hole in the center of the substrate in each hole, with a depth of 0.7cm and a diameter matching the stem segment of the tissue culture seedling. After that, vertically insert the rootless and leafless tissue culture seedlings obtained in Step S1 into the holes, ensuring that the root neck is completely buried in the substrate hole and the stem segment is upright. Gently compact the substrate around the seedling to fix the seedling and prevent it from falling over.

[0064] Finally, the cultivation is carried out in four stages according to time: root primordia differentiation cultivation stage, adventitious root construction cultivation stage, root system stress-resistant tillering cultivation stage, and strong seedling cultivation stage. The root primordia differentiation cultivation stage is 1 to 7 days after transplanting, the adventitious root construction cultivation stage is 8 to 20 days after transplanting, the root system stress-resistant tillering cultivation stage is 21 to 35 days after transplanting, and the strong seedling cultivation stage is 36 to 40 days after transplanting.

[0065] The root primordia differentiation cultivation stage is carried out in an environment with a temperature of 25℃, an air humidity of not less than 80%RH, a light intensity of 2500Lux, and a light duration of 12h / d (using cool white LED lights for supplemental lighting, avoiding direct strong light, and a light quality of red to blue light ratio of 7:3). Root primordia differentiation nutrient solution is used for irrigation: the first irrigation after transplanting involves thoroughly watering the substrate with the root primordia differentiation nutrient solution (until just as nutrient solution seeps out from the bottom tray of the seedling tray), and then emptying any excess liquid from the tray; when the humidity of the mixed substrate is lower than 55%RH, supplemental irrigation is carried out by evenly spraying the nutrient solution along the surface of the substrate using a fine-nozzle sprayer, directly pouring the nutrient solution into the substrate.

[0066] The root primordium differentiation nutrient solution includes 1 / 10 MS medium (i.e., the concentration is diluted to 1 / 10 of the original MS medium concentration), pH-sensitive chitosan-coated bound amino acid nitrogen, N-(1-naphthyl)-o-carbamoylbenzoic acid (CAS: 132-66-1), 24-epibrassinolide (CAS: 78821-43-9), 1,4-butanediamine (CAS: 110-60-1), tea tree oil nanoemulsion, urea peroxide (CAS: 124-43-6), and γ-polyglutamic acid (molecular weight 100,000–700,000 Da). (CAS: 25513-46-6), potassium humate; among which, the concentrations of pH-sensitive chitosan-encapsulated bound amino acid nitrogen, N-(1-naphthyl)-o-carbamoylbenzoic acid, 24-epibrassinolide, 1,4-butanediamine, tea tree oil nanoemulsion, urea peroxide, γ-polyglutamic acid, and potassium humate were 0.8 g / L, 0.1 μmol / L, 0.2 μmol / L, 1 μmol / L, 0.1 g / L, 5 mg / L, 0.3 g / L, and 0.2 g / L, respectively.The specific preparation method of pH-sensitive chitosan-coated amino acid nitrogen is as follows: First, chitosan is added to a 2.5% glacial acetic acid aqueous solution, with a mass-to-volume ratio of 3g:80mL. The solution is stirred at 300rpm at room temperature (25±1℃) until dissolved, obtaining a transparent solution. This solution is then sonicated at 200W power and 20kHz frequency for 15min. The pH is then adjusted to 4.7 using a 0.1mol / L NaOH solution. Next, the composite amino acids, EDC, and NHS are added to the solution... In deionized water, the mass-to-volume ratio of the amino acids was 8 g: 3.5 g: 2.1 g: 200 mL. The mixture was stirred at 500 rpm for 30 min at room temperature to obtain an activated amino acid solution. The complex amino acid solution consisted of L-lysine hydrochloride (CAS: 657-27-2), L-arginine (CAS: 74-79-3), and L-glutamic acid (CAS: 56-86-0) in a mass ratio of 7:2:1. Subsequently, under light-protected conditions at room temperature, the activated amino acid solution was added dropwise to a chitosan solution at a rate of 1 drop / s. The mixture was stirred at 800 rpm for 1 hour and then at 200 rpm for 12 hours. Sodium tripolyphosphate (CAS; 7758-29-4) was then added, with a mass ratio of sodium tripolyphosphate to the composite amino acids of 0.3:2. The mixture was stirred continuously at 180–220 rpm for 2 hours to obtain the coupling reaction solution. The coupling reaction solution was then diluted with deionized water to a solid content of 10% (w / v), ultrasonicated for 10 minutes to ensure homogeneity, and then spray-dried (inlet temperature 125℃, outlet temperature 57℃). The feed rate was 6.5 mL / min, the atomization pressure was 0.25 MPa, and the fan speed was 3000 rpm. The dried powder was collected and vacuum dried at 60℃ and -0.085 MPa for 4 h to obtain pH-sensitive chitosan-amino acid composite powder. Finally, the spray-dried powder was soaked in 95% ethanol aqueous solution for 2.2 h, then the pH was adjusted to 6 with 0.1 mol / L NaOH, and vacuum dried at 60℃ and -0.085 MPa to constant weight (moisture content less than 5%) to obtain the final product.The specific preparation method of tea tree oil nanoemulsion is as follows: First, Tween-80 and anhydrous ethanol are mixed at a mass ratio of 3:1 and stirred evenly at 300 rpm at room temperature to form a transparent emulsion system. Then, tea tree oil is added to the transparent emulsion system at a mass ratio of 1.5:3 to Tween-80, while stirring at 300 rpm for 12.5 min to obtain a transparent oil-emulsion mixture. Next, the oil-emulsion mixture is placed in a high-speed shear mill and sheared at 9000 rpm, while deionized water is added dropwise at a rate of 1.5 g / min for 7.5 min to obtain a pre-emulsion. Then, the pre-emulsion is transferred to an ultrasonic disruptor and disrupted at a power of 180 W and a temperature below 25℃ for 7.5 min to obtain a nanoemulsion. Finally, the nanoemulsion is filtered through a 0.45 μm filter membrane and then sterilized through a 0.22 μm filter membrane to obtain the final product.

[0067] The adventitious root establishment and cultivation stage was carried out in an environment with a temperature of 25℃, an air humidity of 77%RH, a light intensity of 3500Lux, and a light duration of 14h / d (using cool white LED lights for supplemental lighting, avoiding direct strong light, and a light quality of red to blue light ratio of 7:3). Adventitious root establishment nutrient solution was used for irrigation: the first irrigation was performed on the 8th day after transplanting, thoroughly soaking the substrate with the nutrient solution (until just enough nutrient solution seeped out from the bottom tray of the seedling tray), and then emptying the excess liquid from the tray; thereafter, supplemental irrigation was performed once every 5 days, with each irrigation amount being 60% of the substrate's water holding capacity;

[0068] The nutrient solution for adventitious root construction includes 1 / 10 MS medium (i.e., the concentration is diluted to 1 / 10 of the original MS medium concentration), N6-isopentenyl adenosine (CAS: 7724-76-7), L-tryptophan (CAS: 73-22-3), strigolactone analogue (GR24, CAS: 76974-79-3), sodium borate, zinc sulfate, salicin (CAS: 138-52-3), humic acid chelated complex trace elements, γ-polyglutamic acid, chitosan oligosaccharide, and Bacillus subtilis (purchased from China General Microbiological Culture Collection Center, accession number: CGMCC). 1.1086); wherein, the concentrations of N6-isopentenyl adenosine, L-tryptophan, strigolactone analog, sodium borate, zinc sulfate, salicin, humic acid chelated trace elements, γ-polyglutamic acid, and chitosan oligosaccharide (low degree of polymerization, such as degree of polymerization 2-10) were 0.6 μmol / L, 0.1 μmol / L, 0.3 μmol / L, 2 μmol / L, 0.1 μmol / L, 0.5 μmol / L, 1 mL / L, 0.23 g / L, and 0.35 g / L, respectively; the concentration of Bacillus subtilis was 2 × 10⁻⁶. 8CFU / mL. The specific preparation method of humic acid chelated complex trace elements is as follows: First, add 5g of sodium humate to 800mL of deionized water, adjust the pH to 8.5 with 1mol / L NaOH solution, and stir until completely dissolved; then add 2.49g of FeSO4 sequentially. 7H2O, 0.88g ZnSO4 7H2O, 0.67g MnSO4 4H₂O, 0.2g CuSO₄ 5H2O and 0.57g of H3BO3 were stirred at 400rpm for 2 hours in a constant temperature water bath at 60℃. After the reaction was completed, the pH of the solution was lowered to 6 and the volume was adjusted to 1L to obtain the final product.

[0069] During the root-resistant tillering cultivation stage, the plants were cultivated in an environment with a daytime temperature of 26℃ and a diurnal temperature range of 4℃, an air humidity of 65%RH, a light intensity of 4000Lux, and a light duration of 16h / d. Root-resistant tillering nutrient solution was used for irrigation: the first irrigation was performed on the 21st day after transplanting, thoroughly soaking the substrate with the root-resistant tillering nutrient solution (until just enough nutrient solution seeped out from the bottom tray of the seedling tray), and then emptying any excess liquid from the tray. Subsequent irrigations were performed every 4 days, with each irrigation amount being 50% of the substrate's water holding capacity.

[0070] The root stress-resistant tillering nutrient solution includes 1 / 10 MS medium (i.e., the concentration is diluted to 1 / 10 of the original MS medium concentration), methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex, 6-benzyladenine (CAS: 4294-16-0), potassium dihydrogen phosphate, nano silica (30-50nm) solution (i.e., hydrophilic nano-fumed silica is added to sterile water, ultrasonically dispersed, and then sterilized through a 0.22μm filter membrane to obtain a solution of the corresponding concentration), γ-polyglutamic acid, and potassium humate; wherein the concentrations of methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex, 6-benzyladenine, potassium dihydrogen phosphate, nano silica, γ-polyglutamic acid, and potassium humate are 0.5μmol / L, 1.2μmol / L, 0.8g / L, 0.3g / L, 0.2g / L, and 0.17g / L, respectively. The specific preparation method of methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex is as follows: First, 0.1 mmol of methyl jasmonate (CAS: 1211-29-6) was dissolved in 1 mL of anhydrous ethanol to obtain a stock solution; then, 1 mmol of hydroxypropyl-β-cyclodextrin (CAS: 128446-35-5) was dissolved in 80 mL of sterile water and stirred at 300 rpm at room temperature until completely dissolved to obtain a mother liquor; then, under a constant temperature water bath at 40℃, the stock solution was added dropwise to the mother liquor at a rate of 1.5 mL / min while stirring at 400 rpm. After the addition was completed, the mixture was stirred for 4 h, and then ultrasonicated (power 200 W, frequency 20 kHz) for 15 min at room temperature; finally, the mixture was filtered through a 0.22 μm filter membrane to obtain a 1 mmol / L methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex mother liquor.

[0071] During the seedling cultivation stage, seedlings were grown in an environment with a daytime temperature of 26–30℃ and a diurnal temperature range of 8℃, an air humidity of 65%RH, a light intensity of 5000 Lux, and a light duration of 14 h / d. Seedling nutrient solution was used for irrigation: the first irrigation was performed on the 36th day after transplanting, using the seedling nutrient solution to moisten the substrate but not to saturate it; thereafter, supplemental irrigation was performed every 3 days, with each irrigation amount being 20% ​​of the substrate's water holding capacity.

[0072] The seedling nutrient solution consisted of 1 / 10 MS medium (i.e., diluted to 1 / 10 of the original MS medium concentration), trehalose, potassium dihydrogen phosphate, potassium nitrate, betaine, L-proline, potassium humate, Bacillus subtilis (purchased from the China General Microbiological Culture Collection Center, accession number: CGMCC 1.1086), and Bacillus megaterium (purchased from the China General Microbiological Culture Collection Center, accession number: CGMCC 1.6721). The concentrations of trehalose, potassium dihydrogen phosphate, potassium nitrate, *Glomus mosy* spore suspension, betaine, L-proline, and potassium humate were 2 g / L, 1 g / L, 0.5 g / L, 0.1 g / L, 0.1 g / L, and 0.3 g / L, respectively; the concentration of Bacillus subtilis was 2.5 × 10⁻⁶. 7 The internal activity of Bacillus megaterium is 2.5 × 10⁻⁶ CFU / mL. 7 CFU / mL.

[0073] Example 3:

[0074] A method for cultivating rootless and leafless ginger tissue culture seedlings includes:

[0075] Step S1, Material Preparation: Select ginger virus-free tissue culture seedlings that have grown for 37 days, are 6.5cm tall, have 4 fully expanded leaves, have thick stems, are vigorous, and are free from vitrification and obvious pests and diseases. After removing the ginger virus-free tissue culture seedlings from the culture bottle, rinse them 4 times with sterile water to wash away the culture medium attached to the roots. Handle them gently throughout the process to avoid damaging the stems, leaves, and root neck.

[0076] Remove all original roots and callus tissue from the ginger tissue culture seedlings, leaving only the root neck connecting the above-ground part and the root system; at the same time, cut off the excessively long stems and all leaves from the above-ground part, leaving only a 2.5cm healthy stem segment with a clean cut and no tears (after completion, place the rootless individual plant on sterile damp gauze to keep it moist and prevent it from wilting due to water loss).

[0077] Step S2, Mixed Matrix Preparation: Prepare the mixed matrix in advance, and after non-destructive sterilization, put the mixed matrix into a container to make the surface of the mixed matrix smooth.

[0078] The mixed matrix includes hydrophobically modified expanded perlite microspheres (30-50 mesh), aminated silica aerogel microparticles (100-200 mesh), bacterial humic acid-grafted vermiculite composite carrier (200 mesh), and enzymatically hydrolyzed peat fiber (0.5-1 mm short fiber), with their mass percentages being 30.5%, 8.5%, 38.5%, and 22.5%, respectively. The specific preparation method of hydrophobically modified expanded perlite microspheres is as follows: 30-50 mesh perlite is washed and dried (temperature 108℃, drying time 1h), then immersed in a 2.2% concentration KH570 (3-(methacryloyloxy)propyltrimethoxysilane, CAS: 2530-85-0)-anhydrous ethanol solution, with a perlite to solution mass-volume ratio of 1.1g:5.5mL. The mixture is first shaken at 42℃ and 150rpm (rotary shaker) for 3.8h, then filtered, washed, and dried at 108℃ for 2h to obtain the microspheres. The specific preparation method of aminated silica aerogel particles is as follows: 100-200 mesh silica aerogel is vacuum dried (temperature 122℃, vacuum degree -0.09MPa, drying time 1.8h), and then immersed in a 3.2% KH550 (3-aminopropyltriethoxysilane, CAS: 919-30-2)-anhydrous ethanol solution. The mass-volume ratio of silica aerogel to solution is 1.1g:4.4mL. The mixture is first shaken at 130rpm (rotary shaker) for 5.8h under light-protected conditions at 52℃, then filtered and washed, and finally vacuum dried at 62℃ and -0.09MPa for 4h to obtain the final product. The preparation method of the humic acid-grafted vermiculite composite carrier is as follows: 200-mesh vermiculite is added to a 1 mol / L hydrochloric acid solution, with a mass-to-volume ratio of vermiculite to hydrochloric acid solution of 1.1 g: 5.5 mL. The mixture is first stirred at 82℃ and 160 rpm for 3.8 h, then filtered and washed until neutral (pH 7.0), and dried at 108℃ for 2 h. Afterward, the activated vermiculite is mixed with humic acid at a mass ratio of 5.25: 2.1, and EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide salt) is added. A mixed crosslinking agent of vermiculite (CAS: 25952-53-8) and NHS (N-hydroxysuccinimide, CAS: 6066-82-6), with a molar ratio of EDS to NHS of 1:1, and the mixed crosslinking agent accounting for 0.52% of the total mass of vermiculite and humic acid, was stirred at 220 rpm for 5.8 h in a constant temperature water bath at 52 °C. After filtration and washing, the mixture was vacuum dried at 62 °C and -0.09 MPa for 3 h to obtain the carrier. Finally, the sterilized carrier was placed in a sterile vacuum adsorption bottle, and a bacterial solution concentration of 1×10⁻⁶ was added. 9A CFU / mL suspension of *Candida langiocarpa* (purchased from the China General Microbiological Culture Collection Center, accession number: CGMCC 2.1763), with a carrier-to-suspension solid-liquid ratio of 1.1 g: 3.3 mL, was maintained under a vacuum of -0.09 MPa for 15 min, followed by slow introduction of sterile air. The bacterial suspension permeated into the carrier pores under the pressure difference, and this process was maintained for 10 min. After removal, the suspension was air-dried at 29°C until the moisture content reached 30%. The preparation method for enzymatically hydrolyzed peat fiber is as follows: Peat is pulverized and added to a buffer solution composed of a pH 4.8 acetic acid solution and a sodium acetate solution, with a peat-to-buffer volume ratio of 1.1 g: 11 mL. The mixture is stirred at 220 rpm to form a peat suspension. Then, cellulase (enzyme activity not less than 1000 U / g) is added to the peat suspension at an amount of 2.2% of the peat mass, and the mixture is heated at 52°C. Stir at 160 rpm for 3.8 hours; then, place the enzymatically hydrolyzed suspension in a constant temperature wastewater bath at 102℃ and keep it at that temperature for 8 minutes, and repeatedly wash the peat fibers with deionized water until the pH of the filtrate stabilizes at 6.0; finally, filter and collect the fiber filter cake and air dry it to semi-dry (moisture content of about 40%), and then sieve it for classification (first pass it through a 1 mm standard sieve to remove long fibers and impurities; then use a 0.5 mm standard sieve to remove fine powder and collect short fibers of 0.5-1 m), and dry the classified fibers at 62℃ to constant weight (moisture content not greater than 10%) to obtain the final product.

[0079] The specific preparation method of the mixed matrix is ​​as follows: The four components are poured into a horizontal mixer according to the mass ratio and dry-mixed at 40 rpm for 14 minutes to obtain the mixed matrix; then, the mixed matrix is ​​subjected to a 500 mg / m³... 3 The gaseous sterilization process involves chlorine dioxide, followed by a 6-hour sealed sterilization at room temperature and a 2-hour ventilation period to remove any residue.

[0080] Step S3, Transplanting of Tissue Culture Seedlings: Select a seedling tray with water absorption holes at the bottom and a matching tray as the transplanting container. Evenly fill each hole of the seedling tray with the mixed substrate, filling it to a height of 1cm above the top edge of the hole. Gently scrape it flat and lightly compact it. Then, vertically punch a hole in the center of the substrate in each hole, with a depth of 0.7cm and a diameter matching the stem segment of the tissue culture seedling. After that, vertically insert the rootless and leafless tissue culture seedlings obtained in Step S1 into the holes, ensuring that the root neck is completely buried in the substrate hole and the stem segment is upright. Gently compact the substrate around the seedling to fix the seedling and prevent it from falling over.

[0081] Finally, the cultivation is carried out in four stages according to time: root primordia differentiation cultivation stage, adventitious root construction cultivation stage, root system stress-resistant tillering cultivation stage, and strong seedling cultivation stage. The root primordia differentiation cultivation stage is 1 to 7 days after transplanting, the adventitious root construction cultivation stage is 8 to 20 days after transplanting, the root system stress-resistant tillering cultivation stage is 21 to 35 days after transplanting, and the strong seedling cultivation stage is 36 to 40 days after transplanting.

[0082] The root primordia differentiation cultivation stage is carried out in an environment with a temperature of 26℃, an air humidity of not less than 80%RH, a light intensity of 2600Lux, and a light duration of 12h / d (using cool white LED lights for supplemental lighting, avoiding direct strong light, and a light quality of red to blue light ratio of 7:3). Root primordia differentiation nutrient solution is used for irrigation: the first irrigation after transplanting involves thoroughly watering the substrate with the root primordia differentiation nutrient solution (until just as nutrient solution seeps out from the bottom tray of the seedling tray), and then emptying any excess liquid from the tray; when the humidity of the mixed substrate is lower than 55%RH, supplemental irrigation is carried out by evenly spraying the nutrient solution along the surface of the substrate using a fine-nozzle sprayer, directly pouring the nutrient solution into the substrate;

[0083] The root primordium differentiation nutrient solution includes 1 / 10 MS medium (i.e., the concentration is diluted to 1 / 10 of the original MS medium concentration), pH-sensitive chitosan-coated bound amino acid nitrogen, N-(1-naphthyl)-o-carbamoylbenzoic acid (CAS: 132-66-1), 24-epibrassinolide (CAS: 78821-43-9), 1,4-butanediamine (CAS: 110-60-1), tea tree oil nanoemulsion, urea peroxide (CAS: 124-43-6), and γ-polyglutamic acid (molecular weight 100,000–700,000 Da, CAS: 25513-46-6), potassium humate; wherein, the concentrations of pH-sensitive chitosan-coated bound amino acid nitrogen, N-(1-naphthyl)-o-carbamoylbenzoic acid, 24-epibrassinolide, 1,4-butanediamine, tea tree oil nanoemulsion, urea peroxide, γ-polyglutamic acid, and potassium humate are 0.82 g / L, 0.11 μmol / L, 0.22 μmol / L, 1.1 μmol / L, 0.11 g / L, 5.2 mg / L, 0.32 g / L, and 0.22 g / L, respectively.The specific preparation method of pH-sensitive chitosan-coated amino acid nitrogen is as follows: First, chitosan is added to a 2.5% glacial acetic acid aqueous solution, with a mass-to-volume ratio of 3g:80mL. The solution is stirred at 320rpm at room temperature (25±1℃) until dissolved to obtain a transparent solution. This solution is then sonicated at 210W power and 22kHz frequency for 14min. The pH is then adjusted to 4.8 using a 0.1mol / L NaOH solution. Finally, the composite amino acids, EDC, and NHS are added to the deionization... In a solution containing chitosan, the mass-to-volume ratio of the amino acids was 8 g: 3.5 g: 2.1 g: 200 mL. The mixture was stirred at 520 rpm for 28 min at room temperature to obtain an activated amino acid solution. The complex amino acid solution consisted of L-lysine hydrochloride (CAS: 657-27-2), L-arginine (CAS: 74-79-3), and L-glutamic acid (CAS: 56-86-0) in a mass ratio of 7:2:1. Subsequently, under light-protected conditions at room temperature, the activated amino acid solution was added dropwise to the chitosan solution at a rate of 1 drop / s. The mixture was stirred at 820 rpm for 0.8 h and then at 220 rpm for 11.5 h. Sodium tripolyphosphate (CAS; 7758-29-4) was then added, with a mass ratio of sodium tripolyphosphate to complex amino acids of 0.3:2. The mixture was stirred continuously at 220 rpm for 1.8 h to obtain the coupling reaction solution. The coupling reaction solution was then diluted with deionized water to a solid content of 10% (w / v), ultrasonicated for 12 min to ensure homogeneity, and then spray-dried (inlet temperature 130℃, outlet temperature 60℃). The feed rate was 8 mL / min, the atomization pressure was 0.25 MPa, and the fan speed was 3200 rpm. The dried powder was collected and vacuum dried at 62℃ and -0.09 MPa for 3.8 h to obtain pH-sensitive chitosan-amino acid composite powder. Finally, the spray-dried powder was soaked in 95% ethanol aqueous solution for 2.5 h, and then the pH was adjusted to 6.1 with 0.1 mol / L NaOH. It was then vacuum dried at 62℃ and -0.09 MPa to constant weight (moisture content less than 5%) to obtain the final product.The specific preparation method of tea tree oil nanoemulsion is as follows: First, Tween-80 and anhydrous ethanol are mixed at a mass ratio of 3:1 and stirred evenly at 320 rpm at room temperature to form a transparent emulsion system. Then, tea tree oil is added to the transparent emulsion system at a mass ratio of 2:3 to Tween-80, and stirred at 320 rpm for 10 min to obtain a transparent oil-emulsion mixture. Next, the oil-emulsion mixture is placed in a high-speed shear mill and sheared at 10,000 rpm while adding deionized water dropwise at a rate of 2 g / min for 5 min to obtain a pre-emulsion. Then, the pre-emulsion is transferred to an ultrasonic disruptor and disrupted for 5 min at a power of 200 W and a temperature below 25°C to obtain a nanoemulsion. Finally, the nanoemulsion is filtered through a 0.45 μm filter membrane and then sterilized through a 0.22 μm filter membrane to obtain the final product.

[0084] The adventitious root establishment and cultivation stage was carried out in an environment with a temperature of 26℃, an air humidity of 80%RH, a light intensity of 3600Lux, and a light duration of 14h / d (using cool white LED lights for supplemental lighting, avoiding direct strong light, and a light quality of red to blue light ratio of 7:3). Adventitious root establishment nutrient solution was used for irrigation: the first irrigation was performed on the 8th day after transplanting, thoroughly soaking the substrate with the nutrient solution (until just enough nutrient solution seeped out from the bottom tray of the seedling tray), and then emptying the excess liquid from the tray; thereafter, supplemental irrigation was performed once every 5 days, with each irrigation amount being 60% of the substrate's water holding capacity;

[0085] The nutrient solution for adventitious root construction includes 1 / 10 MS medium (i.e., the concentration is diluted to 1 / 10 of the original MS medium concentration), N6-isopentenyl adenosine (CAS: 7724-76-7), L-tryptophan (CAS: 73-22-3), strigolactone analogue (GR24, CAS: 76974-79-3), sodium borate, zinc sulfate, salicin (CAS: 138-52-3), humic acid chelated complex trace elements, γ-polyglutamic acid, chitosan oligosaccharide, and Bacillus subtilis (purchased from China General Microbiological Culture Collection Center, accession number: CGMCC). 1.1086); wherein, the concentrations of N6-isopentenyl adenosine, L-tryptophan, strigolactone analog, sodium borate, zinc sulfate, salicin, humic acid chelated complex trace elements, γ-polyglutamic acid, and chitosan oligosaccharide (low degree of polymerization, such as degree of polymerization 2-10) were 0.62 μmol / L, 0.12 μmol / L, 0.32 μmol / L, 2.1 μmol / L, 0.12 μmol / L, 0.52 μmol / L, 1.05 mL / L, 0.32 g / L, and 0.37 g / L, respectively; the concentration of Bacillus subtilis was 2 × 10⁻⁶. 8CFU / mL. The specific preparation method of humic acid chelated complex trace elements is as follows: First, add 5g of sodium humate to 800mL of deionized water, adjust the pH to 9 with 1mol / L NaOH solution, and stir until completely dissolved; then add 2.49g of FeSO4 sequentially. 7H2O, 0.88g ZnSO4 7H2O, 0.67g MnSO4 4H₂O, 0.2g CuSO₄ 5H2O and 0.57g of H3BO3 were stirred in a constant temperature water bath at 62℃ at a speed of 420rpm for 1.8h. After the reaction was completed, the pH of the solution was lowered to 6.5 and the volume was adjusted to 1L to obtain the final product.

[0086] During the root-resistant tillering cultivation stage, the plants were cultivated in an environment with a daytime temperature of 27℃ and a diurnal temperature range of 4℃, an air humidity of 70%RH, a light intensity of 4100Lux, and a light duration of 16h / d. Root-resistant tillering nutrient solution was used for irrigation: the first irrigation was performed on the 21st day after transplanting, thoroughly soaking the substrate with the root-resistant tillering nutrient solution (until just enough nutrient solution seeped from the bottom tray of the seedling tray), and then emptying any excess liquid from the tray. Subsequent irrigations were performed every 4 days, with each irrigation amount being 50% of the substrate's water holding capacity.

[0087] The root stress-resistant tillering nutrient solution includes 1 / 10 MS medium (i.e., the concentration is diluted to 1 / 10 of the original MS medium concentration), methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex, 6-benzyladenine (CAS: 4294-16-0), potassium dihydrogen phosphate, nano silica (30-50nm) solution (i.e., hydrophilic nano-fumed silica is added to sterile water, ultrasonically dispersed, and then sterilized through a 0.22μm filter membrane to obtain a solution of the corresponding concentration), γ-polyglutamic acid, and potassium humate; wherein the concentrations of methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex, 6-benzyladenine, potassium dihydrogen phosphate, nano silica, γ-polyglutamic acid, and potassium humate are 0.52μmol / L, 1.22μmol / L, 0.82g / L, 0.32g / L, 0.22g / L, and 0.2g / L, respectively. The specific preparation method of methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex is as follows: First, 0.1 mmol of methyl jasmonate (CAS: 1211-29-6) was dissolved in 1 mL of anhydrous ethanol to obtain a stock solution; then, 1 mmol of hydroxypropyl-β-cyclodextrin (CAS: 128446-35-5) was dissolved in 80 mL of sterile water and stirred at 320 rpm at room temperature until completely dissolved to obtain a mother liquor; then, under a constant temperature water bath at 42℃, the stock solution was added dropwise to the mother liquor at a rate of 2 mL / min while stirring at 420 rpm. After the addition was completed, the mixture was stirred for 3.8 h, and then ultrasonicated (power 200 W, frequency 20 kHz) for 16 min at room temperature; finally, the mixture was filtered through a 0.22 μm filter membrane to obtain a 1 mmol / L methyl jasmonate hydroxypropyl-β-cyclodextrin inclusion complex mother liquor.

[0088] During the seedling cultivation stage, seedlings were grown in an environment with a daytime temperature of 30℃ and a diurnal temperature range of 8℃, an air humidity of 70%RH, a light intensity of 5100Lux, and a light duration of 14h / d. Seedling nutrient solution was used for irrigation: the first irrigation was performed on the 36th day after transplanting, using the seedling nutrient solution to moisten the substrate but not to saturate it; thereafter, supplemental irrigation was performed every 3 days, with each irrigation amount being 22% of the substrate's water holding capacity.

[0089] The seedling nutrient solution consisted of 1 / 10 MS medium (i.e., diluted to 1 / 10 of the original MS medium concentration), trehalose, potassium dihydrogen phosphate, potassium nitrate, betaine, L-proline, potassium humate, Bacillus subtilis (purchased from the China General Microbiological Culture Collection Center, accession number: CGMCC 1.1086), and Bacillus megaterium (purchased from the China General Microbiological Culture Collection Center, accession number: CGMCC 1.6721). The concentrations of trehalose, potassium dihydrogen phosphate, potassium nitrate, *Glomus mosy* spore suspension, betaine, L-proline, and potassium humate were 2.1 g / L, 1.1 g / L, 0.52 g / L, 0.12 g / L, 0.12 g / L, and 0.32 g / L, respectively; the concentration of Bacillus subtilis was 2.5 × 10⁻⁶. 7 The internal activity of Bacillus megaterium is 2.5 × 10⁻⁶ CFU / mL. 7 CFU / mL.

[0090] Comparative Example 1:

[0091] A method for cultivating ginger tissue culture seedlings, comprising:

[0092] Step S1, Material Preparation: Same as Step S1 in Example 2.

[0093] Step S2, Preparation of Mixed Matrix: A mixed matrix is ​​prepared in advance and sterilized non-destructively before being placed into a container to ensure a smooth surface. The mixed matrix comprises perlite microspheres (30-50 mesh), aminated silica aerogel particles (100-200 mesh), a bacterial humic acid-grafted vermiculite composite carrier (200 mesh), and enzymatically hydrolyzed peat fibers (0.5-1 mm short fibers), with mass percentages of 30%, 9%, 38%, and 23%, respectively. The preparation methods for the aminated silica aerogel particles, the bacterial humic acid-grafted vermiculite composite carrier, and the enzymatically hydrolyzed peat fibers are consistent with step S2 in Example 2.

[0094] The specific preparation method of the mixed matrix is ​​the same as step S2 in Example 2.

[0095] Step S3, transplanting of tissue culture seedlings: consistent with step S3 in Example 2.

[0096] Comparative Example 2:

[0097] A method for cultivating ginger tissue culture seedlings, comprising:

[0098] Step S1, Material Preparation: Same as Step S1 in Example 2.

[0099] Step S2, Preparation of the Mixed Matrix: The mixed matrix is ​​prepared in advance and sterilized non-destructively before being placed into a container to ensure a smooth surface. The mixed matrix includes hydrophobically modified expanded perlite microspheres (30-50 mesh), silica aerogel microparticles (100-200 mesh), a bacterial humic acid-grafted vermiculite composite carrier (200 mesh), and enzymatically hydrolyzed peat fiber (0.5-1 mm short fiber), with a mass percentage of 30%, 9%, 38%, and 23%, respectively. The preparation methods of the hydrophobically modified expanded perlite microspheres, bacterial humic acid-grafted vermiculite composite carrier, and enzymatically hydrolyzed peat fiber are consistent with step S2 in Example 2.

[0100] The specific preparation method of the mixed matrix is ​​the same as step S2 in Example 2.

[0101] Step S3, transplanting of tissue culture seedlings: consistent with step S3 in Example 2.

[0102] Comparative Example 3:

[0103] A method for cultivating ginger tissue culture seedlings, comprising:

[0104] Step S1, Material Preparation: Same as Step S1 in Example 2.

[0105] Step S2, Preparation of Mixed Matrix: A mixed matrix is ​​prepared in advance and sterilized non-destructively before being placed into a container to ensure a smooth surface. The mixed matrix comprises hydrophobically modified expanded perlite microspheres (30-50 mesh), aminated silica aerogel microparticles (100-200 mesh), vermiculite (200 mesh), and enzymatically hydrolyzed peat fiber (0.5-1 mm short fiber), with mass percentages of 30%, 9%, 38%, and 23%, respectively. The preparation methods for the hydrophobically modified expanded perlite microspheres, aminated silica aerogel microparticles, and enzymatically hydrolyzed peat fiber are consistent with step S2 in Example 2.

[0106] The specific preparation method of the mixed matrix is ​​the same as step S2 in Example 2.

[0107] Step S3, transplanting of tissue culture seedlings: consistent with step S3 in Example 2.

[0108] Comparative Example 4:

[0109] A method for cultivating ginger tissue culture seedlings, comprising:

[0110] Step S1, Material Preparation: Same as Step S1 in Example 2.

[0111] Step S2, Preparation of the Mixed Matrix: The mixed matrix is ​​prepared in advance and sterilized non-destructively before being placed into a container to ensure a smooth surface. The mixed matrix includes hydrophobically modified expanded perlite microspheres (30-50 mesh), aminated silica aerogel microparticles (100-200 mesh), a bacterial humic acid-grafted vermiculite composite carrier (200 mesh), and peat fiber (0.5-1 mm short fiber), with a mass percentage of 30%, 9%, 38%, and 23%, respectively. The preparation methods of the hydrophobically modified expanded perlite microspheres, aminated silica aerogel microparticles, and bacterial humic acid-grafted vermiculite composite carrier are consistent with step S2 in Example 2.

[0112] The specific preparation method of the mixed matrix is ​​the same as step S2 in Example 2.

[0113] Step S3, transplanting of tissue culture seedlings: consistent with step S3 in Example 2.

[0114] Comparative Example 5:

[0115] A method for cultivating ginger tissue culture seedlings, comprising:

[0116] Step S1, Material Preparation: Same as Step S1 in Example 2.

[0117] Step S2, Preparation of mixed matrix: Same as step S2 in Example 2.

[0118] Step S3, Transplanting of Tissue Culture Seedlings: Select a seedling tray with water absorption holes at the bottom and a matching tray as the transplanting container. Evenly fill each hole of the seedling tray with the mixed substrate, filling it to a height of 1cm above the top edge of the hole. Gently scrape it flat and lightly compact it. Then, vertically punch a hole in the center of the substrate in each hole, with a depth of 0.7cm and a diameter matching the stem segment of the tissue culture seedling. After that, vertically insert the rootless and leafless tissue culture seedlings obtained in Step S1 into the holes, ensuring that the root neck is completely buried in the substrate hole and the stem segment is upright. Gently compact the substrate around the seedling to fix the seedling and prevent it from falling over.

[0119] Finally, the cultivation is carried out in four stages according to time: root primordia differentiation cultivation stage, adventitious root construction cultivation stage, root system stress-resistant tillering cultivation stage, and strong seedling cultivation stage. The root primordia differentiation cultivation stage is 1 to 7 days after transplanting, the adventitious root construction cultivation stage is 8 to 20 days after transplanting, the root system stress-resistant tillering cultivation stage is 21 to 35 days after transplanting, and the strong seedling cultivation stage is 36 to 40 days after transplanting.

[0120] The adventitious root construction and cultivation stage, the root system stress-resistant tillering cultivation stage, and the seedling cultivation stage are completely consistent with step S3 of Example 2; the root primordia differentiation cultivation stage directly uses 1 / 10 MS medium as nutrient solution, and the cultivation method (including cultivation environment, irrigation method, etc.) is consistent with step S3 of Example 2.

[0121] Comparative Example 6:

[0122] A method for cultivating ginger tissue culture seedlings, comprising:

[0123] Step S1, Material Preparation: Same as Step S1 in Example 2.

[0124] Step S2, Preparation of mixed matrix: Same as step S2 in Example 2.

[0125] Step S3, Transplanting of Tissue Culture Seedlings: Select a seedling tray with water absorption holes at the bottom and a matching tray as the transplanting container. Evenly fill each hole of the seedling tray with the mixed substrate, filling it to a height of 1cm above the top edge of the hole. Gently scrape it flat and lightly compact it. Then, vertically punch a hole in the center of the substrate in each hole, with a depth of 0.7cm and a diameter matching the stem segment of the tissue culture seedling. After that, vertically insert the rootless and leafless tissue culture seedlings obtained in Step S1 into the holes, ensuring that the root neck is completely buried in the substrate hole and the stem segment is upright. Gently compact the substrate around the seedling to fix the seedling and prevent it from falling over.

[0126] Finally, the cultivation is carried out in four stages according to time: root primordia differentiation cultivation stage, adventitious root construction cultivation stage, root system stress-resistant tillering cultivation stage, and strong seedling cultivation stage. The root primordia differentiation cultivation stage is 1 to 7 days after transplanting, the adventitious root construction cultivation stage is 8 to 20 days after transplanting, the root system stress-resistant tillering cultivation stage is 21 to 35 days after transplanting, and the strong seedling cultivation stage is 36 to 40 days after transplanting.

[0127] The root primordia differentiation cultivation stage, root stress-resistant tillering cultivation stage, and seedling cultivation stage are completely consistent with step S3 of Example 2; the adventitious root construction cultivation stage directly uses 1 / 10 MS medium as nutrient solution, and the cultivation method (including cultivation environment, irrigation method, etc.) is consistent with step S3 of Example 2.

[0128] Comparative Example 7:

[0129] A method for cultivating ginger tissue culture seedlings, comprising:

[0130] Step S1, Material Preparation: Same as Step S1 in Example 2.

[0131] Step S2, Preparation of mixed matrix: Same as step S2 in Example 2.

[0132] Step S3, Transplanting of Tissue Culture Seedlings: Select a seedling tray with water absorption holes at the bottom and a matching tray as the transplanting container. Evenly fill each hole of the seedling tray with the mixed substrate, filling it to a height of 1cm above the top edge of the hole. Gently scrape it flat and lightly compact it. Then, vertically punch a hole in the center of the substrate in each hole, with a depth of 0.7cm and a diameter matching the stem segment of the tissue culture seedling. After that, vertically insert the rootless and leafless tissue culture seedlings obtained in Step S1 into the holes, ensuring that the root neck is completely buried in the substrate hole and the stem segment is upright. Gently compact the substrate around the seedling to fix the seedling and prevent it from falling over.

[0133] Finally, the cultivation is carried out in four stages according to time: root primordia differentiation cultivation stage, adventitious root construction cultivation stage, root system stress-resistant tillering cultivation stage, and strong seedling cultivation stage. The root primordia differentiation cultivation stage is 1 to 7 days after transplanting, the adventitious root construction cultivation stage is 8 to 20 days after transplanting, the root system stress-resistant tillering cultivation stage is 21 to 35 days after transplanting, and the strong seedling cultivation stage is 36 to 40 days after transplanting.

[0134] The root primordia differentiation and cultivation stage, adventitious root construction and cultivation stage, and seedling cultivation stage are completely consistent with step S3 of Example 2; the root system stress-resistant tillering cultivation stage directly uses 1 / 10 MS medium as nutrient solution, and the cultivation method (including cultivation environment, irrigation method, etc.) is consistent with step S3 of Example 2.

[0135] Comparative Example 8:

[0136] A method for cultivating ginger tissue culture seedlings, comprising:

[0137] Step S1, Material Preparation: Same as Step S1 in Example 2.

[0138] Step S2, Preparation of mixed matrix: Same as step S2 in Example 2.

[0139] Step S3, Transplanting of Tissue Culture Seedlings: Select a seedling tray with water absorption holes at the bottom and a matching tray as the transplanting container. Evenly fill each hole of the seedling tray with the mixed substrate, filling it to a height of 1cm above the top edge of the hole. Gently scrape it flat and lightly compact it. Then, vertically punch a hole in the center of the substrate in each hole, with a depth of 0.7cm and a diameter matching the stem segment of the tissue culture seedling. After that, vertically insert the rootless and leafless tissue culture seedlings obtained in Step S1 into the holes, ensuring that the root neck is completely buried in the substrate hole and the stem segment is upright. Gently compact the substrate around the seedling to fix the seedling and prevent it from falling over.

[0140] Finally, the cultivation is carried out in four stages according to time: root primordia differentiation cultivation stage, adventitious root construction cultivation stage, root system stress-resistant tillering cultivation stage, and strong seedling cultivation stage. The root primordia differentiation cultivation stage is 1 to 7 days after transplanting, the adventitious root construction cultivation stage is 8 to 20 days after transplanting, the root system stress-resistant tillering cultivation stage is 21 to 35 days after transplanting, and the strong seedling cultivation stage is 36 to 40 days after transplanting.

[0141] The root primordia differentiation and cultivation stage, the adventitious root construction and cultivation stage, and the root system stress-resistant tillering cultivation stage are consistent with step S3 in Example 2; the seedling cultivation stage directly uses 1 / 10 MS medium as nutrient solution, and the cultivation method (including cultivation environment, irrigation method, etc.) is consistent with step S3 in Example 2.

[0142] The transplanted tissue culture seedlings obtained in Examples 1-3 and Comparative Examples 1-8 were tested for seedling survival rate, adventitious root formation rate 7 days after transplanting, seedling vigor index, seedling disease incidence, and field transplant survival rate (each group had at least 50 transplanted tissue culture seedlings transplanted under the same greenhouse conditions; all groups were transplanted to the field 40 days after transplanting). The test results are as follows:

[0143] Seedling survival rate = (Number of surviving seedlings at the end of the seedling period / Total number of seedlings planted) × 100%;

[0144] Adventitious root formation rate = (Number of seedlings that formed qualified adventitious roots within 7 days / Total number of seedlings planted) × 100%;

[0145] Seedling vigor index = (stem diameter / plant height + number of effective tillers) × total plant dry weight;

[0146] Seedling disease incidence = (cumulative number of diseased seedlings throughout the entire seedling period / total number of seedlings after transplanting) × 100%;

[0147] Field transplant survival rate = (Number of healthy seedlings surviving after the field seedling establishment period / Total number of transplanted seedlings) × 100%;

[0148]

[0149] As shown in the table above, cultivation using the specific mixed substrate and four-stage nutrient solution of this invention can achieve a high adventitious root formation rate and segment differentiation time, ensuring the survival rate of transplanted tissue culture seedlings. At the same time, sufficient phosphorus and potassium nutrition increases the seedling vigor index, thereby ensuring the robustness of ginger tissue culture seedlings, the level of root-crown coordination, and the potential for field planting. In addition, it also reduces the incidence of disease in the seedling stage, achieving a balance of strong roots, vigorous seedlings, and low disease incidence.

Claims

1. A method for cultivating rootless and leafless ginger tissue culture seedlings, characterized in that: include: Step S1, Material Preparation: Remove all original roots and callus tissue at the base of the ginger virus-free tissue culture seedlings, leaving only the root neck connecting the above-ground part and the root system; at the same time, cut off the excessively long stems and all leaves of the above-ground part, leaving only a 2-3cm healthy stem segment, with a clean cut without tearing. Select ginger virus-free tissue culture seedlings that have grown for 30-45 days, are 5-8 cm tall, have 3-5 fully expanded leaves, have thick stems, are vigorous, and are free from vitrification and obvious pests and diseases; after removing the ginger virus-free tissue culture seedlings from the culture bottle, rinse them 3-4 times with sterile water. Step S2, Preparation of Mixed Matrix: The mixed matrix is ​​prepared in advance and sterilized non-destructively before being placed into a container to ensure a smooth surface. The mixed matrix includes hydrophobically modified expanded perlite microspheres, aminated silica aerogel microparticles, bacterial-loaded humic acid-grafted vermiculite composite carrier, and enzymatically hydrolyzed peat fiber, with their mass percentages being 29.5–30.5%, 8.5–9.5%, 37.5–38.5%, and 22.5–23.5%, respectively. The specific preparation method of the humic acid-grafted vermiculite composite carrier is as follows: 200-mesh vermiculite is added to a 1 mol / L hydrochloric acid solution, with a mass-to-volume ratio of vermiculite to hydrochloric acid solution of 0.9–1.1 g: 4.5–5.5 mL. The mixture is first stirred at 78–82℃ and 140–160 rpm for 3.8–4.2 h, then filtered and washed until neutral, and dried at 102–108℃ for 2–3 h. Afterwards, the activated vermiculite and humic acid are mixed at a mass ratio of 4.75–5.25: 1.9–2.

1. Mix the ingredients and add a mixed crosslinking agent of EDC and NHS at a molar ratio of 1:

1. The mixed crosslinking agent accounts for 0.48–0.52% of the total mass of vermiculite and humic acid. Stir at 180–220 rpm for 5.8–6.2 h in a constant temperature water bath at 48–52 °C. After filtration and washing, vacuum dry at 58–62 °C and -0.08–-0.09 MPa for 3–5 h to obtain the carrier. Finally, place the sterilized carrier into a sterile vacuum adsorption bottle and add a bacterial solution with a concentration of 1×10⁻⁶. 9 A CFU / mL suspension of Candida langiocarbazin was prepared with a carrier-to-suspension solid-liquid ratio of 0.9–1.1 g: 2.7–3.3 mL. The suspension was maintained under a vacuum of -0.08–0.09 MPa for 15–20 min, followed by slow introduction of sterile air. The bacterial suspension permeated into the pores of the carrier under the pressure difference. This process was maintained for 10–12 min, and the suspension was then air-dried at 27–29 °C until the water content reached 30%. Step S3, Transplanting of Tissue Culture Seedlings: Select a seedling tray with drainage holes at the bottom and a matching tray as the transplanting container. Evenly fill each hole of the seedling tray with the mixed substrate, filling it to a height of 1 cm above the top edge of the hole. Gently scrape it level and lightly compact it. Then, vertically punch a hole in the center of the substrate in each hole, with a depth of 0.5-1 cm and a diameter matching the stem segment of the tissue culture seedling. Next, vertically insert the rootless and leafless tissue culture seedlings obtained in Step S1 into the hole, ensuring that the root neck is completely buried in the substrate hole and the stem segment is upright. Gently compact the substrate around the seedling to fix it and prevent it from falling over. Finally, carry out the four stages of cultivation in sequence according to the time, and the transplanted tissue culture seedlings will be obtained.

2. The cultivation method for rootless and leafless ginger tissue culture seedlings according to claim 1, characterized in that: The preparation method of the hydrophobically modified expanded perlite microspheres is as follows: 30-50 mesh perlite is washed, dried, and then immersed in a 1.8-2.2% KH570-anhydrous ethanol solution. The mass-to-volume ratio of perlite to solution is 0.9-1.1 g: 4.5-5.5 mL. The mixture is first shaken at 38-42℃ and 120-150 rpm for 3.8-4.2 h, then filtered, washed, and dried at 102-108℃ for 2-3 h to obtain the microspheres.

3. The cultivation method for rootless and leafless ginger tissue culture seedlings according to claim 1, characterized in that: The preparation method of the aminated silica aerogel particles is as follows: 100-200 mesh silica aerogel is vacuum dried and then immersed in a 2.8-3.2% KH550-anhydrous ethanol solution. The mass-to-volume ratio of silica aerogel to solution is 0.9-1.1 g: 3.6-4.4 mL. The mixture is first shaken at 100-130 rpm for 5.8-6.2 h under light-protected conditions at 48-52℃. After filtration and washing, the mixture is vacuum dried at 58-62℃ and -0.08--0.09 MPa for 4-6 h to obtain the final product.

4. The cultivation method for rootless and leafless ginger tissue culture seedlings according to claim 1, characterized in that: The specific method for preparing enzymatically hydrolyzed peat fiber is as follows: Peat is pulverized and added to a buffer solution composed of a pH 4.8 acetic acid solution and a sodium acetate solution. The mass-to-volume ratio of peat to buffer solution is 0.9–1.1 g: 9–11 mL. The mixture is stirred at 180–220 rpm to form a peat suspension. Then, cellulase is added to the peat suspension at an amount of 1.8–2.2% of the peat mass, and the mixture is stirred at 48–52°C and 140–160 rpm for 3.8–4.2 h. Afterward, the enzymatically hydrolyzed suspension is placed in a constant temperature water bath at 98–102°C for 8–10 min, and the peat fiber is repeatedly washed with deionized water until the pH of the filtrate stabilizes at 5.5–6.

0. ​​Finally, the fiber filter cake is collected by filtration, air-dried to semi-dry, sieved, and graded. The graded fibers are then dried at 58–62°C to constant weight to obtain the final product.

5. The cultivation method for rootless and leafless ginger tissue culture seedlings according to claim 1, characterized in that: In step S3, there are four stages: root primordia differentiation and cultivation stage, adventitious root construction and cultivation stage, root system stress-resistant tillering cultivation stage, and seedling cultivation stage. The root primordia differentiation and cultivation stage lasts for 1 to 7 days after transplanting, the adventitious root construction and cultivation stage lasts for 8 to 20 days after transplanting, the root system stress-resistant tillering cultivation stage lasts for 21 to 35 days after transplanting, and the seedling cultivation stage lasts for 36 to 40 days after transplanting.

6. The cultivation method for rootless and leafless ginger tissue culture seedlings according to claim 5, characterized in that: The root primordia differentiation cultivation stage is carried out in an environment with a temperature of 24-26℃, an air humidity of not less than 80%RH, a light intensity of 2400-2600Lux, and a light duration of 12h / d. Root primordia differentiation nutrient solution is used for irrigation: the first irrigation after transplanting involves thoroughly watering the substrate with the root primordia differentiation nutrient solution and then emptying any excess liquid from the tray; when the mixed substrate humidity is below 55%, supplemental irrigation is performed by evenly spraying the nutrient solution along the substrate surface using a fine-nozzle sprayer, directly pouring the nutrient solution into the substrate; The nutrient solution for root primordia differentiation included 1 / 10 MS medium, pH-sensitive chitosan-coated amino acid nitrogen, N-(1-naphthyl)-o-carbamoylbenzoic acid, 24-epibrassinolide, 1,4-butanediamine, tea tree oil nanoemulsion, urea peroxide, γ-polyglutamic acid, and potassium humate; among these, pH-sensitive chitosan-coated amino acid nitrogen, N-(1-naphthyl)-o-carbamoylbenzoic acid, 24-epibrassinolide, 1,4-butanediamine, and tea tree oil nanoemulsion were included. The concentrations of tree essential oil nanoemulsion, urea peroxide, γ-polyglutamic acid, and potassium humate were 0.78–0.82 g / L, 0.09–0.11 μmol / L, 0.18–0.22 μmol / L, 0.9–1.1 μmol / L, 0.09–0.11 g / L, 4.8–5.2 mg / L, 0.28–0.32 g / L, and 0.18–0.22 g / L, respectively.

7. The cultivation method for rootless and leafless ginger tissue culture seedlings according to claim 5, characterized in that: The adventitious root establishment and cultivation stage was carried out in an environment with a temperature of 24-26℃, an air humidity of 75%-80%RH, a light intensity of 3400-3600Lux, and a light duration of 14h / d. Adventitious root establishment nutrient solution was used for irrigation: the first irrigation was performed on the 8th day after transplanting, thoroughly soaking the substrate with the nutrient solution, and then emptying any excess liquid from the tray; thereafter, supplemental irrigation was performed every 5 days, with each irrigation amounting to 60% of the substrate's water holding capacity. The nutrient solution for adventitious root construction included 1 / 10 MS medium, N6-isopentenyl adenosine, L-tryptophan, strigolactone analogue, sodium borate, zinc sulfate, salicin, humic acid chelated trace elements, γ-polyglutamic acid, chitosan oligosaccharide, and Bacillus subtilis; wherein the concentrations of N6-isopentenyl adenosine, L-tryptophan, strigolactone analogue, sodium borate, zinc sulfate, salicin, humic acid chelated trace elements, γ-polyglutamic acid, and chitosan oligosaccharide were 0.5%. 8–0.62 μmol / L, 0.08–0.12 μmol / L, 0.28–0.32 μmol / L, 1.9–2.1 μmol / L, 0.08–0.12 μmol / L, 0.48–0.52 μmol / L, 0.95–1.05 mL / L, 0.28–0.32 g / L, 0.33–0.37 g / L; the concentration of Bacillus subtilis was 2 × 10⁻⁶. 8 CFU / mL.

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