In vitro regeneration and potting method of tomato with fruit cluster

Abstract

The present application relates to a kind of in vitro regeneration potting method of fruit cluster tomato, belong to horticultural plant asexual propagation technical field.The method solves the technical problems that traditional tomato pot cultivation cycle is as long as 70-120 days, in vitro fruit cluster regeneration survival rate is low.The technical scheme points include: the branch cluster containing two node positions, a piece of complete leaf and fruit cluster of more than 80% maturity is taken from field tomato plant;The lower end cut is disinfected and is treated with rooting powder solution;The branch cluster lower end is soaked in water 2-3 centimeters depth;It is placed in semi-shading environment with 50-70% shading rate, temperature 15-30 DEG C;The leaf water content is maintained by foliar spray until rooting;Rooted plant is moved into hydroponic container, and root system is immersed in tomato special nutrient solution and is maintained.The method is used for the rapid preparation of ornamental and edible value pot tomato, realizes 7 days in fruit pot cultivation.

Description

Technical Field

[0001] This invention belongs to the field of asexual reproduction technology of horticultural plants, specifically relating to a method for in vitro regeneration of potted tomatoes with fruit spikes. Background Technology

[0002] Tomatoes, valued for both their ornamental and edible qualities, are often grown in pots. Traditional pot cultivation typically begins with sowing or cuttings to cultivate seedlings, which are then transplanted into pots and allowed to complete their full growth cycle until flowering and fruiting. This process is lengthy, usually taking 70–120 days from seedling stage to fruit maturity. The long cultivation period is due to the fact that tomatoes need to complete physiological stages such as vegetative growth, flower bud differentiation, fruit setting, and fruit coloring in sequence. The limited root growth in a pot further slows down this development. In traditional pot cultivation, plants grow from the seedling stage in a limited volume of soil, physically restricting root development. Compared to field cultivation, potted tomatoes have significantly fewer roots, resulting in an unbalanced root-to-shoot ratio. Limited root absorption capacity reduces the plant's efficiency in utilizing water and fertilizer, thus affecting fruit yield and quality. Specifically, the number of fruits per plant decreases by about 30–40%, the soluble solids content of the fruit decreases by 1.5–2.0°Brix, and the synthesis of flavor compounds (such as hexanal and β-ionone) is only 50–60% of that of field-grown fruit.

[0003] To accommodate the space required for potted plants, tomato plant height needs to be controlled. Conventional methods include spraying plant growth retardants (such as paclobutrazol) to shorten internodes, or restricting vertical growth through pruning and training. However, chemical dwarfing can easily lead to excessive internode compression, causing leaves to cluster and fruit clusters to become crowded, reducing ornamental value; at the same time, pesticide residues may inhibit sugar accumulation in the fruit, resulting in flavor degradation. Physical training requires frequent manual intervention, and improper operation can easily damage the stems and vines.

[0004] Existing techniques for direct transplanting with fruit clusters face the challenge of low survival rates of detached branches and clusters. Detached branches and clusters suffer from disrupted sieve tubes, blocking the transport of organic nutrients, and the interruption of sugar supply accelerates fruit ripening and drop. Simultaneously, the cut surfaces are susceptible to infection by pathogens (such as Fusarium and Erwinia), causing the branches and clusters to rot before rooting. Maintaining water balance in detached tissues is also difficult—transpiration from branches and leaves continuously consumes water, and the lack of roots means water replenishment relies solely on foliar spraying, making them prone to wilting or mold formation when environmental humidity fluctuates.

[0005] Therefore, traditional potted cultivation methods suffer from drawbacks such as long cultivation cycles, limited fruit quality, and high costs associated with controlling plant height. Meanwhile, detached fruit-bearing regeneration technology has not been practically implemented due to difficulties in the survival of the detached fruit clusters. Shortening the cultivation cycle while ensuring fruit quality has become a technical contradiction: directly transplanting mature fruit clusters can save growth time, but the regeneration adaptability of detached organs needs to be addressed; ensuring regeneration success requires sacrificing fruit quality. This contradiction restricts the industrial development of ornamental potted tomatoes. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0007] This invention solves the following technical problems: This research addresses the problems of long cultivation cycles (70-120 days) for traditional potted tomatoes, declining fruit quality due to root system limitations, and low survival rates of regenerated detached fruit-bearing branches. Detached branches struggle to root due to nutrient interruption, pathogen infection, and water imbalance, hindering the industrialization of potted tomato cultivation with fruit.

[0008] This addresses the contradiction between carbon starvation and increased transpiration caused by insufficient photosynthesis in detached shoots under shading conditions. Traditional supplemental lighting cannot balance high photosynthetic efficiency with low transpiration rates, thus affecting the survival of shoots before rooting.

[0009] This addresses the risk of dew and mold growth on fruit clusters caused by continuous foliar spraying due to humidity fluctuations, as well as the problem of water loss or high humidity diseases in branches and leaves caused by differences in transpiration driving forces during supplemental lighting / non-supplemental lighting cycles.

[0010] This method addresses the delayed root primordium differentiation caused by rapid depletion of carbon sources in detached branches. Conventional constant-temperature soaking accelerates respiration and extends rooting time to 7-10 days.

[0011] To resolve the synergistic contradiction between carbon starvation and respiratory metabolic imbalance. A single temperature method cannot simultaneously meet the needs of early cell division activation and later carbon source preservation.

[0012] This method addresses the osmotic pressure surge damage to the root system caused by weakened mechanical tissue during the transition from hydroponics to soil cultivation, resulting in a transplant survival rate of less than 50%.

[0013] This addresses the problem of slow root development and decreased stress resistance caused by the lack of soil microbial signals in hydroponic systems.

[0014] This method addresses the deficiency of inhibited terpene flavor compound synthesis in long-term hydroponics. A sterile environment blocks mycorrhizal symbiotic signals, resulting in fruit volatile matter content that is only 30-40% of that in soil-grown fruit.

[0015] This addresses the issues of uneven ethylene concentration and fruit drop risk caused by direct ethylene introduction, as well as acid-base fluctuations resulting from ethylene hydrolysis.

[0016] To achieve these objectives and other advantages according to the present invention, a method for in vitro regeneration of tomato plants with fruit clusters in pots is provided, comprising the following steps: S1: Take a fruit cluster from a tomato plant grown in the field, the fruit cluster containing at least two nodes, at least one complete leaf and at least one fruit cluster with a maturity of more than 80%. S2: Disinfect the cut at the lower end of the branch and dip the cut in rooting powder solution; S3: Immerse the lower end of the twig in water to a depth of 2-3 cm; S4: Place the soaked branches in a semi-shaded environment with a shading rate of 50%-70% and maintain the ambient temperature at 15-30℃. S5: Maintain the water content of the leaves and fruit clusters of the branches and spikes through foliar spraying until the branches and spikes take root; S6: After the plant has rooted, transfer it to a hydroponic container and immerse the roots in a tomato-specific nutrient solution to cultivate it, thus obtaining a tomato ornamental potted plant with both ornamental and edible value.

[0017] Preferably, in step S4, a controllable spectrum supplementary lighting device is added to the semi-shaded environment. The supplementary lighting device provides a light source with blue light as the main spectrum at 450-460nm, with blue light accounting for 40-50% of the spectral energy distribution, red light at 630-660nm accounting for 20-25%, and the remainder being white light. The supplementary lighting cycle is 12-14 hours per day, and the light intensity is controlled at 80-120μmol•m⁻²•s⁻¹.

[0018] Preferably, in the leaf spraying operation in step S5, an intermittent directional micro-mist system is used. This system monitors the air humidity around the leaves in real time through a humidity sensor. When the humidity is below 65%, the spraying starts, the spraying duration is ≤10 seconds, the droplet size is 30-50μm, and the spraying direction is towards the back of the leaves. The supplemental lighting device and the intermittent directional micro-mist system are linked through a controller to maintain the air humidity around the leaves in the range of 70%±5% during the supplemental lighting cycle and control the humidity in the range of 60%±5% during the non-supplemental lighting cycle.

[0019] Preferably, 0.05-0.1 mmol•L⁻¹ of α-ketoglutarate and 0.1-0.2 μmol•L⁻¹ of brassinolide are added to the soaking water in step S3, and the dissolved oxygen concentration is maintained at 6.0-8.0 mg•L⁻¹.

[0020] Preferably, in the semi-shaded environment of step S4, the lower part of the twig soaked in the gradient cooling zone is placed in the gradient cooling zone so that the temperature at the cut is reduced in stages: specifically, on the first and second days of soaking, the temperature is maintained at 25±1℃; on the third day, it is reduced to 20±1℃; and from the fourth day onwards, it is reduced to 18±0.5℃.

[0021] Preferably, the rooted plants are pretreated with proline before transplanting: the roots are soaked in a 5-10 mmol•L⁻¹ proline solution for 30 minutes at a temperature of 22±1℃.

[0022] Preferably, a sterilized soil extract is added to the tomato-specific nutrient solution at a volume of 3-5% v / v of the nutrient solution. The method for preparing the sterilized soil extract includes the following steps: a) Take rhizosphere soil from field-grown tomatoes, pass it through a 2mm sieve, and mix it with water at a ratio of 1:5; b) Sterilize it at 121℃ for 30 minutes, cool it, and centrifuge to obtain the supernatant; c) Sterilize it through a 0.22μm filter membrane to obtain the sterilized soil extract.

[0023] Preferably, the tomato-specific nutrient solution also contains a water-soluble arbuscular mycorrhizal fungal metabolite extract, added at a rate of 0.8-1.2% v / v of the nutrient solution volume. The preparation method of the water-soluble arbuscular mycorrhizal fungal metabolite extract is as follows: a) *Cyclocarya mosie* is cultured in modified Hoagland liquid medium for 21 days, and the mycelium is collected; b) the mycelium is ground with liquid nitrogen and then extracted with phosphate buffer at pH 6.2 at 4°C for 12 hours; c) the supernatant is collected by centrifugation and then filtered through a 10kDa ultrafiltration membrane to remove macromolecules, obtaining active components with a molecular weight <10kDa.

[0024] Preferably, ethylene slow-release agent is added to the tomato-specific nutrient solution at the time of transplanting, at a dosage of 0.5-1.5 g per liter of nutrient solution, followed by 0.5 g•L⁻¹ every 7 days. The ethylene slow-release agent is prepared by the following steps: 1) Zeolite ZIF-8 particles with a pore size of 4-6 nm and a particle size of 100-200 nm are calcined at 300°C for 2 hours, cooled, and then immersed in a 5% v / v ethanol solution of 3-aminopropyltriethoxysilane. The mixture is shaken at 50°C for 12 hours for modification, and centrifuged and washed to obtain an aminated carrier; 2) The aminated carrier is immersed in a 40% aqueous solution of 2-chloroethylphosphonic acid, and 0.1 mol•L⁻¹ phosphate buffer (pH 5.8) is added. The mixture is vacuum-loaded at 50°C for 4 hours, and centrifuged to remove the residue to obtain an ethephon-carrier complex; 3) The complex is dispersed in a 2% w / v sodium alginate solution and added dropwise to a final volume of 0.5 mol•L⁻¹. Cross-linking in CaCl2 solution forms calcium alginate microcapsules with a diameter of 1-2 mm. After washing and drying, ethylene sustained-release products are obtained.

[0025] This invention has at least the following beneficial effects: By using detached fruit-bearing branches for hydroponic regeneration, the pot cultivation cycle can be shortened from 70-120 days to within 7 days. Detached branches retain the quality of field-grown fruit, avoiding the flavor degradation caused by limited root systems in potted plants. Targeted water immersion combined with foliar spraying maintains moisture balance, resulting in a rooting rate of over 90% for the branches.

[0026] The 450-460nm blue light spectrum increases the net photosynthetic rate to 3.5-4.2 μmol CO2·m⁻²·s⁻¹, while simultaneously increasing it to 3.2-3.8 μmol CO2·m⁻²·s⁻¹, with the transpiration rate increase controlled within 25%, thus alleviating carbon starvation. A red light concentration of 20-25% inhibits excessive transpiration, ensuring the fruit remains intact before rooting.

[0027] During the supplemental lighting period, a humidity level of 70%±5% reduces transpiration by 25%, while a humidity level of 60%±5% during the non-supplemental lighting period inhibits pathogens. 30-50μm droplets target the stomatal zone on the underside of leaves, preventing condensation on the fruit ears and reducing the incidence of gray mold to zero.

[0028] α-Ketoglutarate supplementation of TCA cycle intermediates increased ATP content by 2.3 times; brassinolide (0.1-0.2 μmol·L⁻¹) induced WOX11 gene expression, advancing root primordia differentiation by 48 hours.

[0029] Gradual cooling (25℃→20℃→18℃) reduced respiration intensity by 35% and extended carbon source maintenance time by 2 days; 18℃ induced CBF1 gene expression, increased root proline accumulation by 3 times, and enhanced stress resistance.

[0030] Proline pretreatment enhances cell osmotic pressure, increases root SOD activity by 50% after transplanting, reduces membrane lipid peroxidation damage by 70%, and increases survival rate to 85%.

[0031] Sterilized soil extract provides humic acid and ion chelates, which induce the expression of the expansion protein gene, increase the fresh weight of roots by 40%, and improve the survival rate of hydroponics to soil culture to 80%.

[0032] AMF metabolite extract (<10kDa) contains branching factors, activates LYK3 / CERK1 receptors, restores the expression level of fruit terpene synthase gene to soil culture level, and the total volatile matter content reaches 98%.

[0033] Ethylene slow-release agent is controlled to release ethylene via calcium alginate microcapsules, with an ethylene release fluctuation rate of <5% within 7 days, avoiding fruit drop caused by local concentration exceeding the standard; pH buffer carrier maintains the acid-base stability of nutrient solution, with conductivity change of <10%.

[0034] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0035] Figure 1 This is a photograph of a potted plant obtained by the method of the present invention. Detailed Implementation

[0036] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0037] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0038] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0039] <Example 1> A method for regenerating tomato plants with fruit clusters in detachment and using potted plants includes the following steps: S1: Cut a fruit cluster from a tomato plant grown in the field, the fruit cluster containing two nodes, one complete leaf and one fruit cluster with a maturity of more than 80%; S2: Disinfect the cut at the lower end of the branch with 75% alcohol and dip the cut in 1000mg / L rooting powder solution; S3: Immerse the lower part of the twig in water to a depth of 2.5 cm; S4: Place the soaked branches in a semi-shaded environment with a shading rate of 60% and maintain the ambient temperature at 25℃. S5: Maintain the water content of the leaves and fruit clusters of the branches and spikes through foliar spraying until the branches and spikes take root; S6: After rooting, transfer the plant to a hydroponic container, immersing the roots in a tomato-specific nutrient solution for cultivation, resulting in an ornamental potted tomato plant with both ornamental and edible value. For example... Figure 1 As shown.

[0040] Cut fruit clusters from healthy tomato plants in the field. Use sterilized pruning shears to make a 45° angled cut 1 cm below the base of the node. Commercially available stainless steel pruning shears can be used. Immediately after cutting, place the cuttings in a humidifier for transport.

[0041] Disinfect the cut end of the branch cutting: Immerse the cut end in a 75% alcohol solution for 10 seconds, allow it to air dry, then dip it in a 1000 mg / L indolebutyric acid rooting powder solution for 3 seconds. Next, vertically immerse the lower end of the branch cutting into an opaque container filled with clean water, maintaining a immersion depth of 2.5 cm. A 500 mL brown glass bottle can be used as the immersion container, with the water level precisely controlled using the graduation marks. Change the water every 24 hours during the immersion period.

[0042] Place the soaking container in a greenhouse environment with 60% shading and a constant temperature of 25℃. Use an ultrasonic humidifier to maintain a relative humidity of 65%. Spray the leaves (both sides) three times daily using the ultrasonic humidifier, ensuring each spray forms a fine film of water on the leaves without dripping. Once new white roots ≥1 cm in length have sprouted from the basal cut, transfer the plant to a transparent hydroponic pot, ensuring the roots are completely submerged in tomato-specific nutrient solution (EC value 1.8 mS / cm). A 15 cm diameter polypropylene hydroponic pot equipped with a liquid level observation window can be used.

[0043] Technical effects: This method achieves in vitro regeneration of tomato branches with fruit clusters through standardized selection of branches and scions, precise environmental control, and hydroponic transplanting, shortening the pot cultivation cycle while maintaining the original quality of the fruit.

[0044] <Example 2> A method for in vitro regeneration of tomato plants with fruit clusters in pots is adopted, using the method in Example 1. The difference is that in step S4, a controllable spectrum supplemental lighting device is added to the semi-shaded environment. The supplemental lighting device provides a light source with blue light as the main spectrum at 450-460 nm, with blue light accounting for 45% of the spectral energy distribution, red light at 630-660 nm accounting for 22%, and the remainder being white light. The supplemental lighting cycle is 13 hours per day, and the light intensity is controlled at 100 μmol•m⁻²•s⁻¹.

[0045] Install a supplemental lighting device in a semi-shaded environment with a 60% shading rate. This device provides a light source primarily composed of 455nm blue light, accounting for 45% of the total spectral energy, with 650nm red light accounting for 22%, and the remainder being 4000K white light. Commercially available full-spectrum LED plant grow lights can be used, with the light panel installed 30 cm directly above the branches. The supplemental lighting should be turned on for 13 hours daily, with the start and stop times controlled by a timer. The light intensity should be calibrated to 100 μmol·m⁻²·s⁻¹ using a dimmer and monitored in real-time using a photonics sensor.

[0046] The supplemental lighting system is linked to the temperature and humidity control system. During supplemental lighting, when the humidity sensor detects that the air humidity around the leaves is below 65%, the directional spray system is triggered. The spray nozzle is aimed at the stomatal area on the back of the leaves, spraying water mist with a droplet size of 40μm, with each spray lasting 8 seconds. A piezoelectric micro-mist nozzle can be used, mounted on a bracket 15 cm from the back of the leaf. During non-supplemental lighting periods, the humidity threshold is set to 60% and maintained by a dehumidifier. The controller automatically switches the humidity setpoint based on the supplemental lighting signal.

[0047] The spectrum control uses a programmable LED board, with LED beads arranged in a blue:red:white ratio of 45:22:23. A capacitive probe is used as the humidity sensor, installed at the center of the branch and leaf canopy. The spray system consists of a water tank, a miniature water pump, and ceramic atomizing discs; the pump operates at a pressure of 0.2 MPa. All equipment is connected to a PLC controller and operates according to a preset program: supplemental lighting is activated daily from 6:00 AM to 7:00 PM, maintaining humidity at 70% ± 3% during the supplemental lighting period and 60% ± 3% during the non-supplemental lighting period.

[0048] Technical effects: This supplemental lighting solution maintains the photosynthetic needs of branches and spikelets under shading conditions through specific spectral ratios and synergistic humidity control, while reducing water consumption through transpiration. The linkage control system ensures stable environmental parameters, which is beneficial for maintaining the integrity of the fruit and the physiological activity of the leaves of detached branches and spikelets before rooting.

[0049] <Example 3> A method for regenerating tomato plants with fruit clusters in detachment and using potted plants, adopting the method in <Example 2>, differs in that, in the foliar spraying operation in step S5, an intermittent directional micro-mist system is used. This system monitors the air humidity around the leaves in real time through a humidity sensor. When the humidity is below 65%, the spraying is started, the spraying duration is 8 seconds, the droplet size is 40μm, and the spraying direction is towards the back of the leaves. The supplemental lighting device and the intermittent directional micro-mist system are linked through a controller to maintain the air humidity around the leaves in the range of 70% during the supplemental lighting cycle and control the humidity at 60% during the non-supplemental lighting cycle.

[0050] An intermittent, directional micro-mist system is used to maintain leaf humidity. This system is equipped with a capacitive air humidity sensor to monitor air humidity in real time at a distance of 5 cm from the leaf surface. When the humidity drops below a threshold of 65%, the piezoelectric ceramic atomizing nozzle is activated. The nozzle outputs droplets with a diameter of 40 μm, and each spray lasts for 8 seconds. A commercially available ultrasonic atomizing plate can be used as the core component. The nozzle is mounted on an adjustable bracket, aimed at the densely stomata area on the underside of the leaf, with the nozzle 10 cm from the leaf surface. The water tank is made of polyethylene, has a capacity of 5 L, and is connected to the atomizing device via a silicone hose.

[0051] The supplemental lighting device and the micro-mist system operate in coordination via a PLC controller. During the supplemental lighting period (6:00-19:00 daily), the controller sets the target humidity to 70%, with an allowable fluctuation range of ±3%; during non-supplemental lighting periods, the target humidity is set to 60% ±3%. Humidity sensor data is collected every 30 seconds, and spraying is activated when three consecutive readings are below the set threshold. An industrial-grade PLC controller can be used, with its input port receiving humidity sensor signals and its output port connected to the atomizer relay. The controller program prioritizes the supplemental lighting signal and automatically switches between humidity control modes.

[0052] After the spray system is activated, a miniature water pump delivers water to the atomization chamber at a pressure of 0.15 MPa. A piezoelectric ceramic plate, oscillating at a high frequency of 1.7 MHz, breaks water molecules into 40 μm droplets, which are then focused and sprayed through a conical guide shroud. The guide shroud has an opening angle of 60°, ensuring that the mist covers more than 80% of the leaf underside area. After each spray cycle, the system re-monitors humidity after a 300-second delay. A brass guide shroud with a polished inner wall can be used to reduce droplet adhesion. The entire atomization assembly is mounted on a height-adjustable aluminum alloy bracket, allowing for precise calibration of the spray angle based on the branch / spike position.

[0053] Technical Effects: This micro-mist system, through precise directional spraying and supplemental lighting control, effectively prevents excessive humidity in the fruit cluster area while maintaining the physiological needs of the leaves. Droplets of specific sizes are efficiently absorbed by the stomata on the underside of the leaves, reducing water waste. Graded management of humidity thresholds reduces the risk of fungal diseases and ensures the survival stability of detached branches during the rooting stage.

[0054] <Example 4> A method for in vitro regeneration of tomato plants with fruit clusters, using the method in <Example 3>, differs in that 0.08 mmol•L⁻¹ of α-ketoglutarate and 0.15 μmol•L⁻¹ of brassinolide are added to the soaking water in step S3, and the dissolved oxygen concentration is maintained at 7.0 mg•L⁻¹.

[0055] Add root-promoting substances to the soaking water. Dissolve 0.118 g of α-ketoglutaric acid powder in 1 L of deionized water to prepare a 0.08 mmol / L stock solution; separately dissolve 0.040 mg of brassinolide in 1 L of ethanol to prepare a 0.1 μmol / L stock solution. When using, add 1 mL of α-ketoglutaric acid stock solution and 1.5 mL of brassinolide stock solution per liter of soaking water. Analytical grade chemical reagents can be used. Store the stock solutions in brown glass bottles at 4℃. Accurately measure the amounts using a pipette, inject into the soaking container, and stir magnetically for 5 minutes to mix thoroughly.

[0056] Maintain the dissolved oxygen concentration of the soaking solution at 7.0 mg / L. A miniature air pump connected to a titanium alloy aeration stone is used, with the aeration stone placed at the center of the bottom of the container. The air pump output pressure is 0.025 MPa, and the air intake rate is controlled at 0.1 L / min via a gas flow meter. A DC-powered miniature air pump can be used, paired with a disc-shaped aeration stone with a 10 μm pore size. Dissolved oxygen levels are monitored using a fluorescence dissolved oxygen meter, with the probe fixed 2 cm below the liquid surface. The air pump automatically starts when the dissolved oxygen level drops below 6.8 mg / L and shuts off when it exceeds 7.2 mg / L.

[0057] The soaking container is a 5L cylindrical glass tank, wrapped with aluminum foil for light protection. The soaking solution is changed every 24 hours: first, drain the old solution, rinse the inner wall of the container three times with deionized water, and then refill with the prepared additive solution. Maintain the water temperature at 25℃±0.5℃ during solution changes, achieved through a constant-temperature water bath jacket. A PTFE drain valve can be used, installed on the side wall of the container 1cm from the bottom. After each solution change, check the pH value and adjust it to 6.0±0.2 using a 0.1mol / L citric acid solution or sodium bicarbonate solution.

[0058] Technical Effects: This method provides a suitable rooting environment for detached branches and inflorescences through specific concentrations of additives and stable dissolved oxygen control. α-Ketoglutarate supplements intermediate products of energy metabolism, brassinolide promotes cell differentiation, and dissolved oxygen maintains the necessary respiration. Regular solution changes prevent the accumulation of metabolic products, and shading treatment reduces algae growth, all of which promote the rapid formation of robust root systems at the base of the branches and inflorescences.

[0059] <Example 5> A method for in vitro regeneration of tomato plants with fruit clusters, using the method in Example 4, differs in that, in the semi-shaded environment of step S4, the lower part of the branch cluster is placed in a gradient cooling zone to gradually reduce the temperature at the cut: specifically, on the first day of soaking, the temperature is maintained at 25±1℃; on the third day, it is reduced to 20±1℃; and from the fourth day onwards, it is reduced to 18±0.5℃.

[0060] A three-stage temperature control system is employed to achieve gradient cooling. This system consists of a constant-temperature water bath, titanium alloy heat exchange coils, and a temperature controller. The heat exchange coils are spiral-shaped, with an inner diameter of 8mm, and are installed 1cm above the bottom of the immersion container. A semiconductor cooling chip with a cooling capacity of 200W can be used as the cold source, with heat dissipation through an aluminum heat sink. The temperature controller is programmed with three stages: stage one (0-48 hours) with a target temperature of 25.0℃, stage two (48-96 hours) with 20.0℃, and stage three (after 96 hours) with 18.0℃. The controller samples temperature every 10 seconds.

[0061] The heat exchange coil is connected to the circulating water pump via a silicone hose, with the pump speed adjusted to 1.5 L / min. A PT100 platinum resistance temperature sensor is used, with the probe fixed at the cut of the branch, 2 mm from the cut surface. When the detected temperature deviates from the set value by ±0.5℃, the controller automatically adjusts the power of the cooling element. A PID control algorithm can be used, with a proportional gain of 5 and an integral time of 120 seconds. A 2-hour gradual change period is set for the cooling transition phase (e.g., 25℃→20℃), with a cooling rate of 2.5℃ / hour to avoid sudden temperature changes.

[0062] The immersion container is placed inside an insulated chamber with walls made of 3cm thick polyurethane foam. Air circulation within the chamber is maintained by a miniature fan at a speed of 0.3m / s to prevent localized temperature stratification. An 8cm diameter axial fan can be used, installed on the side wall of the chamber 20cm from the bottom. Temperature curve data is recorded daily and exported to a computer via USB. When the ambient temperature exceeds 30℃, the backup cooling module automatically activates to ensure stable execution of the gradient cooling program.

[0063] Technical Effects: This gradient cooling scheme promotes cell division in the early stages of root development in detached shoots through phased temperature regulation, while reducing respiration consumption in the later stages. Precise temperature control reduces energy waste, and a gradual cooling transition avoids cold stress on plant tissues. Combined with the use of root-promoting additives in the early stages, it helps detached shoots to efficiently form new root systems while maintaining fruit quality.

[0064] <Example 6> A method for in vitro regeneration of tomato plants with fruit clusters in pots is adopted, using the method in <Example 5>, except that the rooted plants are pretreated with proline before transplanting: the roots are soaked in an 8 mmol•L⁻¹ proline solution for 30 minutes, and the temperature is maintained at 22±1℃.

[0065] Sterile soil extract is added to a tomato-specific nutrient solution at a volume of 4% v / v. The preparation method of the sterilized soil extract includes the following steps: a) Take rhizosphere soil from field-grown tomatoes, pass it through a 2mm sieve, and mix it with water at a ratio of 1:5; b) Sterilize at 121℃ for 30 minutes, cool, and centrifuge to obtain the supernatant; c) Sterilize through a 0.22μm filter membrane to obtain the sterilized soil extract.

[0066] Prepare an 8 mmol / L L-proline solution. Weigh 0.920 g of L-proline powder and dissolve it in 1 L of deionized water. Stir magnetically for 20 minutes until completely dissolved. Adjust the pH to 6.0 ± 0.1 using 0.1 mol / L sodium hydroxide solution. Store the solution in a 25°C constant temperature water bath for later use. Analytical grade L-proline reagent can be used; the water bath should have a volume of 10 L and an internal quartz heating tube.

[0067] Immerse the roots of the rooting shoots in the pretreatment solution, ensuring the solution completely covers the roots. Use a 2L glass beaker placed in a constant-temperature water bath to maintain the solution temperature at 22.0℃±0.5℃. Immerse for 30 minutes, controlled by a digital timer. A PT100 temperature sensor can be used to monitor the solution temperature, with the probe positioned at the center of the beaker. After treatment, remove the plants, drain for 30 seconds, and then transplant.

[0068] The constant temperature water bath is equipped with a circulating water pump, with a flow rate of 0.8L / min to ensure uniform temperature. The outside of the bath is wrapped with a 5mm thick polystyrene insulation layer. The temperature controller is set with PID parameters: proportional band 2.0, integral time 90 seconds, derivative time 30 seconds. Stainless steel treatment beakers, 15cm in diameter and 20cm deep, can be used, with no more than 5 plants treated at a time.

[0069] Technical effects: This pretreatment enhances the osmotic regulation capacity of root cells, helps alleviate water stress during transplanting, and promotes rapid adaptation of plants to the hydroponic environment.

[0070] Take 200g of rhizosphere soil from the tomato plant (topsoil within 10cm of the main stem) and pass it through a 2mm stainless steel sieve. Add 1000mL of deionized water at a soil-to-water ratio of 1:5 and stir magnetically for 30 minutes. Dispense into high-pressure resistant glass bottles and autoclave at 121℃ for 30 minutes. After cooling, centrifuge at 3000g for 15 minutes, and filter the supernatant through a 0.22μm aqueous filter membrane. A polyethersulfone membrane can be used, and the filtration device should be equipped with a vacuum pump to maintain a negative pressure of 0.08MPa.

[0071] The tomato-specific nutrient solution was prepared according to the Hoagland formula, with the EC value adjusted to 1.8 mS / cm. Sterilized soil extract was added at a ratio of 4% (v / v), i.e., 40 mL of extract per liter of nutrient solution. The mixture was stirred using a magnetic stirrer at 300 rpm for 10 minutes. A 20L polyethylene storage tank equipped with an online pH / EC monitoring probe can be used.

[0072] Store the prepared nutrient solution in a dark environment at a temperature maintained between 18-25℃. Aerate for 30 minutes before use to ensure dissolved oxygen reaches 7.0 mg / L. When transplanting, pour the nutrient solution into the hydroponic pot to a depth of 10 cm, ensuring the roots are completely submerged. A black, opaque storage container with an aeration disc (20 μm pore size) can be used.

[0073] Technical effects: The addition of sterilized soil extract provides soil-derived active substances to the hydroponic system, supporting root development and nutrient absorption, and helping to maintain the stability of plant physiological functions.

[0074] <Example 7> A method for in vitro regeneration of tomato plants with fruit clusters in pots, using the method in <Example 6>, differs in that a water-soluble arbuscular mycorrhizal fungal metabolite extract is added to the tomato-specific nutrient solution at a volume of 1.0% v / v. The preparation method of the water-soluble arbuscular mycorrhizal fungal metabolite extract is as follows: a) *Cyclocarya mosie* is cultured in modified Hoagland liquid medium for 21 days, and the mycelium is collected; b) The mycelium is ground with liquid nitrogen and then extracted with phosphate buffer at pH 6.2 at 4°C for 12 hours; c) The supernatant is collected by centrifugation and then filtered through a 10kDa ultrafiltration membrane to remove macromolecules, obtaining active components with a molecular weight <10kDa.

[0075] *Mesosphaeria mosierifolia* was inoculated onto a modified Hoagland broth containing 20 g of sucrose per liter. The culture was incubated on a shaker at 25℃ ± 0.5℃ and 120 rpm for 21 days. After incubation, the mycelium was collected by filtering through a 200-mesh nylon screen and rinsed three times with sterile deionized water. A 2L glass conical flask with 800 mL of liquid can be used as the culture container. After draining the mycelium, it was immediately flash-frozen in liquid nitrogen.

[0076] Grind the mycelium into powder in liquid nitrogen, and add 10 mL of pH 6.2 phosphate buffer (0.05 mol / L) per 1 g wet weight of mycelium. Extract magnetically with stirring at 150 rpm for 12 hours in a 4°C cold room. Centrifuge the extract at 5000 g for 20 minutes, and filter the supernatant through a 0.45 μm filter membrane. A polytetrafluoroethylene (PTFE) grinding bowl can be used, and the grinding time should be controlled within 3 minutes. Maintain the centrifuge rotor temperature at 4°C ± 1°C.

[0077] The filtrate was treated with a 10 kDa ultrafiltration membrane at an operating pressure of 0.4 MPa, and the permeate with a molecular weight <10 kDa was collected. This permeate was added to the tomato nutrient solution at a ratio of 1.0% (v / v). A tangential flow ultrafiltration system with a membrane area of ​​0.1 m² and a flow rate of 30 L / h can be used. The permeate was stored in a -20°C freezer and thawed at 4°C for 6 hours before use. The nutrient solution was mixed at a temperature of 18°C ​​to prevent deactivation of active substances.

[0078] Technical effects: This metabolite extract provides mycorrhizal symbiotic signaling molecules to the hydroponic system, supports root development and activation of secondary metabolic pathways, and helps maintain the ability to synthesize fruit flavor substances.

[0079] <Example 8> A method for in vitro regeneration of tomato plants with fruit clusters in pots, using the method in <Example 7>, differs in that an ethylene slow-release agent is added to the tomato-specific nutrient solution at the time of transplanting, at a dosage of 1.0 g per liter of nutrient solution, followed by 0.5 g•L⁻¹ every 7 days. The ethylene slow-release agent is prepared through the following steps: 1) Zeolite ZIF-8 particles with a pore size of 4-6 nm and a particle size of 100-200 nm are calcined at 300°C for 2 hours, cooled, and then immersed in a 5% v / v ethanol solution of 3-aminopropyltriethoxysilane. The mixture is then shaken at 50°C for 12 hours for modification, centrifuged, and washed to obtain an aminated carrier; 2) The aminated carrier is immersed in a 40% aqueous solution of 2-chloroethylphosphonic acid, with the addition of 0.1 mol•L⁻¹ phosphate buffer (pH 5.8), and vacuum-loaded at 50°C for 4 hours. The residue is removed by centrifugation to obtain an ethephon-carrier complex; 3) The complex is dispersed in 2%... A sodium alginate solution (w / v) was added dropwise to a 0.5 mol•L⁻¹ CaCl₂ solution to crosslink and form calcium alginate microcapsules with a diameter of 1-2 mm. After washing and drying, an ethylene sustained-release compound was obtained.

[0080] Mesoporous silica with a particle size of 150 nm was calcined in a muffle furnace at 300 °C for 2 hours. After cooling, it was immersed in a 5% 3-aminopropyltriethoxysilane ethanol solution and shaken at 50 °C for 12 hours. The solid was collected by centrifugation, washed three times with ethanol, and dried under vacuum at 60 °C. Polytetrafluoroethylene centrifuge tubes can be used, with a centrifugation force of 3000 g × 10 min. The modified carrier was stored in a desiccator for later use.

[0081] The aminated carrier was immersed in a 40% ethephon aqueous solution, and 0.1 mol / L phosphate buffer (pH 5.8) was added. The mixture was then loaded in a vacuum drying oven at 50°C for 4 hours at a vacuum degree of -0.08 MPa. The residue was removed by centrifugation, and the solid was washed twice with deionized water. A vacuum rotary evaporator at 30 rpm can be used to ensure uniform loading.

[0082] The support was dispersed in a 2% sodium alginate solution at a ratio of 1:10 (w / v). A 0.5 mol / L CaCl2 solution was added dropwise at a rate of 2 mL / min using a syringe pump with a needle diameter of 0.5 mm. After cross-linking for 10 minutes, the microcapsules were collected, washed three times with deionized water, and air-dried at 25°C for 24 hours. A stainless steel needle, 15 cm above the liquid surface, can be used. The final particle size was controlled at 1.5 ± 0.3 mm.

[0083] Technical benefits: This slow-release formulation achieves controlled ethylene release through a microcapsule structure, maintains the pH stability of the nutrient solution, and prevents abnormal fruit drop.

[0084] <Comparative Example 1> A method for regenerating tomato plants with fruit clusters in detachment and using potted plants includes the following steps: S1: Cut a fruit cluster from a tomato plant grown in the field, the fruit cluster containing two nodes, one complete leaf and one fruit cluster with a maturity of more than 80%; S2: Disinfect the cut at the lower end of the branch with 75% alcohol and dip the cut in 1000mg / L rooting powder solution; S3: Sow in a substrate of peat:vermiculite = 3:1, and cover with soil to a thickness of 0.5cm; S4: Place in a 25℃ greenhouse for seedling cultivation, with 12 hours of light per day (ordinary LED white light, light intensity 80μmol·m⁻²·s⁻¹), and continue cultivation.

[0085] Data on rooting rate, transplant survival rate, gray mold incidence, and fruit drop were collected and the results are shown in Table 1.

[0086] Table 1 Example 1 83.3% 5.2 86.7% 10.0% Slight fruit drop (2 bunches) Example 2 90.0% 6.5 93.3% 6.7% No shedding Example 3 93.3% 7.1 96.7% 3.3% No shedding Example 4 96.7% 8.0 100.0% 0.0% No shedding Example 5 100.0% 8.3 100.0% 0.0% No shedding Example 6 100.0% 9.7 100.0% 0.0% No shedding Example 7 100.0% 10.4 100.0% 0.0% No shedding Example 8 100.0% 11.3 100.0% 0.0% No shedding Comparative Example 1 0.0% - - 23.3% All fruit clusters fell off (30 clusters) From Example 1 to Example 8, the rooting rate gradually increased from 83.3% to 100%. This change is closely related to the optimization techniques gradually introduced in each example. Example 2 introduced blue light supplementation, which enhanced photosynthetic efficiency and provided more energy for rooting, thus increasing the rooting rate. Example 4 added α-ketoglutarate and brassinolide; the former supplemented intermediate products of energy metabolism, and the latter promoted root primordia differentiation, further improving the rooting rate. Example 5 used gradient cooling, which reduced respiration consumption and induced the expression of stress resistance genes, resulting in a rooting rate of 100%.

[0087] The root elongation rate increased from 5.2 mm / day in Example 1 to 11.3 mm / day in Example 8. The sterilized soil extract added in Example 6 provided substances such as humic acid, which induced the expression of the expansin gene and promoted root growth; the arbuscular mycorrhizal fungal metabolites introduced in Example 7 activated the relevant receptors, further accelerating root development and resulting in a continuous increase in the root elongation rate.

[0088] Improved transplant survival rate and reduced disease rate: The transplant survival rate increased from 86.7% in Example 1 to 100% in Example 8, and the incidence of gray mold decreased from 10.0% to 0. The intermittent directional micro-mist system in Example 3, by precisely controlling humidity, maintained a suitable humidity range during both the supplemental lighting and non-supplemental lighting periods, which not only prevented dew and mold growth on the ears but also inhibited the growth of pathogens, thus reducing the disease rate; the proline pretreatment in Example 6 enhanced the root system's osmotic pressure regulation capacity and improved the transplant survival rate.

[0089] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for the in vitro regeneration of tomato plants with fruit clusters into pots, characterized in that, Includes the following steps: S1: Take a fruit cluster from a tomato plant grown in the field, the fruit cluster containing at least two nodes, at least one complete leaf and at least one fruit cluster with a maturity of more than 80%. S2: Disinfect the cut at the lower end of the branch and the cutting is 2-3 cm long from the lower node. Dip the cut in rooting powder solution. S3: Immerse the lower end of the shoots in water to a depth of 2-3 cm; add 0.05-0.1 mmol·L⁻¹ of α-ketoglutarate and 0.1-0.2 μmol·L⁻¹ of brassinolide to the immersion water, and maintain the dissolved oxygen concentration at 6.0-8.0 mg·L⁻¹. S4: Place the soaked branches and scions in a semi-shaded environment with a shading rate of 50%-70% and an ambient temperature maintained at 15-30℃. A controllable spectrum supplemental lighting device is added to the semi-shaded environment. This device provides a light source with 450-460nm blue light as the main spectral source, accounting for 40-50% of the spectral energy distribution, 630-660nm red light accounting for 20-25%, and the remainder being white light. The supplemental lighting cycle is 12-14 hours per day, with the light intensity controlled at 80-120μmol·m⁻²·s⁻¹. In the semi-shaded environment, place the lower soaked portion of the branches and scions in a gradient cooling zone to gradually reduce the temperature at the cut: specifically, maintain 25±1℃ on the first 1-2 days of soaking; reduce to 20±1℃ on the third day; and reduce to 18±0.5℃ from the fourth day onwards. S5: Maintain the water content of the leaves and fruit clusters of the branches and spikes through foliar spraying until the branches and spikes take root; in the foliar spraying operation, an intermittent directional micro-mist system is used. The system monitors the air humidity around the leaves in real time through a humidity sensor. When the humidity is lower than 65%, the spraying is started. The spraying duration is ≤10 seconds, the droplet size is 30-50μm, and the spraying direction is towards the back of the leaves. The supplemental lighting device is linked with the intermittent directional micro-mist system through a controller to maintain the air humidity around the leaves in the range of 70%±5% during the supplemental lighting cycle and control the humidity in the range of 60%±5% during the non-supplemental lighting cycle. S6: After the plant has rooted, transfer it to a hydroponic container and immerse the roots in a tomato-specific nutrient solution to cultivate it, thus obtaining a tomato ornamental potted plant with both ornamental and edible value.

2. The method for in vitro regeneration of tomato plants with fruit clusters in pots according to claim 1, characterized in that, Pre-treat rooted plants with proline before transplanting: Soak the roots in a 5-10 mmol·L⁻¹ proline solution for 30 minutes, maintaining the temperature at 22±1℃.

3. The method for in vitro regeneration of tomato plants with fruit clusters in pots according to claim 1, characterized in that, Sterile soil extract is added to tomato-specific nutrient solution at a concentration of 3-5% v / v of the nutrient solution volume. The preparation method of the sterile soil extract includes the following steps: a) Take the rhizosphere soil of field-grown tomatoes, pass it through a 2mm sieve, and mix it with water at a soil-to-water ratio of 1:

5. b) Autoclave at 121℃ for 30 minutes, cool, and centrifuge to collect the supernatant; c) Sterilize the soil extract by passing it through a 0.22 μm filter membrane.

4. The method for in vitro regeneration of tomato plants with fruit clusters in pots according to claim 3, characterized in that, The tomato-specific nutrient solution also contains a water-soluble extract of arbuscular mycorrhizal fungi metabolites, added at a rate of 0.8-1.2% v / v of the nutrient solution volume. The preparation method of the water-soluble arbuscular mycorrhizal fungi metabolite extract is as follows: a) The mycelium of *Cyclocarya mosieri* was cultured in modified Hoagland liquid medium for 21 days and collected; b) The mycelium was ground with liquid nitrogen and then extracted with phosphate buffer at pH 6.2 at 4°C for 12 hours. c) Centrifuge to collect the supernatant, and pass it through a 10kDa ultrafiltration membrane to remove macromolecules and obtain active components with a molecular weight <10kDa.

5. The method for in vitro regeneration of tomato plants with fruit clusters in pots according to claim 4, characterized in that, At the time of transplanting, ethylene slow-release agent was added to the tomato-specific nutrient solution at a rate of 0.5-1.5g per liter of nutrient solution, followed by a supplement of 0.5g·L⁻¹ every 7 days. The ethylene slow-release agent was prepared through the following steps: 1) Zeolite ZIF-8 particles with pore size of 4-6 nm and particle size of 100-200 nm were calcined at 300 °C for 2 hours. After cooling, they were immersed in an ethanol solution of 5% v / v 3-aminopropyltriethoxysilane and modified by shaking at 50 °C for 12 hours. After centrifugation and washing, an aminated carrier was obtained. 2) The aminated carrier was immersed in a 40% aqueous solution of 2-chloroethylphosphonic acid, 0.1 mol·L⁻¹ phosphate buffer was added, pH 5.8, and the mixture was vacuum loaded at 50℃ for 4 hours. The residue was removed by centrifugation to obtain the ethephon-carrier complex. 3) The complex was dispersed in a 2% w / v sodium alginate solution and added dropwise to a 0.5 mol·L⁻¹ CaCl₂ solution for cross-linking to form calcium alginate microcapsules with a diameter of 1-2 mm. After washing and drying, the ethylene sustained-release body was obtained.

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