A method for recovering acer truncatum bunge seeds and application thereof

Abstract

The application relates to the technical field of germplasm resource protection, and particularly relates to a method for recovering Acer truncatum Bunge seeds and application. The recovery method comprises the following steps: using a thawing device to gradient-thaw the ultra-low-temperature-preserved Acer truncatum Bunge seeds, to obtain thawed seeds; the transport time of the gradient-thawing is less than or equal to 5 min; using a composite washing liquid to wash the thawed seeds, to obtain sterile seeds; using a repair culture medium to culture the sterile seeds in a dark environment, to obtain primary recovery seeds; using a basic culture medium to culture the primary recovery seeds, to complete the recovery of the Acer truncatum Bunge seeds; the thawing device comprises a base, a thawing part, a transport part and a fixing frame; the thawing part comprises a thawing partition plate, a first thawing groove, a second thawing groove, a constant-temperature tube, an isolation plate and the fixing frame; the transport part comprises a screw rod, a threaded sliding block, an extension rod, a transport fixing rod and a transport connecting block. The recovery method realizes a relatively high recovery survival rate of the Acer truncatum Bunge seeds by means of the precise temperature control core of the gradient thawing, in combination with the device structure design, the matching processing steps and the repair strategy.

Description

Technical Field

[0001] This application relates to the field of germplasm resource conservation technology, and in particular to a method and application for the revival of Acer truncatum seeds. Background Technology

[0002] Cryopreservation refers to the long-term storage of plant cells or tissues at extreme low temperatures (e.g., -196°C liquid nitrogen). Under these extreme low-temperature conditions, cell differentiation, metabolism, and physiological and biochemical reactions of the preserved germplasm resources almost completely cease, while cell viability and morphogenesis potential are still preserved. Cryopreservation can maximally suppress the physiological metabolic intensity of materials and reduce the frequency of deterioration, thereby achieving the goal of long-term germplasm preservation. It has been proven to be a very effective long-term preservation method. Currently, cryopreservation has been successfully applied to various tissues and organs of plants, including seeds, stem segments and shoot tips, meristems, embryos and somatic embryos, callus, and buds. Cryopreservation has the advantages of not requiring subculturing, no variation in the preserved germplasm resources, and high genetic stability of the preserved materials. It is the core method for the long-term safe preservation of recalcitrant seeds and asexually propagated plant germplasm resources, and also an effective, stable, and economical long-term preservation means for biotechnological materials. Currently, various preservation methods based on cryopreservation have been established, such as the two-step cooling method, pre-culture method, embedding and drying method, vitrification method, and embedding and vitrification method. The typical cryopreservation process is as follows: First, the material to be preserved undergoes appropriate pre-cultivation and loading treatments. Then, after the plant material is treated with a cryoprotectant, it is rapidly immersed in liquid nitrogen. At the same time, the cryoprotectant alters the dynamic behavior of water by forming a hydrogen bond network. For example, the strong hydrogen bond interaction between the cryoprotectant and water can inhibit ice crystal nucleation, reduce the free water content in the solution, or slow down the growth kinetics of ice crystals by disrupting the tetrahedral structure of water. This avoids the formation of ice crystals and prevents mechanical damage to the plant material, thus achieving the purpose of protecting the plant material.

[0003] *Acer truncatum* Bunge, also known as the Chinese maple, is a plant belonging to the genus *Acer* of the family Aceraceae. It is highly valued for its ornamental qualities, strong resilience, and the abundance of functional components in its seeds, oil, and leaves. The oil is particularly rich in nervonic acid, making it a unique woody oilseed tree species and a new resource. Currently, *Acer truncatum* plays a unique role in various fields, including medicine, health care, food and oil security, and biodiesel production. In particular, its oil value has attracted widespread attention from the pharmaceutical and food industries. Given the high value of nervonic acid and other high-value oils in its seeds, the preservation of *Acer truncatum* germplasm resources is of great significance.

[0004] However, the cryopreservation and subsequent recovery of Acer truncatum still face multiple technical bottlenecks. At present, the survival rate of Acer truncatum seeds after cryopreservation is less than 50%, which can easily lead to the waste of Acer truncatum germplasm resources. Summary of the Invention

[0005] This application provides a method and application for the revival of Acer truncatum seeds to solve the following technical problem: how to improve the revival survival rate of Acer truncatum seeds preserved at ultra-low temperatures.

[0006] In a first aspect, embodiments of this application provide a method for reviving Acer truncatum seeds, the revival method comprising: The seeds of Acer truncatum stored at ultra-low temperature were thawed in a gradient using a thawing device to obtain thawed seeds; wherein the transfer time of the gradient thawing was ≤5 min. The thawed seeds were washed with a compound washing solution to obtain sterile seeds; The sterile seeds were cultured in a dark environment using a remediation medium to obtain primary resuscitation seeds. The primary revived seeds were cultured using a basal culture medium to complete the revival of Acer truncatum seeds; The defrosting device includes: Base; The thawing section includes a thawing partition plate, a first thawing tank, a second thawing tank, a constant temperature tube, an isolation plate, and a mounting bracket. The first thawing tank and the second thawing tank are connected in series on the surface of the base. The mounting bracket is fixed in the first thawing tank or the second thawing tank. The first thawing tank and the second thawing tank are separated by the thawing partition plate. The constant temperature tube is respectively disposed in the first thawing tank and the second thawing tank. The mounting bracket is disposed above the constant temperature tube. The mounting bracket is provided with multiple sets of fixing mechanisms for fixing the cryopreservation tube containing the Acer truncatum seeds. The isolation plate is disposed between the mounting bracket and the constant temperature tube. The transfer unit includes a screw, a threaded sliding block, a telescopic rod, a transfer fixing rod, and a transfer connecting block. One end of the rotating connecting block is fixedly connected to one end of the mounting frame, and the other end of the transfer connecting block is fixedly connected to one end of the transfer fixing rod. The other end of the transfer fixing rod is fixedly connected to the telescopic end of the telescopic rod. The telescopic rod is fixed on the threaded sliding block, and the threaded sliding block slides relative to the screw. The screw is symmetrically arranged on both sides of the base, and the length of the screw is greater than the sum of the lengths of the first thawing groove and the second thawing groove, causing the mounting frame to undergo gradient thawing in the first thawing groove or the second thawing groove.

[0007] Optionally, the fixing mechanism includes a frame bracket, a support rod, a first sliding block, a second sliding block, a spring, a clamping unit, and a limiting sleeve. The frame bracket is disposed within the mounting frame, and the support rod is disposed within the frame bracket. The first sliding block and the second sliding block are symmetrically disposed on the support rod and slide on the support rod. The spring is disposed between the first sliding block and the second sliding block. The clamping unit is fixedly connected to both the first sliding block and the second sliding block, and the two clamping units form a clamping space for clamping the cryopreservation tube. One end of the clamping unit abuts against the limiting sleeve, causing the clamping unit to move in the horizontal direction.

[0008] Optionally, the clamping unit includes a clamping fixing rod, a clamping connecting block, an arc-shaped fixing block, and a limiting rod. One end of the clamping fixing rod is fixedly connected to the first sliding block or the second sliding block, and the other end of the clamping fixing rod is connected to one end of the clamping connecting block. The other end of the clamping connecting block is connected to the arc-shaped fixing block, and one end of the arc-shaped fixing block is fixedly connected to the limiting rod. The limiting rod abuts against the limiting sleeve. The two arc-shaped fixing blocks form a clamping space for clamping the cryopreservation tube.

[0009] Optionally, the transfer unit further includes a support block, a slide rod, and a control motor. The output end of the control motor is fixedly connected to the screw to drive the screw to rotate. The support block is fixed to one side of the base, and two support blocks are inserted through both ends of the screw. The slide rod is provided between the two support blocks and is fixed to the base. The threaded sliding block is provided with a sliding groove that matches the slide rod to enable the threaded sliding block to slide on the screw.

[0010] Optionally, the defrosting device further includes a control unit, which includes a temperature controller, a temperature sensor group, and a time controller. The temperature controller is located inside the defrosting partition plate, and the temperature sensor group is located inside the first defrosting tank and the second defrosting tank. The temperature sensors are connected to the temperature controller via electrical signals, and the time controller is connected to the control motor via electrical signals.

[0011] Optionally, the gradient thawing includes a first thawing and a second thawing, wherein the time for the first thawing is the same as the time for the second thawing; The first thawing temperature is 35°C to 45°C, and the first thawing time is 5 min to 10 min; The second thawing temperature is 20°C to 30°C, and the second thawing time is 5 min to 10 min.

[0012] Optionally, the components of the composite detergent include sucrose, trehalose, proline, and vitamin C, wherein the molar concentration of sucrose is 0.25 mol / L to 0.35 mol / L, the molar concentration of trehalose is 0.08 mol / L to 1.2 mol / L, the molar concentration of proline is 25 mmol / L to 35 mmol / L, and the mass concentration of vitamin C is 20 mg / L to 30 mg / L.

[0013] Secondly, embodiments of this application provide an application of the resuscitation method described in the first aspect in the cryopreservation of Acer truncatum seeds, the application including: The seeds of Acer truncatum are dried to obtain dried seeds with a preset moisture content of 8.1% to 8.2%. The dried seeds with a preset moisture content are subjected to gradient protection treatment using a cryoprotectant to obtain vitrified seeds; The vitrified seeds were cryopreserved to obtain cryopreserved Acer truncatum seeds; The cryopreserved Acer truncatum seeds are subjected to the revival method described in the first aspect to obtain revival seeds.

[0014] Optionally, the dried seeds at a preset moisture content are subjected to a gradient protection treatment using a cryoprotectant to obtain vitrified seeds, including the following steps: The dried seeds with a preset moisture content were first soaked using a cryoprotectant with a volume concentration of 60% to obtain first soaked seeds; The first soaked seeds were soaked a second time using the cryoprotectant with a volume concentration of 80% to obtain second soaked seeds; The second soaked seeds were then soaked a third time using the cryoprotectant at a volume concentration of 100% to obtain vitrified seeds; The first soaking time is 18 to 22 minutes, the second soaking time is 35 to 45 minutes, and the third soaking time is 55 to 65 minutes.

[0015] Optionally, the cryoprotectant comprises dimethyl sulfoxide, trehalose, vitamin C, glycerol, ethylene glycol, and sucrose, wherein the volume concentration of dimethyl sulfoxide is 5%, the mass concentration of trehalose is 10 g / L to 15 g / L, the mass concentration of vitamin C is 0.005 g / L to 0.015 g / L, the mass concentration of glycerol is ≥30 g / L, the volume concentration of ethylene glycol is 10% to 15%, and the molar concentration of sucrose is 0.4 mol / L.

[0016] The technical solutions provided in this application have the following advantages compared with the prior art:

[0017] This application provides a method for reviving Acer truncatum seeds. The method first utilizes a gradient thawing system within a thawing device: a first thawing tank and a second thawing tank are connected in series, each equipped with an independent thermostat tube, allowing for a preset, continuous cooling gradient from high to low temperatures. The mounting frame is controlled to remain in the two tanks sequentially, ensuring the Acer truncatum seeds undergo a gradual process from slow thawing to stabilization of osmotic pressure, maximizing the protection of seed cell integrity. Simultaneously, the transfer time between the two thawing tanks is controlled to within 5 minutes, minimizing transfer time and ensuring the seeds are almost never exposed to room temperature during transfer, guaranteeing the continuity and stability of the gradient thawing. Furthermore, the combination of a fixing mechanism and an isolation plate within the thawing device ensures uniform heating of the seeds. Secondly, a specific composite washing solution is used to wash away residual cryoprotectant, eliminating toxicity to seed cells and providing a buffering effect. Next, a repair culture medium is used for dark environment culture to reduce light damage and restore seed cell physiological function. Finally, a basal culture medium provides comprehensive nutrition to complete seed metabolic reconstruction, achieving complete revival. The entire recovery method forms a complete protection and repair system, effectively solving the problem of low survival rate of Acer truncatum seeds after recovery, and making the survival rate of seeds after cryopreservation as high as 82%. Attached Figure Description

[0018] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0019] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of a method for reviving Acer truncatum seeds provided in an embodiment of this application; Figure 2 This is a schematic diagram of the defrosting device provided in the embodiments of this application; Figure 3 This is a schematic diagram of the transfer section in the thawing device provided in the embodiments of this application; Figure 4 This is a schematic diagram of the fixing mechanism in the thawing device provided in the embodiments of this application; Figure 5 This is a schematic diagram of the structure of the fixing part in the defrosting device provided in the embodiments of this application. Figure 6 This application provides a flowchart illustrating the application of the resuscitation method in the cryopreservation of Acer truncatum seeds. Figure 7 This application provides a detailed flowchart illustrating the application of the resuscitation method in the cryopreservation of Acer truncatum seeds. Figure 8 The statistical results of dehydration time, moisture content, and TTC activity value of Acer truncatum seeds provided in the embodiments of this application are shown in the figure. Figure 8 Figure A shows the statistical results of dehydration time and moisture content of Acer truncatum seeds. Figure 8 B is a graph showing the statistical results of dehydration time and TTC activity value of Acer truncatum seeds; Figure 9 This is a diagram showing the germination of Acer truncatum seeds cultured in a dark environment for 15 days in Example 1 of this application. Figure 10 The germination of Acer truncatum seeds cultured in basal medium for 15 days in Example 1 of this application is shown. Figure 11 A physical model diagram of the defrosting device provided in the embodiments of this application; Among them, 1-base, 2-first thawing tank, 3-second thawing tank, 4-thawing partition plate, 5-thermostatic tube, 6-motor, 7-support block, 8-screw, 9-threaded sliding block, 10-telescopic rod, 11-transfer fixing rod, 12-transfer connecting block, 13-mounting bracket, 14-fixing mechanism, 15-time controller, 16-isolation plate, 17-temperature controller, 1701-temperature sensor, 18-sliding rod, 19-sliding groove, 20-freeze tube, 141-frame bracket, 142-support rod, 143-first sliding block, 144-second sliding block, 145-spring, 146-clamping fixing rod, 147-clamping connecting block, 148-arc-shaped fixing block, 149-limiting sleeve, 1410-limiting rod. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0022] The range descriptions used in this application, such as numerical ranges and proportional ranges, include all possible sub-ranges and single numerical values ​​within that range. For example, the range descriptions of "1 to 6" or "1~6" cover all sub-ranges (such as 1 to 3, 2 to 5, etc.) and single numbers (such as 1, 2, 3, 4, 5, 6) between 1 and 6. Unless otherwise specified, the terms "comprising" and others used herein mean "including but not limited to"; relational terms such as "first" and "second" are used only to distinguish different entities or operations and do not imply an actual order or relationship; "and / or" indicates that multiple situations can exist individually or simultaneously; expressions such as "at least one," "multiple," and "at least one" refer to any combination of the corresponding objects, including combinations of single or multiple objects. The proportional relationships involved in this document, such as mass ratios and molar ratios, should be understood as the correspondence between the first and second terms of a proportional formula, according to the order of description. The raw materials, reagents, instruments, and equipment used herein can all be obtained by purchasing from the market or by existing methods.

[0023] It should be noted that the survival rate of Acer truncatum seeds after cryopreservation is currently less than 50%, mainly due to the following reasons: (1) The inherent challenges of the physiological characteristics of Acer truncatum seeds: Acer truncatum seeds are stubborn seeds, with a water content as high as 20% to 25% at maturity. They are sensitive to dehydration and low-temperature storage conditions. If directly frozen at ultra-low temperature, the free water in the cells of Acer truncatum seeds is prone to form ice crystals, which leads to cell membrane rupture and organelle damage. If directly dehydrated, it will lead to cell damage or even death of Acer truncatum seeds. Therefore, it is necessary to find a balance between dehydration damage (water content below 4%) and ice crystal damage (water content above 10%), that is, the water content should be within a safe range. At the same time, Acer truncatum seeds have an oil content as high as 45% to 48%, and the main oil is unsaturated fatty acid. During ultra-low temperature freezing and thawing, reactive oxygen species (ROS) bursts are easily induced in Acer truncatum seeds, causing unsaturated fatty acids to oxidize and degrade, and destroying the cell membrane integrity of Acer truncatum seeds. (2) Insufficient compatibility of cryoprotectants: Existing cryoprotectants (such as dimethyl sulfoxide and glycerol) used for cryopreservation have low penetration rates on Acer truncatum seeds with high oil content, and composite cryoprotectants (such as PVS2 vitrifying solution) also cannot play an effective role on Acer truncatum seeds. (3) Lack of standardization system: At present, the standard method for cryopreservation is the "General Technical Rules for Cryopreservation of Resilient Seeds of Medicinal Plants" (T / CACM 1326.1–2019). However, this standard method is not applicable to Acer truncatum seeds. Moreover, the parameters of this standard method (such as dehydration time) depend on experience adjustment, and the subsequent revival operation also depends on experience adjustment, resulting in unstable preservation and revival effects of Acer truncatum seeds. (4) Unstable recovery process: At present, germplasm resources preserved by ultra-low temperature rely on mild heating methods such as water bath heating for recovery. However, during the water bath heating process, the cryopreservation tubes for preserving Acer truncatum seeds are prone to floating in the water. Therefore, a specific device is needed to fix the cryopreservation tubes. At the same time, uneven heating during the water bath heating process can easily induce ROS bursts in Acer truncatum seeds, which can damage the cell membrane integrity of Acer truncatum seed cells, damage proteins and DNA, and directly lead to the death of Acer truncatum seed cells, affecting the subsequent recovery and survival of Acer truncatum seeds.

[0024] These factors have all contributed to a survival rate of less than 50% for Acer truncatum seeds, making it difficult to meet the current requirements for Acer truncatum seeds.

[0025] In view of the deficiencies of the prior art, the present application provides the following technical solution: Figure 1 An exemplary schematic diagram of a method for reviving Acer truncatum seeds provided in an embodiment of this application is shown. Figure 2 An exemplary schematic diagram of the defrosting device provided in an embodiment of this application is shown; Figure 3 An exemplary schematic diagram of the transfer section in the thawing device provided in this application embodiment is shown; Figure 11 An exemplary physical model diagram of the defrosting device provided in the embodiments of this application is shown; like Figure 1 , Figure 2 , Figure 3 as well as Figure 11 As shown in the embodiment of this application, a method for reviving Acer truncatum seeds is provided, the revival method comprising: S1. Use a thawing device to perform gradient thawing on the ultra-low temperature stored Acer truncatum seeds to obtain thawed seeds; wherein, the transfer time of the gradient thawing is ≤5min; S2. Wash the thawed seeds with a compound washing solution to obtain sterile seeds; S3. The sterile seeds are cultured in a dark environment using a remediation culture medium to obtain primary resuscitation seeds; S4. The primary revived seeds are cultured using a basal culture medium to complete the revival of Acer truncatum seeds; Among them, such as Figure 2 and Figure 3 As shown, the defrosting device includes: Base 1; The thawing section includes a thawing partition plate 4, a first thawing tank 2, a second thawing tank 3, a constant temperature tube 5, an isolation plate 16, and a mounting bracket 13. The first thawing tank 2 and the second thawing tank 3 are connected in series on the surface of the base 1. The mounting bracket 13 is fixed in the first thawing tank 2 or the second thawing tank 3. The first thawing tank 2 and the second thawing tank 3 are separated by the thawing partition plate 4. The constant temperature tube 5 is respectively disposed in the first thawing tank 2 and the second thawing tank 3. The mounting bracket 13 is disposed above the constant temperature tube 5. The mounting bracket 13 is provided with multiple sets of fixing mechanisms 14. The fixing mechanisms 14 are used to fix the cryopreservation tube 20 containing the Acer truncatum seeds. The isolation plate 16 is disposed between the mounting bracket 13 and the constant temperature tube 5. The transfer unit includes a screw 8, a threaded sliding block 9, a telescopic rod 10, a transfer fixing rod 11, and a transfer connecting block 12. One end of the rotating connecting block is fixedly connected to one end of the mounting frame 13, and the other end of the transfer connecting block 12 is fixedly connected to one end of the transfer fixing rod 11. The other end of the transfer fixing rod 11 is fixedly connected to the telescopic end of the telescopic rod 10. The telescopic rod 10 is fixed on the threaded sliding block 9, and the threaded sliding block 9 slides relative to the screw 8. The screw 8 is symmetrically arranged on both sides of the base 1, and the length of the screw 8 is greater than the sum of the lengths of the first thawing groove 2 and the second thawing groove 3, so that the mounting frame 13 performs gradient thawing in the first thawing groove 2 or the second thawing groove 3.

[0026] It should be noted that the specific process of this gradient thawing is as follows: First, the first thawing tank 2, the second thawing tank 3, and the thawing partition plate 4 are assembled on the base 1. Then, the constant temperature tube 5 is fixed on both sides of the thawing partition plate 4. Next, the partition plate 16 is placed on the constant temperature tube 5, and then the mounting bracket 13 is placed above the partition plate 16. Water is injected into the first thawing tank 2 and the second thawing tank 3. Through the heating of the constant temperature tube 5, the temperature in the first thawing tank 2 and the second thawing tank 3 reaches the expected value. At this time, the cryopreservation tube 20 is placed in the fixing mechanism 14, so that the cryopreservation tube 20 is fixed in the mounting bracket 13. After the mounting bracket 13 has been in the first thawing tank 2 for a certain period of time, the telescopic rod 10 is adjusted so that the telescopic rod 10... The transfer fixing rod 11 contracts, and at this time, the transfer fixing rod 11 descends vertically, thereby driving the transfer connecting block 12 to rise. This causes the mounting frame 13, which is fixedly connected to the transfer connecting block 12, to rise from the first thawing tank 2. Then, the control motor 6 drives the screw 8 to rotate. The rotating screw 8 drives the threaded sliding block 9 to move horizontally on the screw 8, thereby moving the mounting frame 13 from the first thawing tank 2 to the top of the second thawing tank 3. Then, the telescopic end of the telescopic rod 10 is adjusted to extend the telescopic rod 10. At this time, the transfer fixing rod 11 rises vertically, thereby driving the transfer connecting block 12 to descend, causing the mounting frame 13 to enter the second thawing tank 3. Thus, through this series of structures, the gradient thawing of Acer truncatum seeds stored at ultra-low temperatures is achieved.

[0027] It should be noted that this repair medium is based on MS medium, with the addition of 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), vitamin C, and sucrose. The mass concentration of 6-BA can be 1.0 mg / L, the mass concentration of NAA can be 0.2 mg / L, the mass concentration of vitamin C can be 100 mg / L, and the mass concentration of sucrose can be 30 g / L.

[0028] It should be noted that this basal culture medium is formed by adding gibberellin and sucrose to 1 / 2 MS medium. The mass concentration of gibberellin can be 0.5 mg / L, and the mass concentration of sucrose can be 20 g / L.

[0029] It should be noted that the washing can be performed 3 times, with each washing session lasting 10 minutes. After washing, filter paper can be used to absorb the moisture on the surface of the thawed seeds. Then, soak the thawed seeds in 70% ethanol for 1 minute, rinse them 3 times with sterile water, and then filter paper can be used to absorb the moisture on the surface of the thawed seeds. Finally, use tweezers to remove the seed coat of the thawed seeds on a clean bench.

[0030] It should be noted that the temperature for this dark environment incubation can be between 24℃ and 26℃, and the incubation can be terminated when the sterile seeds show significant enlargement. The incubation period can be 2 weeks.

[0031] It should be noted that this application provides a method for the revival of Acer truncatum seeds. This revival method, through precise temperature control of gradient thawing, combined with device structural design, supporting processing steps, and repair strategies, addresses the problem of low seed survival rate after cryopreservation from four dimensions: "reducing thawing damage, removing toxic residues, repairing cell damage, and ensuring revival continuity," ultimately achieving a survival rate of up to 82% for Acer truncatum seeds. The specific mechanism is as follows: I. The structural design of the gradient thawing device achieves "precise temperature gradient + undisturbed transport", reducing ice crystal recrystallization damage from the source.

[0032] During the thawing process of cryopreserved Acer truncatum seeds, ice crystal recrystallization and sudden temperature changes are the core causes of cell rupture and organelle damage. This resuscitation method avoids this problem from two aspects through device structure and parameter control: 1. A constant temperature gradient consisting of multiple thawing tanks connected in series enables gradual heating and thawing: The thawing section is configured as a first thawing tank 2 and a second thawing tank 3. Each thawing tank is equipped with an independent constant temperature tube 5, which can preset a continuous temperature gradient from high temperature to low temperature. The mounting frame 13 carries the cryopreservation tube 20 and keeps it in different tanks in sequence, so that the Acer truncatum seeds undergo a gradient thawing process of "slow heating-smooth transition". This avoids the cell membrane rupture and organelle damage caused by the rapid melting of small ice crystals in Acer truncatum seed cells due to traditional rapid thawing (such as direct high temperature water bath). At the same time, it can also avoid the dehydration damage caused by the cells being in a hypertonic environment for a long time due to slow thawing.

[0033] 2. The automated transfer system ensures that the transfer time between the first thawing tank 2 and the second thawing tank 3 is ≤5 minutes, eliminating interference from temperature fluctuations between the tanks. The transfer unit, through the linkage of screw 8, threaded sliding block 9 and telescopic rod 10, realizes the automated and linear transfer of the mounting frame 13 between the first thawing tank 2 and the second thawing tank 3, replacing the time-consuming and error-prone manual operation. The length of screw 8 is greater than the total length of the first thawing tank 2 and the second thawing tank 3, ensuring that the movement trajectory of the mounting frame 13 can smoothly cover all thawing tanks. The rigid connection between the transfer connecting block 12 and the fixed rod can prevent the cryopreservation tube 20 from shaking in the fixed mechanism 14. The short transfer time ensures that the Acer truncatum seeds are almost not exposed to room temperature when transferred between the first thawing tank 2 and the second thawing tank 3, eliminating cell stress damage caused by the sudden drop in temperature in the cryopreservation tube 20, and ensuring the continuity and stability of gradient thawing.

[0034] 3. The design of the fixing mechanism 14 + isolation plate 16 ensures that the seeds of Acer truncatum are heated evenly: The fixing mechanism 14 inside the mounting frame 13 vertically fixes the cryopreservation tube 20, and the isolation plate 16 separates the constant temperature tube 5 from the mounting frame 13, so that the cryopreservation tube 20 only comes into contact with the constant temperature medium in the first thawing tank 2 or the second thawing tank 3, avoiding local temperature differences caused by uneven high temperature in the constant temperature tube 5, ensuring that each Acer truncatum seed is in a consistent gradient temperature environment, and reducing the failure of some seeds to recover due to individual thawing differences.

[0035] 2. Washing with compound washing solution: Removes cryoprotectant residues and eliminates cytotoxic damage.

[0036] After gradient thawing, cryoprotectants (such as DMSO and glycerol) used during ultra-low temperature storage still remain on the surface of the thawed seeds and in the intercellular spaces. If these substances are not removed in time, they can produce cytotoxicity in subsequent culture and inhibit the metabolic activity of Acer truncatum seeds. The role of the compound washing solution is reflected in: 1. Targeted dissolution and elution of cryoprotectants to reduce the residual concentration of cryoprotectants in Acer truncatum seed cells and avoid continuous damage to cell membranes and enzyme systems by cryoprotectants; 2. The sterile components added to the compound washing solution can simultaneously remove microorganisms that may adhere during the thawing process, resulting in sterile seeds. This creates a pollution-free environment for subsequent dark-environment culture and ordinary culture, avoiding microbial contamination that could lead to the death of Acer truncatum seeds and indirectly improving the survival rate of the resuscitation.

[0037] III. Dark environment + repair culture medium culture: targeted repair of oxidative damage to cells caused by thawing.

[0038] While gradient thawing reduces physical damage, Acer truncatum seeds still generate a large amount of reactive oxygen species (ROS) during cryopreservation and thawing, triggering oxidative stress and damaging biomolecules such as DNA, proteins, and lipids in the seed cells. This is another key reason for the low survival rate of traditional Acer truncatum seed resuscitation. This step reverses the damage through "environmental control + nutrient remediation": 1. Dark environment cultivation: Avoids photo-oxidation reactions caused by light, reduces further ROS generation, and reduces the continuous accumulation of oxidative damage; 2. Optimization of the repair culture medium formulation: Adding antioxidants (such as vitamin C), cell membrane repair substances (such as 6-BA and NAA), and energy substances (such as sucrose) to the culture medium can target and repair the cell membrane structure damaged by ROS, restore enzyme activity, and replenish the energy required for cell metabolism, so that the physiological functions of the initially revived seeds can be quickly restored, laying the foundation for subsequent complete revival.

[0039] IV. Basic culture medium culture: Complete physiological metabolic reconstruction and achieve complete seed revival.

[0040] After initial thawing and repair, the cell structure and function of the seeds have been preliminarily restored. At this time, they are transferred to a basal culture medium for further cultivation. This provides the comprehensive nutrition required for the germination of Acer truncatum seeds (such as mineral elements like nitrogen, phosphorus, and potassium, and organic nutrients like amino acids and vitamins). This meets the needs of Acer truncatum seeds to transition from "dormant thawing" to "metabolic activity," gradually restoring physiological processes such as cell division and water absorption and swelling. Ultimately, this completes the entire thawing process, ensuring that the repaired Acer truncatum seeds can continue to survive and germinate, thus translating the effects of the initial gradient thawing and repair into a high actual survival rate.

[0041] In summary, this application provides a method for the resuscitation of Acer truncatum seeds. This method uses a thawing device for gradient thawing as its core. Through the precise temperature control and efficient transport of the thawing device, it solves the problem of physical damage to Acer truncatum seeds stored at ultra-low temperatures during the traditional thawing stage. Furthermore, it employs a combination of steps: a compound washing solution to remove toxic residues from the thawing process, a dark environment culture medium to repair oxidative damage, and a basal culture medium to complete metabolic reconstruction. This forms a complete processing chain of "thawing to prevent damage + subsequent detoxification + rapid damage repair + full metabolic recovery," systematically solving the problem of low survival rate after long-term ultra-low temperature storage of Acer truncatum seeds, ultimately achieving a survival rate of up to 82%.

[0042] In some optional embodiments, the fixing mechanism 14 includes a frame bracket 141, a support rod 142, a first sliding block 143, a second sliding block 144, a spring 145, a clamping unit, and a limiting sleeve 149. The frame bracket 141 is disposed within the mounting frame 13, and the support rod 142 is disposed within the frame bracket. The first sliding block 143 and the second sliding block are symmetrically disposed on the support rod 142 and slide on the support rod 142. The spring 145 is disposed between the first sliding block 143 and the second sliding block. The clamping unit is fixedly connected to both the first sliding block 143 and the second sliding block. The two clamping units form a clamping space for clamping the cryopreservation tube 20. One end of the clamping unit abuts against the limiting sleeve 149, causing the clamping unit to move in the horizontal direction.

[0043] In these embodiments, a fixing mechanism 14 is formed using a frame bracket 141, a support rod 142, a first sliding block 143, a second sliding block 144, a spring 145, a clamping unit, and a limiting sleeve 149. The frame bracket 141 can fix each structure of the fixing mechanism within the mounting frame 13, while the limiting sleeve 149 can fix the clamping unit in the horizontal direction, ensuring the stable existence of the cryopreservation tube 20 within the clamping unit. In addition, the first sliding block 143 and the second sliding block 144 are stacked opposite each other on the support rod 142 to form a relative sliding space. At the same time, the spring 145 is provided on the first sliding block 143 and the second sliding block 144, which can play a role in tension adjustment, so that the cryopreservation tube 20 can be stably fixed between the two clamping units. Specifically: In the relaxed state, the spring 145 can keep the distance between the first sliding block 143 and the second sliding slider relatively stable, so that the opening distance between the two clamping units is slightly smaller than that of the cryopreservation tube 20. After the cryopreservation tube 20 enters the two clamping units, the two clamping units will tend to expand outward. At this time, under the action of the limiting sleeve 149, the clamping unit will undergo a slight displacement. The limiting sleeve 149 can prevent the clamping unit from moving in the vertical direction and can only move in the horizontal direction. At the same time, the clamping unit will drive the relative sliding between the first sliding block 143 and the second sliding block 144, so that the spring 145 between the first sliding block 143 and the second sliding slider will extend. Under the elastic force of the spring 145, the first sliding block 143 and the second sliding slider will tend to retract, so that the two clamping units have a large clamping force, and achieve stable fixation between the clamping unit and the cryopreservation tube 20.

[0044] In some optional embodiments, the clamping unit includes a clamping fixing rod 146, a clamping connecting block 147, an arc-shaped fixing block 148, and a limiting rod 1410. One end of the clamping fixing rod 146 is fixedly connected to the first sliding block 143 or the second slider, and the other end of the clamping fixing rod 146 is connected to one end of the clamping connecting block 147. The other end of the clamping connecting block 147 is connected to the arc-shaped fixing block 148, and one end of the arc-shaped fixing block 148 is fixedly connected to the limiting rod 1410. The limiting rod 1410 abuts against the limiting sleeve 149. The two arc-shaped fixing blocks 148 form a clamping space for clamping the cryopreservation tube 20.

[0045] In these embodiments, the clamping unit, employing a clamping fixing rod 146, a clamping connecting block 147, an arc-shaped fixing block 148, and a limiting rod 1410, uses the arc-shaped fixing block 148 as the clamping port to match the curvature of the clamping surface of the cryopreservation tube 20. Simultaneously, the clamping connecting block 147 fixes the arc-shaped fixing block 148 and the limiting rod 1410 together. The combination of the limiting rod 1410 and the limiting sleeve 149 ensures that the clamping connecting block 147 can only move horizontally, preventing vertical movement and improving the clamping stability of the clamping unit. Finally, the clamping fixing rod 146 fully fixes the clamping connecting block 147 to either the first sliding block 143 or the second sliding block 144, facilitating the formation of a clamping space for the cryopreservation tube 20 between the arc-shaped fixing blocks 148.

[0046] In some optional embodiments, the transfer unit further includes a support block 7, a slide rod 18, and a control motor 6. The output end of the control motor 6 is fixedly connected to the screw 8 to drive the screw 8 to rotate. The support block 7 is fixed to one side of the base 1, and two support blocks 7 are inserted through both ends of the screw 8. The slide rod 18 is arranged between the two support blocks 7 and is fixed to the base 1. The threaded sliding block 9 is provided with a sliding groove 19 that matches the slide rod 18 to enable the threaded sliding block 9 to slide on the screw 8.

[0047] In these embodiments, a support block 7, a slide rod 18, and a control motor 6 are introduced into the transfer section. The rotation of the control motor 6 can drive the screw 8 to rotate, thereby driving the threaded sliding block 9 to move. At the same time, support blocks 7 are set at both ends of the screw 8, and the slide rod 18 is fixed on the base 1. The horizontal sliding between the slide rod 18 and the threaded sliding block 9, combined with the threaded sliding between the screw 8 and the threaded sliding block 9, enables the telescopic rod 10 to slide quickly and stably in the horizontal direction. This allows the mounting frame 13 to operate quickly and stably between the first thawing tank 2 and the second thawing tank 3, and achieves a transfer time of less than 5 minutes.

[0048] In some optional embodiments, the defrosting device further includes a control unit, which includes a temperature controller 17, a group of temperature sensors 1701, and a time controller 15. The temperature controller 17 is disposed inside the defrosting partition plate 4, and the group of temperature sensors 1701 is disposed inside the first defrosting tank 2 and the second defrosting tank 3. The temperature sensors 1701 are connected to the temperature controller 17 via electrical signals, and the time controller 15 is connected to the control motor 6 via electrical signals.

[0049] In these embodiments, a temperature controller 17, a group of temperature sensors 1701, and a time controller 15 are used as the control unit. The temperature sensors 1701 collect real-time temperature data in the first thawing tank 2 and the second thawing tank 3, and then transmit the real-time temperature data to the PLC chip or control unit of the temperature controller 17 via electrical signals. By matching the real-time temperature data with the preset temperature standard value, the temperature of the thermostat tube 5 can be controlled by the temperature controller 17 to ensure the temperature gradient in the first thawing tank 2 and the second thawing tank 3, which is conducive to the smooth thawing of Acer truncatum seeds.

[0050] It should be noted that the temperature controller 17 can be an AI-516P type temperature controller 17, and its control panel and control unit can be installed on the defrosting partition plate 4. The temperature sensor 1701 can be a PT1000 waterproof temperature sensor. The control motor 6 can be a 42HS08 type stepper motor. The time controller 15 can be an H3CR-A8 type Omron controller.

[0051] In some optional implementations, the gradient thawing includes a first thawing and a second thawing, wherein the time for the first thawing is the same as the time for the second thawing; The first thawing temperature is 35°C to 45°C, and the first thawing time is 5 min to 10 min; The second thawing temperature is 20°C to 30°C, and the second thawing time is 5 min to 10 min.

[0052] In these embodiments, the first thawing time is controlled to be the same as the second thawing time, and the temperature of the first thawing is controlled to be 35°C to 45°C, and the temperature of the second thawing is controlled to be 20°C to 30°C. The first thawing can melt the ice crystals inside the Acer truncatum seeds, and the second thawing can stabilize the osmotic pressure of the Acer truncatum seeds in the cryopreservation tube, thus avoiding cell damage to the Acer truncatum seeds caused by excessively high osmotic pressure.

[0053] The first thawing temperature can be 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃ or 45℃.

[0054] The first thawing time can be 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min.

[0055] The second thawing temperature can be 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, or 30℃.

[0056] The second thawing time can be 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min.

[0057] In some optional embodiments, the composite detergent comprises sucrose, trehalose, proline, and vitamin C, wherein the molar concentration of sucrose is 0.25 mol / L to 0.35 mol / L, the molar concentration of trehalose is 0.08 mol / L to 1.2 mol / L, the molar concentration of proline is 25 mmol / L to 35 mmol / L, and the mass concentration of vitamin C is 20 mg / L to 30 mg / L.

[0058] In these embodiments, a composite washing solution comprising sucrose, trehalose, proline, and vitamin C is used. The molar concentrations of sucrose, trehalose, proline, and vitamin C are controlled at 0.25 mol / L to 0.35 mol / L, trehalose at 0.08 mol / L to 1.2 mol / L, proline at 25 mmol / L to 35 mmol / L, and vitamin C at 20 mg / L to 30 mg / L. This composite washing solution can elute the cryoprotectant, reducing its residual concentration in the Acer truncatum seed cells and preventing continued damage to the cell membrane and enzyme system. Simultaneously, the composite washing solution can remove microorganisms that may adhere during thawing, preventing microbial contamination that could lead to seed necrosis and indirectly improving the survival rate after thawing.

[0059] The molar concentration of the sucrose can be 0.25 mol / L, 0.26 mol / L, 0.27 mol / L, 0.28 mol / L, 0.29 mol / L, 0.30 mol / L, 0.31 mol / L, 0.32 mol / L, 0.33 mol / L, 0.34 mol / L, or 0.35 mol / L.

[0060] The molar concentration of trehalose can be 0.08 mol / L, 0.085 mol / L, 0.09 mol / L, 0.095 mol / L, 0.10 mol / L, 0.105 mol / L, 0.110 mol / L, 0.115 mol / L, or 0.12 mol / L.

[0061] The molar concentration of proline can be 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L, 30 mmol / L, 31 mmol / L, 32 mmol / L, 33 mmol / L, 34 mmol / L, or 35 mmol / L.

[0062] The concentration of vitamin C can be 20 mg / L, 21 mg / L, 22 mg / L, 23 mg / L, 24 mg / L, 25 mg / L, or 30 mg / L.

[0063] Figure 6An exemplary flowchart of an embodiment of this application is shown, illustrating the application of the resuscitation method in the cryopreservation of Acer truncatum seeds; Based on a general inventive concept, such as Figure 6 As shown in the figure, this application provides an application of the resuscitation method in the cryopreservation of Acer truncatum seeds, the application including: S1. The seeds of Acer truncatum are dried to obtain dried seeds with a preset moisture content; wherein the preset moisture content is 8.1% to 8.2%; S2. The dried seeds with a preset moisture content are subjected to gradient protection treatment using a cryoprotectant to obtain vitrified seeds; S3. The vitrified seeds are cryopreserved to obtain cryopreserved Acer truncatum seeds; S4. The cryopreserved Acer truncatum seeds are subjected to the revival method described above to obtain revival seeds.

[0064] This application is based on the above-described resuscitation method. The specific steps of the resuscitation method can be referred to the above embodiments. Since this application adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0065] It should be noted that this drying process can be performed at a low temperature of 45°C for 10 minutes using a crown-and-spot moisture analyzer. For ultra-low temperature preservation, vitrified seeds can be directly immersed in a liquid nitrogen tank (-196°C) for ultra-low temperature storage.

[0066] Figure 7 An exemplary embodiment of this application provides a detailed flowchart of the application of the resuscitation method in the cryopreservation of Acer truncatum seeds; In some alternative implementations, such as Figure 7 As shown, the dried seeds with a preset moisture content are subjected to gradient protection treatment using a cryoprotectant to obtain vitrified seeds, including the following steps: S201. The dried seeds with a preset moisture content are soaked for the first time using a cryoprotectant with a volume concentration of 60% to obtain first soaked seeds; S202. The first soaked seeds are soaked a second time using the cryoprotectant with a volume concentration of 80% to obtain second soaked seeds; S203. The second soaked seeds are soaked a third time using the cryoprotectant with a volume concentration of 100% to obtain vitrified seeds; The first soaking time is 18 to 22 minutes, the second soaking time is 35 to 45 minutes, and the third soaking time is 55 to 65 minutes.

[0067] In these embodiments, the dried seeds are first soaked in a 60% cryoprotectant solution for 18 to 22 minutes to initially displace the water in the dried seeds and allow them to slowly dehydrate. Then, the seeds are soaked in a 80% cryoprotectant solution for 35 to 45 minutes to enhance the cell permeability of the first-soaked seeds, allowing the cryoprotectant to enter the cells smoothly. Finally, the seeds are soaked in a 100% cryoprotectant solution for 55 to 65 minutes to rapidly displace the water in the second-soaked seeds, causing the cells to completely dehydrate and inducing the seeds to enter a vitrified state, thereby completing the displacement and protection by the cryoprotectant.

[0068] The first soaking time can be 18 min, 18.5 min, 19.0 min, 19.5 min, 20.0 min, 20.5 min, 21.0 min, 21.5 min, or 22 min.

[0069] The second soaking time can be 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, or 45 min.

[0070] The third soaking time can be 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, 61 min, 62 min, 63 min, 64 min, or 65 min.

[0071] In some optional embodiments, the cryoprotectant comprises dimethyl sulfoxide, trehalose, vitamin C, glycerol, ethylene glycol, and sucrose, wherein the dimethyl sulfoxide has a volume concentration of 5%, the trehalose has a mass concentration of 10 g / L to 15 g / L, the vitamin has a mass concentration of 0.005 g / L to 0.015 g / L, the glycerol has a mass concentration ≥30 g / L, the ethylene glycol has a volume concentration of 10% to 15%, and the sucrose has a molar concentration of 0.4 mol / L.

[0072] In these embodiments, dimethyl sulfoxide, trehalose, vitamin C, glycerol, ethylene glycol, and sucrose are used as cryoprotectants. By controlling the volume concentration of dimethyl sulfoxide to 5%, the mass concentration of trehalose to 10 g / L to 15 g / L, the mass concentration of vitamin C to 0.005 g / L to 0.015 g / L, the mass concentration of glycerol to ≥30 g / L, the volume concentration of ethylene glycol to 10% to 15%, and the molar concentration of sucrose to 0.4 mol / L, the cryoprotectants can effectively displace water from the seed cells of Acer truncatum and avoid damage to the seed cells.

[0073] The mass concentration of trehalose can be 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L or 15 g / L.

[0074] The concentration of this vitamin can be 0.005 g / L, 0.006 g / L, 0.007 g / L, 0.008 g / L, 0.009 g / L, 0.010 g / L, 0.011 g / L, 0.012 g / L, 0.013 g / L, 0.014 g / L, or 0.015 g / L.

[0075] The volume concentration of the ethylene glycol can be 10%, 11%, 12%, 13%, 14%, or 15%.

[0076] The present application is further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national / industry standards; if there is no corresponding national / industry standard, they are performed according to general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0077] Example 1 I. Precise control of the moisture content of Acer truncatum seeds.

[0078] Select Acer truncatum seeds of uniform maturity, air dry them naturally in a cool, ventilated place for one week, and then put them into breathable cloth bags. A portion of the Acer truncatum seeds were hung in a cool, dry place as a control group, while the other portion was stored in a standard seed bank (temperature 4℃, relative humidity 30%) for later use as the experimental group.

[0079] Remove the seed wings from two portions of Acer truncatum seeds, and select healthy, plump seeds with no surface damage. First, wash these seeds, then disinfect them with a 5% sodium hypochlorite solution for 5 minutes (gently shake during disinfection to avoid cell damage). Then rinse them with sterile water 3 to 5 times to remove residual disinfectant, obtaining disinfected seeds. These disinfected seeds are in a dormancy-broken state with an initial moisture content >10%, so they need to be further dried to adjust the moisture content of the disinfected seeds to a safe range of 4% to 10%.

[0080] The sterilized seeds from the control group were placed in petri dishes at a rate of 25 seeds per dish, and then dried at 45°C using a crown-and-spot moisture analyzer. The seed moisture content and cell viability were monitored in real time using the TTC method. The drying time was determined to be within 30 minutes to ensure that the moisture content was within a safe range. Finally, it was confirmed that Acer truncatum seeds with a moisture content of about 8% were selected. At this time, the TTC activity values ​​of Acer truncatum seeds were 437.31 μg TTC / (g·h), 438.14 μg TTC / (g·h), and 440.47 μg TTC / (g·h), which are close to the initial activity levels of Acer truncatum seeds before drying, which were 458.97 μg TTC / (g·h), 452.39 μg TTC / (g·h), and 449.04 μg TTC / (g·h). This ensured that the cell viability of the Acer truncatum seeds was maintained in a good state, which could meet the requirements for subsequent cryopreservation. The above steps were repeated for the disinfected seeds in the experimental group to determine the optimal drying time as 10 minutes, with the moisture content of the Acer truncatum seeds around 8% (8.04%, 8.23%, and 8.52%). The dehydration time, moisture content, and TTC activity value of the Acer truncatum seeds were correlated as follows: Figure 8 As shown.

[0081] Depend on Figure 8 It is known that the moisture content of Acer truncatum seeds must be strictly controlled during storage, as this is one of the core factors determining the storage life, germination rate, and stress resistance of Acer truncatum seeds. Using the drying and weight reduction method to dehydrate Acer truncatum seeds and testing their real-time viability, the viability of the seeds gradually decreased with decreasing moisture content, from an initial 458.97 μg TTC / (g·h) to 364.24 μg TTC / (g·h). Acer truncatum seeds dehydrated for approximately 10 minutes maintained high viability while simultaneously reducing moisture content, which is beneficial for long-term germplasm preservation.

[0082] II. Screening of highly efficient cryoprotectants for Acer truncatum seeds.

[0083] Based on the high oil content and osmotic pressure sensitivity of Acer truncatum seeds, the basic cryoprotectant formula was designed as follows: DMSO at a volume concentration of 5% to 10%, trehalose at a mass concentration of 8 g / L to 12 g / L, vitamin C at a mass concentration of 0.005 g / L to 0.015 g / L, glycerol at a mass concentration of 30 g / L, ethylene glycol at a volume concentration of 15%, and sucrose at a molar concentration of 0.4 mol / L.

[0084] The preparation process of this cryoprotectant is as follows: heat deionized water to 40°C, then add glycerin and vitamin C, and stir until completely dissolved to obtain a solution; after cooling the solution, add ethylene glycol, DMSO and sucrose, stir evenly, and adjust the pH value to 5.8.

[0085] An orthogonal experimental design was adopted, selecting three factors: DMSO (5%, 10%, 15%), trehalose (8 g / L, 10 g / L, 12 g / L), and vitamin C (0.005 g / L, 0.010 g / L, 0.015 g / L), and setting up multiple sets of experiments. Acer truncatum seeds were divided into group A (dried seeds with a moisture content of 8.1% to 8.2% obtained after drying) and group B (control group air-dried Acer truncatum seeds). Ten to twelve Acer truncatum seeds from each group were placed into 5 mL cryovials 20. The experiments were then conducted according to the above multiple sets of ratios. After adding cryoprotectant to the cryovials 20, they were directly placed in a liquid nitrogen tank for freezing for one week. Subsequently, they were placed in the thawing device provided in this application embodiment for gradient thawing, and the TTC activity value was measured. Each set of ratios was performed in triplicate. The results are shown in Table 1.

[0086] Table 1. The concentration ratio of DMSO, trehalose and vitamin C in the cryoprotectant and their cell viability values ​​after one week of cryopreservation.

[0087] As shown in Table 2, DMSO with a volume concentration of 15% penetrated too quickly into the seeds of Acer truncatum, causing a significant osmotic pressure shock to the cell structure of the seeds. This resulted in a significant decrease in cell viability of the seeds after cryopreservation compared to before cryopreservation. When the volume concentration of DMSO was reduced to 5%, the TTC activity value of group A was significantly higher than that of group B. For example, in experimental group 5, the TTC activity value of group A was about 15 μg TTC / (g·h) higher, while in experimental group 8, the TTC activity value of group A was about 20 μg TTC / (g·h) higher. With the cryoprotectant formulation consisting of 5% DMSO (v / L), 12 g / L trehalose (w / L), 0.010 g / L vitamin C (w / L), 30 g / L glycerol (w / L), 15% ethylene glycol (v / L), and 0.4 mol / L sucrose, the highest TTC activity value of Acer truncatum seeds in Group A after cryopreservation reached 424.65 μg TTC / (g·h), which is close to the TTC activity values ​​of Acer truncatum seeds before cryopreservation (458.97 μg TTC / (g·h), 452.39 μg TTC / (g·h), and 449.04 μg TTC / (g·h). This indicates that the cell viability of the Acer truncatum seeds was optimally maintained, and the cryoprotectant formulation was determined to be the optimal cryoprotectant.

[0088] III. Cryoprotectant gradient treatment and ultra-low temperature preservation.

[0089] Seeds of *Acer truncatum* with a dehydration time of 10 min and a moisture content of approximately 8%, along with the optimal cryoprotectant formulation (CPA), were selected. After washing, sterilization, drying, and cooling to room temperature, they were placed into 5 mL cryovials (each cryovial containing 10 to 12 *Acer truncatum* seeds) and subjected to gradient cryoprotection treatment according to the following gradient process: Step 1, Pretreatment: Soaking in 60% CPA for 20 min to initiate slow dehydration of the *Acer truncatum* seed cells; Step 2, Transition Treatment: Soaking in 80% CPA for 40 min to enhance the osmotic adaptability of the *Acer truncatum* seed cells; Step 3, Final Treatment: Soaking in 100% CPA for 60 min to achieve complete dehydration of the *Acer truncatum* seed cells and induce vitrification of the seeds. After the gradient cryoprotection treatment was completed, the cryovials were directly immersed in a liquid nitrogen tank (-196℃) for ultra-low temperature preservation.

[0090] IV. Gradual thawing process for Acer truncatum seeds.

[0091] The cryopreservation tube 20 containing Acer truncatum seeds, which had been stored at ultra-low temperatures for a period of time in Example 3, was taken out and subjected to a first thawing and a second thawing using the thawing device provided in this application embodiment. The specific process is as follows: First, assemble the first thawing tank 2, the second thawing tank 3, and the thawing partition plate 4 on the base 1. Then, fix the thermostatic tube 5 on both sides of the thawing partition plate 4. Next, place the partition plate 16 on the thermostatic tube 5 and place the mounting bracket 13 above the partition plate 16. Inject water into the first thawing tank 2 and the second thawing tank 3. Set the preset heating temperature through the temperature controller 17. Once the thermostatic tube 5 reaches the preset temperature, the temperatures in the first thawing tank 2 and the second thawing tank 3 will reach the expected levels (40°C for the water in the first thawing tank 2 and 25°C for the water in the second thawing tank 3). At this time, the spring 145 of the fixing mechanism 14 inside the mounting bracket 13 is in a relaxed state. In the relaxed state, the spring 145 can keep the distance between the first sliding block 143 and the second sliding slider relatively stable. The opening distance between the two clamping units is slightly smaller than that between the cryopreservation tube 20 and the two clamping units. After the cryopreservation tube 20 enters the two clamping units, the two clamping units tend to expand outward. At this time, under the action of the limiting sleeve 149, the clamping units will undergo a slight displacement. The limiting sleeve 149 can prevent the clamping units from moving in the vertical direction and can only move in the horizontal direction. At the same time, the clamping units will drive the relative sliding between the first sliding block 143 and the second sliding block 144, causing the spring 145 between the first sliding block 143 and the second sliding slider to extend. Under the elastic force of the spring 145, the first sliding block 143 and the second sliding slider will tend to retract, thereby giving the two clamping units a large clamping force and achieving stable fixation between the clamping units and the cryopreservation tube 20. The mounting frame 13 undergoes a first thawing of 5 minutes in the first thawing tank 2. The telescopic rod 10 is adjusted to retract, causing the transfer fixing rod 11 to descend vertically, thereby raising the transfer connecting block 12. This allows the mounting frame 13, which is fixedly connected to the transfer connecting block 12, to rise from the first thawing tank 2. Then, the screw 8 is driven to rotate by the control motor 6. The rotating screw 8 causes the threaded sliding block 9 to move horizontally on the screw 8, thus transferring the mounting frame 13 from the first thawing tank 2 to the top of the second thawing tank 3. The telescopic end of the telescopic rod 10 is then adjusted to extend, causing the transfer fixing rod 11 to rise vertically, thereby lowering the transfer connecting block 12. This allows the mounting frame 13 to enter the second thawing tank 3 for a second thawing of 5 minutes. The overall transfer time is controlled within 3 minutes. This series of structures achieves the gradient thawing of Acer truncatum seeds stored at ultra-low temperatures.

[0092] V. Standardized revival process for Acer truncatum seeds.

[0093] After the Acer truncatum seeds have thawed and formed thawed seeds, unscrew the cap of cryopreservation tube 20 and pour the thawed seeds into a sterile filter. Then place it in a laminar flow hood and use a compound washing solution of 0.3 mol / L sucrose, 0.1 mol / L trehalose, 30 mmol / L proline and 25 mg / L vitamin C to wash the thawed seeds three times at room temperature, each wash lasting 10 minutes. After washing, use filter paper to absorb the moisture on the surface of the thawed seeds, then soak them in 70% ethanol for 1 minute, and then rinse them three times with sterile water. After absorbing the moisture on the surface of the rinsed thawed seeds, use tweezers to peel off the seed coat to obtain sterile seeds. Then, sterile seeds were inoculated into repair medium (MS medium as the base, with the addition of 1.0 mg / L 6-BA, 0.2 mg / L NAA, 100 mg / L vitamin C, and 30 g / L sucrose) and cultured in the dark at 24°C to 26°C until the seeds of Acer truncatum showed significant enlargement. At this point, the cell membrane system damage of Acer truncatum seeds was repaired, and physiological metabolic activities were restarted, resulting in primary revived seeds. The primary revived seeds were then inoculated into basal medium (1 / 2 MS medium as the base, with the addition of 0.5 mg / L gibberellin and 20 g / L sucrose) and cultured for two weeks. The growth was observed, and the survival rate of these primary revived seeds was counted.

[0094] Example 2

[0095] Compared to Example 1, the differences in this example are as follows, while the rest are the same: The first thawing temperature is 35℃, and the first thawing time is 10 minutes; The second thawing temperature is 20℃, and the second thawing time is 10 minutes.

[0096] Example 3

[0097] Compared to Example 2, the differences in this example are as follows, while the rest are the same: The first thawing temperature is 45℃, and the first thawing time is 8 minutes; The second thawing temperature is 30℃, and the second thawing time is 8 minutes.

[0098] Comparative Example 1

[0099] Compared to Example 1, the differences in this comparative example are as follows, while the rest are the same: Without using a defrosting device, the first defrosting temperature is used directly, and the process is carried out using a single water bath heating method.

[0100] Comparative Example 2

[0101] Compared to Example 1, the differences in this comparative example are as follows, while the rest are the same: The first thawing temperature is 30℃, and the first thawing time is 15 minutes; The second thawing temperature is 15℃, and the second thawing time is 20 minutes.

[0102] Comparative Example 3

[0103] Compared to Example 1, the differences in this comparative example are as follows, while the rest are the same: The first thawing temperature is 60℃, and the first thawing time is 3 minutes; The second thawing temperature is 40℃, and the second thawing time is 3 minutes.

[0104] Relevant experimental and effect data: The germination of Acer truncatum seeds cultured in the dark environment for 15 days in Example 1, and the germination of Acer truncatum seeds cultured in basal medium for 15 days were observed. The results are as follows: Figure 9 and Figure 10 As shown, this indicates that the seeds of Acer truncatum can germinate normally after being treated by the revival method provided in the embodiments of this application.

[0105] The survival rate of Acer truncatum seeds in each embodiment and comparative example was statistically analyzed, and the results are shown in Table 2.

[0106] Table 2. Survival rate of Acer truncatum seeds in each embodiment and comparative example.

[0107] As shown in Table 2, the method for reviving Acer truncatum seeds provided in this application embodiment solves the problem of low seed survival rate after cryopreservation by using a precise temperature control core of gradient thawing, combined with device structure design, supporting processing steps and repair strategies, from four dimensions: "reducing thawing damage, removing toxic residues, repairing cell damage, and ensuring the continuity of revival". Ultimately, it achieves a survival rate of up to 82% for Acer truncatum seeds.

[0108] Compared to Example 1, Comparative Example 1 used a traditional water bath heating method and controlled a higher water bath heating temperature. This caused the ice crystals inside the Acer truncatum seed cells to melt too quickly, resulting in cell membrane rupture and organelle damage, which affected the survival rate of Acer truncatum seeds.

[0109] Compared to Example 1, Comparative Example 2, which used lower first and second thawing temperatures and longer first and second thawing times, resulted in a slower melting rate of ice crystals in Acer truncatum seeds, causing cell membrane damage and affecting the survival rate of Acer truncatum seeds after recovery. In contrast, Comparative Example 3, which used higher first and second thawing temperatures and lower first and second thawing times, resulted in a faster melting rate of ice crystals in Acer truncatum seeds, directly causing a large number of Acer truncatum seed cells to rupture or even die, thus affecting the survival rate of Acer truncatum seeds after recovery.

[0110] In summary, the embodiment of this application provides a method for reviving Acer truncatum seeds. This revival method, through precise temperature control of gradient thawing, combined with device structural design, supporting processing steps and repair strategies, solves the problem of low seed survival rate after cryopreservation from four dimensions: "reducing thawing damage, removing toxic residues, repairing cell damage, and ensuring revival continuity". Ultimately, it achieves a survival rate of up to 82% for Acer truncatum seeds.

[0111] Furthermore, this application provides a method for reviving Acer truncatum seeds. This method addresses the three core bottlenecks restricting the survival rate of Acer truncatum seeds after cryopreservation (ice crystal damage, dehydration sensitivity, and lipid peroxidation). By precisely controlling the moisture content of Acer truncatum seeds, optimizing the formulation of efficient cryoprotectants, gradient protection treatment, designing a gradient thawing device, and implementing antioxidant repair during the revival stage, this method solves the problem of low seed survival rate after cryopreservation. The survival rate of Acer truncatum seeds after cryopreservation reaches approximately 80%, solving the current problem of difficult survival rate after cryopreservation and achieving long-term safe and stable preservation of Acer truncatum seeds.

[0112] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed in this application.

Claims

1. A method for reviving Acer truncatum seeds, characterized in that, The recovery methods include: The seeds of Acer truncatum stored at ultra-low temperature were thawed in a gradient using a thawing device to obtain thawed seeds; wherein the transfer time of the gradient thawing was ≤5 min. The thawed seeds were washed with a compound washing solution to obtain sterile seeds; The sterile seeds were cultured in a dark environment using a remediation medium to obtain primary resuscitation seeds. The primary revived seeds were cultured using a basal culture medium to complete the revival of Acer truncatum seeds; The defrosting device includes: Base; The thawing section includes a thawing partition plate, a first thawing tank, a second thawing tank, a constant temperature tube, an isolation plate, and a mounting bracket. The first thawing tank and the second thawing tank are connected in series on the surface of the base. The mounting bracket is fixed in the first thawing tank or the second thawing tank. The first thawing tank and the second thawing tank are separated by the thawing partition plate. The constant temperature tube is respectively disposed in the first thawing tank and the second thawing tank. The mounting bracket is disposed above the constant temperature tube. The mounting bracket is provided with multiple sets of fixing mechanisms for fixing the cryopreservation tube containing the Acer truncatum seeds. The isolation plate is disposed between the mounting bracket and the constant temperature tube. The transfer unit includes a screw, a threaded sliding block, a telescopic rod, a transfer fixing rod, and a transfer connecting block. One end of the rotating connecting block is fixedly connected to one end of the mounting frame, and the other end of the transfer connecting block is fixedly connected to one end of the transfer fixing rod. The other end of the transfer fixing rod is fixedly connected to the telescopic end of the telescopic rod. The telescopic rod is fixed on the threaded sliding block, and the threaded sliding block slides relative to the screw. The screw is symmetrically arranged on both sides of the base, and the length of the screw is greater than the sum of the lengths of the first thawing groove and the second thawing groove, causing the mounting frame to undergo gradient thawing in the first thawing groove or the second thawing groove.

2. The resuscitation method according to claim 1, characterized in that, The fixing mechanism includes a frame bracket, a support rod, a first sliding block, a second sliding block, a spring, a clamping unit, and a limiting sleeve. The frame bracket is disposed within the mounting frame, and the support rod is disposed within the frame bracket. The first sliding block and the second sliding block are symmetrically disposed on the support rod and slide on the support rod. The spring is disposed between the first sliding block and the second sliding block. The clamping unit is fixedly connected to both the first sliding block and the second sliding block, and the two clamping units form a clamping space for clamping the cryopreservation tube. One end of the clamping unit abuts against the limiting sleeve, causing the clamping unit to move in the horizontal direction.

3. The resuscitation method according to claim 2, characterized in that, The clamping unit includes a clamping fixing rod, a clamping connecting block, an arc-shaped fixing block, and a limiting rod. One end of the clamping fixing rod is fixedly connected to the first sliding block or the second sliding block, and the other end of the clamping fixing rod is connected to one end of the clamping connecting block. The other end of the clamping connecting block is connected to the arc-shaped fixing block, and one end of the arc-shaped fixing block is fixedly connected to the limiting rod. The limiting rod abuts against the limiting sleeve. The two arc-shaped fixing blocks form a clamping space for clamping the cryopreservation tube.

4. The resuscitation method according to claim 1, characterized in that, The transfer unit also includes a support block, a slide bar, and a control motor. The output end of the control motor is fixedly connected to the screw to drive the screw to rotate. The support block is fixed to one side of the base, and two support blocks are inserted through both ends of the screw. The slide bar is provided between the two support blocks and is fixed to the base. The threaded sliding block is provided with a sliding groove that matches the slide bar to enable the threaded sliding block to slide on the screw.

5. The resuscitation method according to claim 1, characterized in that, The defrosting device further includes a control unit, which includes a temperature controller, a temperature sensor group, and a time controller. The temperature controller is located inside the defrosting partition plate, and the temperature sensor group is located inside the first defrosting tank and the second defrosting tank. The temperature sensors are connected to the temperature controller via electrical signals, and the time controller is connected to the control motor via electrical signals.

6. The resuscitation method according to claim 1, characterized in that, The gradient thawing includes a first thawing and a second thawing, wherein the first thawing time is the same as the second thawing time. The first thawing temperature is 35°C to 45°C, and the first thawing time is 5 min to 10 min; The second thawing temperature is 20°C to 30°C, and the second thawing time is 5 min to 10 min.

7. The resuscitation method according to claim 1, characterized in that, The composite detergent comprises sucrose, trehalose, proline, and vitamin C, wherein the molar concentration of sucrose is 0.25 mol / L to 0.35 mol / L, the molar concentration of trehalose is 0.08 mol / L to 1.2 mol / L, the molar concentration of proline is 25 mmol / L to 35 mmol / L, and the mass concentration of vitamin C is 20 mg / L to 30 mg / L.

8. An application of the resuscitation method according to any one of claims 1 to 7 in the cryopreservation of Acer truncatum seeds, characterized in that, The applications include: The seeds of Acer truncatum are dried to obtain dried seeds with a preset moisture content of 8.1% to 8.2%. The dried seeds with a preset moisture content are subjected to gradient protection treatment using a cryoprotectant to obtain vitrified seeds; The vitrified seeds were cryopreserved to obtain cryopreserved Acer truncatum seeds; The cryopreserved Acer truncatum seeds are subjected to the revival method described in any one of claims 1 to 7 to obtain revival seeds.

9. The application according to claim 8, characterized in that, The dried seeds at a predetermined moisture content are subjected to gradient protection treatment using a cryoprotectant to obtain vitrified seeds, including the following steps: The dried seeds with a preset moisture content were first soaked using a cryoprotectant with a volume concentration of 60% to obtain first soaked seeds; The first soaked seeds were soaked a second time using the cryoprotectant with a volume concentration of 80% to obtain second soaked seeds; The second soaked seeds were then soaked a third time using the cryoprotectant at a volume concentration of 100% to obtain vitrified seeds; The first soaking time is 18 to 22 minutes, the second soaking time is 35 to 45 minutes, and the third soaking time is 55 to 65 minutes.

10. The application according to claim 8, characterized in that, The cryoprotectant comprises dimethyl sulfoxide, trehalose, vitamin C, glycerol, ethylene glycol, and sucrose. The dimethyl sulfoxide has a volume concentration of 5%, the trehalose has a mass concentration of 10 g / L to 15 g / L, the vitamin has a mass concentration of 0.005 g / L to 0.015 g / L, the glycerol has a mass concentration ≥30 g / L, the ethylene glycol has a volume concentration of 10% to 15%, and the sucrose has a molar concentration of 0.4 mol / L.

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