A hybrid breeding method for breeding cold-resistant dwarfing rootstocks by using malus sieversii

Abstract

The application relates to the technical field of fruit tree breeding, and discloses a hybrid breeding method for breeding cold-resistant dwarf rootstocks by using Xinjiang wild apples, which comprises the following steps: Xinjiang wild apples are used as female parents, and conventional dwarf rootstocks M9 are used as male parents to cross and extract seeds; the seeds are put into a reaction kettle, and a composite seed soaking liquid containing a citric acid-disodium hydrogen phosphate buffer solution, polyethylene glycol, dimethyl sulfoxide, tert-butyl hydrogen peroxide and gibberellin is pumped in for constant-temperature stirring reaction; then linear cooling is carried out, and citric acid aqueous solution is synchronously added dropwise to lower the pH value; after constant-temperature and constant-pH reaction, liquid is discharged, the seeds are leached, soaked and rehydrated, and finally, the seeds are placed in a dark environment for static buffering recovery and germination acceleration. The application breaks the dormancy of hybrid seeds by using chemical stress combined with cold and acid double environments, shortens the breeding period, and completes early screening of cold resistance adaptability before germination, so that the acquisition rate of cold-resistant excellent single plants is obviously improved.

Description

Technical Field

[0001] This invention relates to the field of fruit tree breeding technology, specifically a hybridization breeding method using Xinjiang wild apple to select cold-resistant and dwarfing rootstocks. Background Technology

[0002] Dwarf and high-density apple cultivation is the main model in the modern apple industry. While conventional dwarfing rootstocks have good dwarfing and flowering-promoting effects, they lack cold resistance when planted in cold northern regions, making them susceptible to root or winter frost damage. Xinjiang wild apples possess outstanding cold-resistant and stress-resistance genetic resources. Artificial hybridization between Xinjiang wild apples and conventional dwarfing rootstocks is an effective way to breed cold-resistant dwarfing rootstocks.

[0003] However, existing apple seeds exhibit deep physiological dormancy. Conventional breeding methods primarily rely on long-term low-temperature stratification to break dormancy, a time-consuming process that directly leads to lengthy breeding cycles and makes it difficult to control seed germination uniformity. Furthermore, the screening for cold resistance in existing breeding systems is severely lagging. Conventional methods typically require observing natural overwintering in the field or conducting artificial freezing tests on plants after hybrid seeds have germinated and matured, or even after transplanting, to assess cold resistance. This delayed screening model results in the consumption of significant land resources, greenhouse space, and labor costs in the early stages of breeding to maintain a large population of ineffective individuals lacking cold resistance traits. Current technologies lack an early physical and chemical stress screening mechanism that can intervene before seed germination, failing to eliminate individuals with poor stress tolerance in advance, leading to low efficiency and significant resource waste in the breeding and screening of cold-resistant dwarfing rootstocks. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a hybrid breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples. This method solves the problems of long breeding cycles due to the long dormancy period of hybrid seeds and low screening efficiency and resource waste caused by the lag in cold resistance screening to the seedling stage in existing apple rootstock hybrid breeding methods.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples includes the following steps:

[0007] Xinjiang wild apple was used as the female parent and conventional dwarfing rootstock M9 was used as the male parent for artificial hybridization. The hybrid fruits were collected, washed and the hybrid seeds were extracted. The hybrid seeds were then air-dried and shriveled and mechanically damaged seeds were removed.

[0008] The hybrid seeds are placed into a reaction vessel equipped with a temperature control device and a stirring device, and a composite soaking solution is pumped in for constant temperature stirring reaction. The composite soaking solution contains citrate-disodium hydrogen phosphate buffer base solution, polyethylene glycol 6000, dimethyl sulfoxide, tert-butyl hydrogen peroxide and gibberellin.

[0009] After the constant temperature stirring reaction, linear cooling is performed. During the synchronous period of the linear cooling temperature drop, citric acid aqueous solution is added dropwise to the reaction vessel at a uniform rate to adjust the pH value in the reaction vessel.

[0010] After the linear cooling and the addition of the citric acid aqueous solution are stopped, the reaction is carried out at a constant temperature and constant pH value.

[0011] The composite soaking solution was drained, and the hybrid seeds were rinsed with phosphate buffer solution. Then the hybrid seeds were transferred to sterile pure water for rehydration.

[0012] The hybrid seeds, after being soaked and rehydrated, were buried in a sterile vermiculite substrate, placed in a completely dark environment for static buffering and recovery, and then transferred to an incubator for routine germination.

[0013] By adopting the above technical solution, the dormancy restriction of hybrid seeds is broken by using a compound soaking solution combined with temperature and acid-lowering processes, and cold resistance screening is completed before germination. Therefore, the breeding cycle is shortened and the acquisition rate of superior cold-resistant individual plants is increased. The reaction process and mechanism of action of this invention are divided into the following steps:

[0014] Step 1: Homogeneous Osmosis and Swelling Initiation. Hybrid seeds are placed in a composite composition containing polyethylene glycol 6000. The polyethylene glycol creates external osmotic pressure, limiting the rapid entry of water into the seed and preventing cell membrane rupture due to water absorption and swelling. Dimethyl sulfoxide dissolves the waxy coating on the seed coat, increasing its permeability and allowing gibberellins and tert-butyl hydroperoxide in the composition to penetrate the seed coat barrier and enter the endosperm and embryo tissue.

[0015] Step Two: Chemical Oxidation and Dormancy Breaking. Tert-butyl hydroperoxide, as an organic peroxide, slowly decomposes within the seed, generating alkoxy radicals and hydroxyl radicals. These free radicals oxidize and inhibit the germination endogenous hormone abscisic acid (ABA) and trigger the oxidative breakage of cell wall polysaccharide macromolecules, reducing their binding force. The free radicals also act as signaling molecules, activating the antioxidant enzyme structure within the seed and initiating the dormancy-breaking metabolic process. Gibberellins simultaneously activate hydrolytic enzyme activity, promoting the degradation of stored substances.

[0016] Step 3: Cooling and Acid Stress and Early Screening. After the isothermal reaction, linear cooling is initiated, and citric acid solution is added dropwise at a uniform rate to adjust the pH. Under the dual stress of cold and acid, hybrid seeds lacking the ability to express cold-resistance genes or with poor stress tolerance undergo cell membrane phase transition damage and are eliminated before germination. Seeds carrying cold-resistance genes survive and enter the germination process.

[0017] Step 4: After rinsing, rehydration, and buffering to terminate the reaction, residual chemicals are washed away using phosphate buffer to block the oxidation reaction. The seeds are then transferred to sterile pure water for rehydration, followed by complete darkness in sterile vermiculite to promote seed membrane structure repair and cytoskeleton reconstruction, ensuring uniform seedling emergence during the germination stage.

[0018] Preferably, the preparation process of the composite soaking solution includes: preparing a citrate-disodium hydrogen phosphate buffer solution with a concentration of 0.05 mol / L to 0.15 mol / L; adjusting the initial pH of the citrate-disodium hydrogen phosphate buffer solution to 6.2 to 6.8 by adding a 1 mol / L NaOH solution or HCl solution dropwise; maintaining the temperature of the citrate-disodium hydrogen phosphate buffer solution at 25°C to 30°C; and adding polyethylene glycol 6000 in batches while stirring at 100 rpm to 200 rpm, controlling the amount of polyethylene glycol 6000... The final mass fraction of 000 is 15% to 25%, and the mixture is stirred until clear. The dimethyl sulfoxide is added, and the volume fraction of the dimethyl sulfoxide is controlled to be 0.3% to 0.8%. The mixture is stirred for another 30 minutes. After the mixture containing the dimethyl sulfoxide has cooled naturally to 20°C, the tert-butyl hydroperoxide is added, and the final concentration of the tert-butyl hydroperoxide is controlled to be 5 mmol / L to 10 mmol / L. The gibberellin is added, and the final concentration of the gibberellin is controlled to be 1.5 mg / L to 3.0 mg / L. The mixture is stirred at 50 rpm for 15 minutes under light-protected conditions.

[0019] By adopting the above technical solution, and by controlling the concentration and preparation order, the degradation of tert-butyl hydroperoxide at higher temperatures and speeds is avoided. Polyethylene glycol is dissolved first, followed by the addition of dimethyl sulfoxide to maintain the polymer's dispersion in the buffer solution. Finally, the oxidant and hormone are added under light-protected conditions at 20°C to maintain the molecular structure of the active ingredient. Therefore, the stability of the active ingredient in the seed soaking composition is improved.

[0020] Preferably, in the constant temperature stirring reaction, the solid-liquid mass ratio of the hybrid seed to the composite soaking solution is 1:8 to 1:12; the temperature control device is set to maintain the temperature inside the reactor at 12°C to 18°C, the stirring device is turned on and the speed is set to 40 rpm to 60 rpm, and the reaction is continued for 10 to 15 hours.

[0021] By employing the above technical solution, the seeds are kept in a suspended state within the reactor due to the controlled solid-liquid ratio and stirring speed, thus reducing mass transfer resistance. Maintaining the temperature at 12℃ to 18℃ controls the penetration rate of the soaking solution into the seeds, preventing premature germination metabolism caused by excessively high temperatures. Therefore, the effect of promoting safe water absorption and swelling of hybrid seeds is achieved.

[0022] Preferably, in the linear cooling process, the temperature inside the reactor is reduced from 12°C to 18°C ​​to 1°C to 4°C at a rate of 0.8°C / h to 1.5°C / h; in the pH adjustment process, a citric acid aqueous solution with a concentration of 0.5 mol / L to 1.0 mol / L is added dropwise at a uniform rate to adjust the pH value inside the reactor from the initial 6.2 to 6.8 to 4.0 to 5.0 linearly; in the process of maintaining constant temperature and constant pH, the temperature inside the reactor is maintained at 1°C to 4°C, the pH value inside the reactor is controlled at 4.0 to 5.0, the stirring speed of the stirring device is maintained at 40 rpm to 60 rpm, and the reaction is carried out for 36 hours to 60 hours.

[0023] By employing the above technical solution, and by matching the cooling rate of 0.8℃ / h to 1.5℃ / h with the pH adjustment, the seeds are given a period to adapt to changes in temperature and acidity, preventing the physical structure of the hybrid seeds from cracking due to sudden temperature drops. Maintaining the final temperature at 1℃ to 4℃ and reacting for 36 to 60 hours achieves the low-temperature accumulation required for the hybrid seeds to break dormancy. The acidic environment with a pH of 4.0 to 5.0 promotes the decomposition of peroxides, thus achieving the effect of screening cold-resistant hybrid seeds.

[0024] Preferably, in the rinsing process, the concentration of the phosphate buffer is 0.1 mol / L, the pH value is 7.0, and the solid-liquid ratio of the hybrid seeds to the phosphate buffer is 1:5; the hybrid seeds are centrifuged and rinsed at a speed of 400 rpm to 600 rpm for 10 to 15 minutes each time, and the operation is repeated 3 to 5 times; the soaking and rehydration time is 3 to 5 hours.

[0025] By employing the above technical solution, the acidic substances on the surface of hybrid seeds are neutralized by using a neutral phosphate buffer solution combined with centrifugal rinsing. Fluid shear force is then used to remove polymers and solvents adhering to the seed coat. Soaking and rehydration restores the internal moisture content of the hybrid seeds to their germination state. Therefore, the effect of removing residual chemicals and restoring seed moisture content is achieved.

[0026] Preferably, in the static buffer recovery process, the moisture content of the sterile vermiculite substrate is 55% to 65%, the temperature of the dark environment is controlled at 12°C to 18°C, and the static buffer recovery time is 4 to 7 days; in the conventional germination process, the temperature of the incubator is 22°C to 28°C, the relative humidity is 80% to 90%, and the light cycle is set to alternate between 16 hours of light and 8 hours of darkness per day.

[0027] By employing the above technical solutions, the use of a completely dark and constant-temperature substrate environment allows seeds subjected to chemical and low-temperature stress to repair their cell structure and prevents oxidative stress caused by light. The temperature, humidity, and alternating light and dark conditions during the germination stage promote seed emergence, thus improving the germination rate of hybrid seeds.

[0028] Preferably, in the air-drying process, the ambient temperature is 20°C to 25°C, and the hybrid seeds are air-dried to an absolute moisture content of 8% to 12%.

[0029] By adopting the above technical solution, the moisture content of hybrid seeds decreases and the seed coat shrinks and hardens due to the use of constant ambient temperature for air drying. Therefore, it is easier to remove poorly developed and mechanically damaged hybrid seeds.

[0030] This invention provides a hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apple. It has the following beneficial effects:

[0031] 1. This invention employs a composite soaking solution containing polyethylene glycol 6000, dimethyl sulfoxide, tert-butyl hydrogen peroxide, and gibberellin to treat hybrid seeds at a constant temperature with stirring. Dimethyl sulfoxide dissolves the seed coat wax, increasing permeability and allowing tert-butyl hydrogen peroxide to enter the seed interior to generate free radicals that oxidize endogenous dormant hormones. In conjunction with gibberellin, hydrolytic enzymes are activated, thus breaking the dormancy state of the hybrid seeds and shortening the long-cycle low-temperature stratification time required for conventional breeding.

[0032] 2. This invention constructs a dual physical and chemical stress environment of low temperature and acidity by linearly cooling the reaction after constant temperature stirring and by uniformly adding citric acid aqueous solution to the reaction vessel to adjust the pH value during the synchronous temperature drop period. Hybrid seeds lacking the ability to express cold-resistant genes or with poor stress tolerance suffer cell membrane damage and are eliminated under cold and acid stress, while hybrid seeds with cold-resistant genes survive. Early screening of cold-resistant traits is completed before seed germination, which improves the acquisition rate of superior cold-resistant individual plants.

[0033] 3. This invention uses phosphate buffer to centrifuge and rinse hybrid seeds that have undergone stress response, then transfers them to sterile pure water for rehydration, and then embeds them in a sterile vermiculite matrix and allows them to stand in complete darkness for buffering and recovery. This process removes residual chemical substances from the surface of the hybrid seeds and restores their internal moisture. The complete darkness process provides time for the hybrid seeds to repair their cell structure, prevents oxidative stress caused by light, and ensures the germination rate and uniformity of the selected hybrid seeds during the germination stage. Attached Figure Description

[0034] Figure 1 The graph shows the linear relationship between the macroscopic dynamic viscosity of each composite seed soaking system and the decrease in temperature under extreme low temperature conditions.

[0035] Figure 2 for Figure 1 A semi-logarithmic coordinate system plot of the corresponding data;

[0036] Figure 3 A comparison chart of the changes in absolute redox potential of each system during the programmed cooling cycle;

[0037] Figure 4 Comparison chart of internal physiological and biochemical indicators of seeds from different treatment groups;

[0038] Figure 5 A comparison of the average internode length and dispersion of seedlings in each treatment group;

[0039] Figure 6 A comparison chart of the semi-lethal temperatures of seedlings in each treatment group. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, preparation examples, embodiments, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Examples 1-3:

[0042] Example 1:

[0043] This embodiment provides a hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples, including the following steps:

[0044] Step 1: Parental hybridization and seed pretreatment:

[0045] Artificial hybridization was carried out using Xinjiang wild apple as the female parent and conventional dwarfing rootstock M9 as the male parent. The hybrid fruits were collected in autumn, the hybrid seeds were extracted by washing with water, and dried in the shade at room temperature of 20℃ until the absolute moisture content of the seeds was 8%. Shriveled and mechanically damaged seeds were removed for later use.

[0046] Step 2, Preparation of the compound soaking solution:

[0047] Prepare a 0.05 mol / L citrate-disodium hydrogen phosphate buffer solution in a mixing tank, and precisely adjust its initial pH value to 6.2 by adding 1 mol / L NaOH solution or HCl solution.

[0048] Maintain the temperature of the mixing tank at 25°C, and slowly add polyethylene glycol PEG-6000 to the buffer solution in batches while stirring at 100 rpm, so that the final mass fraction is 15%, and stir until the system is completely clear and free of particles.

[0049] Add 0.3% (v / v) of dimethyl sulfoxide (DMSO) to the above system and continue stirring for 30 minutes;

[0050] After the system cooled naturally to room temperature (20°C), tert-butyl hydroperoxide (t-BHP) was added to bring the final concentration to 5 mmol / L, and gibberellin (GA3) was added to bring the final concentration to 1.5 mg / L. The mixture was stirred at a low speed of 50 rpm for 15 minutes under dark conditions to obtain the composite soaking solution.

[0051] Step 3: Establishment of isothermal swelling and primary osmosis:

[0052] The pretreated hybrid seeds from step 1 were placed into a reaction vessel equipped with a jacketed temperature control and mechanical stirring device. The composite soaking solution prepared in step 2 was pumped in, and the solid-liquid mass ratio was controlled at 1:8. The jacketed temperature control was set to maintain the temperature inside the vessel at a constant 12°C. The mechanical stirring was turned on, and the speed was set to 40 rpm. The reaction was continued for 10 hours.

[0053] Step 4: Programmed cooling and mass transfer oxidation targeted screening:

[0054] The jacket cooling program is started, and the temperature of the system inside the vessel is slowly reduced from 12℃ to 4℃ at a linear cooling rate of 0.8℃ / h.

[0055] During the synchronous period of linear temperature decrease, 0.5 mol / L citric acid aqueous solution was uniformly added to the reaction vessel system using a dosing pump, so that the pH value of the system was linearly and smoothly adjusted from the initial 6.2 to 5.0.

[0056] Once the temperature inside the reactor reaches 4°C and the pH value reaches 5.0, stop cooling and adding acid solution, maintain a stirring speed of 40 rpm, and maintain this constant physicochemical state for 36 hours.

[0057] Step 5: Termination of reaction and elution to remove toxins:

[0058] Drain the soaking solution from the reactor, add isotonic 0.1 mol / L phosphate buffer (pH 7.0) to the reactor, and control the solid-liquid ratio to 1:5. Centrifuge the seeds at 400 rpm for 10 minutes each time, and repeat the operation 3 times. After rinsing, transfer the seeds to sterile pure water for 3 hours to rehydrate.

[0059] Step 6, Physiological recovery and routine germination:

[0060] After washing and rehydrating the seeds in step 5, bury them in a sterile vermiculite substrate with a moisture content of 55% and place them in an artificial climate chamber. Let them stand and buffer for 4 days in a completely dark environment at 12℃. After the recombination period, take out the seeds and transfer them to a culture chamber with a temperature of 22℃, relative humidity of 80%, and a light cycle of 16h / 8h (light / dark) for conventional germination. After they germinate and sprout, transplant them to their final location.

[0061] Example 2:

[0062] This embodiment provides a hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples, including the following steps:

[0063] Step 1: Parental hybridization and seed pretreatment:

[0064] Artificial hybridization was carried out using Xinjiang wild apple as the female parent and conventional dwarfing rootstock M9 as the male parent. The hybrid fruits were collected in autumn, the hybrid seeds were extracted by washing with water, and then air-dried at room temperature of 22℃ until the absolute moisture content of the seeds was 10%. Shriveled and mechanically damaged seeds were removed for later use.

[0065] Step 2, Preparation of the compound soaking solution:

[0066] Prepare a 0.1 mol / L citrate-disodium hydrogen phosphate buffer solution in a mixing tank, and precisely adjust its initial pH value to 6.5 by adding 1 mol / L NaOH solution or HCl solution.

[0067] Maintain the temperature of the mixing tank at 28°C, and slowly add polyethylene glycol PEG-6000 to the buffer solution in batches while stirring at 150 rpm, so that the final mass fraction is 20%, and stir until the system is completely clear and free of particles.

[0068] Add 0.5% (v / v) of dimethyl sulfoxide (DMSO) to the above system and continue stirring for 30 minutes;

[0069] After the system cooled naturally to room temperature (20°C), tert-butyl hydroperoxide (t-BHP) was added to bring the final concentration to 8 mmol / L, and gibberellin (GA3) was added to bring the final concentration to 2.0 mg / L. The mixture was stirred at a low speed of 50 rpm for 15 minutes under dark conditions to obtain the composite soaking solution.

[0070] Step 3: Establishment of isothermal swelling and primary osmosis:

[0071] The pretreated hybrid seeds from step 1 were placed into a reaction vessel equipped with a jacketed temperature control and mechanical stirring device. The composite soaking solution prepared in step 2 was pumped in, and the solid-liquid mass ratio was controlled at 1:10. The jacketed temperature control was set to maintain the temperature inside the vessel at a constant 15°C. The mechanical stirring was turned on, and the speed was set to 50 rpm. The reaction was continued for 12 hours.

[0072] Step 4: Programmed cooling and mass transfer oxidation targeted screening:

[0073] The jacket cooling program is started, and the temperature of the system inside the vessel is slowly reduced from 15℃ to 2℃ at a linear cooling rate of 1.0℃ / h.

[0074] During the synchronous period of linear temperature decrease, 0.8 mol / L citric acid aqueous solution was uniformly added to the reactor system using a dosing pump, so that the pH value of the system was linearly and smoothly adjusted from the initial 6.5 to 4.5.

[0075] Once the temperature inside the reactor reaches 2°C and the pH value reaches 4.5, stop cooling and adding acid solution, maintain a stirring speed of 50 rpm, and maintain this constant physicochemical state for 48 hours.

[0076] Step 5: Termination of reaction and elution to remove toxins:

[0077] Drain the soaking solution from the reactor, add isotonic 0.1 mol / L phosphate buffer (pH 7.0) to the reactor, and control the solid-liquid ratio to 1:5. Centrifuge the seeds at 500 rpm for 12 minutes each time, and repeat the operation 4 times. After rinsing, transfer the seeds to sterile pure water for 4 hours to rehydrate.

[0078] Step 6, Physiological recovery and routine germination:

[0079] After washing and rehydrating the seeds in step 5, bury them in a sterile vermiculite substrate with a moisture content of 60% and place them in an artificial climate chamber. Let them stand and buffer for 5 days in a completely dark environment at 15℃. After the recombination period, take out the seeds and transfer them to a culture chamber with a temperature of 25℃, relative humidity of 85%, and a light cycle of 16h / 8h (light / dark) for conventional germination. After they germinate and sprout, transplant them to their final location.

[0080] Example 3:

[0081] This embodiment provides a hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples, including the following steps:

[0082] Step 1: Parental hybridization and seed pretreatment:

[0083] Artificial hybridization was carried out using Xinjiang wild apple as the female parent and conventional dwarfing rootstock M9 as the male parent. The hybrid fruits were collected in autumn, the hybrid seeds were extracted by washing with water, and dried in the shade at room temperature of 25℃ until the absolute moisture content of the seeds was 12%. Shriveled and mechanically damaged seeds were removed for later use.

[0084] Step 2, Preparation of the compound soaking solution:

[0085] A 0.15 mol / L citrate-disodium hydrogen phosphate buffer solution was prepared in a mixing tank, and its initial pH value was precisely adjusted to 6.8 by adding 1 mol / L NaOH solution or HCl solution.

[0086] Maintain the temperature of the mixing tank at 30℃, and slowly add polyethylene glycol PEG-6000 to the buffer solution in batches while stirring at 200 rpm, so that the final mass fraction is 25%, and stir until the system is completely clear and free of particles.

[0087] Add 0.8% (v / v) of dimethyl sulfoxide (DMSO) to the above system and continue stirring for 30 minutes;

[0088] After the system cooled naturally to room temperature (20°C), tert-butyl hydroperoxide (t-BHP) was added to bring the final concentration to 10 mmol / L, and gibberellin (GA3) was added to bring the final concentration to 3.0 mg / L. The mixture was stirred at a low speed of 50 rpm for 15 minutes under dark conditions to obtain the composite seed soaking solution.

[0089] Step 3: Establishment of isothermal swelling and primary osmosis:

[0090] The pretreated hybrid seeds from step 1 were placed into a reaction vessel equipped with a jacketed temperature control and mechanical stirring device. The composite soaking solution prepared in step 2 was pumped in, and the solid-liquid mass ratio was controlled at 1:12. The jacketed temperature control was set to maintain the temperature inside the vessel at a constant 18°C. The mechanical stirring was turned on, and the speed was set to 60 rpm. The reaction was continued for 15 hours.

[0091] Step 4: Programmed cooling and mass transfer oxidation targeted screening:

[0092] The jacket cooling program is started, and the temperature of the system inside the vessel is slowly reduced from 18℃ to 1℃ at a linear cooling rate of 1.5℃ / h.

[0093] During the synchronous period of linear temperature decrease, a 1.0 mol / L citric acid aqueous solution was uniformly added to the reactor system using a dosing pump, so that the pH value of the system was linearly and smoothly adjusted from the initial 6.8 to 4.0.

[0094] Once the temperature inside the reactor reaches 1°C and the pH value reaches 4.0, stop cooling and adding acid solution, maintain a stirring speed of 60 rpm, and maintain this constant physicochemical state for 60 hours.

[0095] Step 5: Termination of reaction and elution to remove toxins:

[0096] Drain the soaking solution from the reactor, add isotonic 0.1 mol / L phosphate buffer (pH 7.0) to the reactor, and control the solid-liquid ratio to 1:5. Centrifuge the seeds at 600 rpm for 15 minutes each time, and repeat the operation 5 times. After rinsing, transfer the seeds to sterile pure water for 5 hours to rehydrate.

[0097] Step 6, Physiological recovery and routine germination:

[0098] After washing and rehydrating the seeds in step 5, bury them in a sterile vermiculite substrate with a moisture content of 65% and place them in an artificial climate chamber. Let them stand and recover for 7 days in a completely dark environment at 18℃. After the recombination period, take out the seeds and transfer them to a culture chamber with a temperature of 28℃, relative humidity of 90%, and a light cycle of 16h / 8h (light / dark) for conventional germination. After they germinate and sprout, transplant them to their final location.

[0099] Comparative Examples 1-5:

[0100] Comparative Example 1:

[0101] Compared with Example 2, the difference is that no chemical soaking or temperature and acid treatment is performed. Instead, the traditional wet sand low-temperature stratification method (mixing hybrid seeds with wet sand and refrigerating them at 2°C for 60 days) is used to break dormancy and natural selection. The remaining steps (such as obtaining parent seeds, germination and transplanting) are the same.

[0102] Comparative Example 2:

[0103] The difference from Example 2 is that the polar shearing and penetration enhancer dimethyl sulfoxide (DMSO) is not added in the composition preparation of step 2, while the rest are the same.

[0104] Comparative Example 3:

[0105] Compared with Example 2, the difference is that no citric acid aqueous solution is added during the temperature program in step 4, and the pH value of the system in the reactor remains constant at the initial 6.5. All other aspects are the same.

[0106] Comparative Example 4:

[0107] The difference from Example 2 is that gibberellin (GA3) is not added in the preparation of the composition in step 2, but everything else is the same.

[0108] Comparative Example 5:

[0109] Compared with Example 2, the difference is that in step 4, the temperature is not programmed to decrease, but the system is kept constant at 15°C for 60 hours. At the same time, the acid reduction operation is not performed. All other steps are the same.

[0110] Test Examples 1-4:

[0111] Test Example 1:

[0112] 60 mL of the composite seed soaking liquid prepared before the second stage reaction of Examples 1, 2, 3 and Comparative Example 2 was extracted as test samples.

[0113] Each group of test samples was transferred into the measuring cylinder of a digital rotational viscometer equipped with a circulating water bath temperature control jacket. A coaxial cylindrical rotor of the same specification was selected, and the initial circulating water temperature of the jacket was set to 15℃. The system was kept at a constant temperature for 20 minutes to allow the internal temperature gradient to balance and eliminate residual fluid shear stress.

[0114] A linear cooling program for the circulating water bath was initiated, with the cooling rate controlled at 1℃ / h, to simulate the reaction cooling process in stage two. The viscometer rotor speed was set to a constant 50 rpm, and a constant shear rate was applied to the fluid for continuous measurement.

[0115] The macroscopic dynamic viscosity of the system was read and recorded when the actual temperature of the system reached 15℃, 12℃, 9℃, 6℃, 4℃, 2℃ and 1℃, respectively. Each sample was measured three times and the average value was taken.

[0116] Table 1. Data on macroscopic dynamic viscosity of various systems under extreme low temperature conditions.

[0117] Temperature node (°C) Example 1 Viscosity (mPa·s) Example 2 Viscosity (mPa·s) Example 3 Viscosity (mPa·s) Comparative Example 2 Viscosity (mPa·s) 15 8.3 14.5 22.1 14.7 12 9.1 15.8 23.6 16.2 9 10.4 17.6 26.3 21.4 6 11.8 19.8 30.1 58.7 4 12.6 21.4 33.5 145.2 2 13.5 23.1 36.4 386.5 1 14.2 24.6 38.8 612.8

[0118] in conclusion:

[0119] Based on the data in Table 1, combined with Figure 1 (The horizontal axis indicates the programmed cooling process, and the vertical axis reflects the actual viscous resistance of the system.) Figure 2 ( Figure 1 The data is presented in a semi-logarithmic coordinate system for analysis. In Comparative Example 2, the viscosity change is relatively gradual as the temperature drops from 15℃ to 6℃; when the system temperature is below 6℃ and continues to drop to 1℃, its macroscopic dynamic viscosity increases from 21.4 mPa·s to 612.8 mPa·s. Figure 2By logarithmizing the ordinate, the exponential mutation characteristic of Comparative Example 2 is revealed. According to polymer physics mechanisms, in high-concentration polyethylene glycol (PEG) aqueous solutions without added polar shear agents, near the freezing point, molecular thermal motion weakens, free water molecules decrease, and hydrogen bonds between PEG molecular chains undergo reconstruction and cross-linking, causing the system to transition from a solution state to a physical gel state. According to the Stokes-Einstein equation, the diffusion coefficient of solute molecules is inversely proportional to the dynamic viscosity of the fluid medium. The high viscosity generated in Comparative Example 2 at low temperatures forms a physical mass transfer barrier, preventing the reagent molecules within the reaction system from maintaining the mass transfer flux to penetrate into the seed interior.

[0120] Figure 2 The low-viscosity, smooth, and stable behavior during the phase-change-free phase in the example group was also amplified. Test results from Examples 1 to 3 show that the macroscopic dynamic viscosity of the system increases linearly and gradually within a cooling cycle from 15°C to 1°C. In Example 3, which contains 25% polyethylene glycol, the extreme viscosity at 1°C is 38.8 mPa·s, and the system maintains the characteristics of a low-viscosity Newtonian fluid. This indicates that dimethyl sulfoxide (DMSO) plays a polar shear role in this low-temperature, high-permeability system. DMSO molecules preferentially occupy the free hydroxyl sites on polyethylene glycol segments through sulfur-oxygen double bonds, forming steric hindrance and blocking the low-temperature crosslinking and recombination of the hydrogen bond network between polyethylene glycol segments. The stability of the fluid's physical state ensures the constant solute diffusion coefficient during the programmed cooling screening phase, providing a basis for mass transfer kinetics.

[0121] Test Example 2:

[0122] The high-precision redox potential composite electrode calibrated with standard buffer solution and the matching measuring instrument were deployed in the reaction vessels of Example 2 and Comparative Example 3, respectively, with the electrode probe submerged at the same depth below the liquid surface.

[0123] In the second stage of the cooling process, both Example 2 and Comparative Example 3 cooled down from 15°C to 2°C at a rate of 1.0°C / h, with a total cooling cycle time of 13 hours.

[0124] During the cooling process, in Example 2, a 0.8 mol / L citric acid aqueous solution was added dropwise at a uniform rate using a dosing pump, causing the system pH to decrease linearly from 6.5 to 4.5; in Comparative Example 3, no dropwise addition was performed, and the system pH remained constant at 6.5. Both groups maintained a set mechanical stirring speed of 50 rpm.

[0125] The cooling program was started at 0 hours. At 2, 4, 6, 8, 10 hours and at 13 hours after the cooling was completed, the absolute redox potential values ​​of the two reaction systems were read and recorded in situ.

[0126] Table 2. Absolute redox potential measurement data of each system during the programmed cooling cycle.

[0127] Test time (h) System temperature (°C) Example 2 ORP (mV) Comparative example 3ORP (mV) 0 15 423.6 421.8 2 13 418.2 386.4 4 11 421.7 342.1 6 9 416.9 305.7 8 7 424.1 273.4 10 5 419.3 241.9 13 2 422.5 218.6

[0128] in conclusion:

[0129] Based on the data in Table 2, combined with Figure 3 (The horizontal axis in the figure represents the test time and simultaneously marks the actual temperature of the reaction system at that time point; the vertical axis represents the absolute redox potential measured in the system and the chemical oxidation potential intensity within the reaction system.) The analysis was conducted.

[0130] The black dashed line in the figure, representing Comparative Example 3 (the group that was cooled but not acidified), shows a clear stepwise decline as the temperature decreases. During the 13-hour period when the system temperature dropped from 15°C to 2°C, without acidity reduction intervention, the absolute redox potential decreased continuously from an initial 421.8 mV to 218.6 mV, exhibiting a steep decline. The redox potential of lipid peroxidation induced by tert-butyl hydroperoxide is governed by the Nernst equation and is positively correlated with the system's hydrogen ion concentration; the rate constant for its decomposition to generate free radicals is governed by the Arrhenius equation and is exponentially positively correlated with the system's absolute temperature. The cooling process weakens molecular thermal motion, relatively increasing the activation energy barrier of the oxidation reaction. Without increasing the hydrogen ion concentration, the temperature decrease directly causes a decrease in the thermodynamic driving force of the reaction system. This decrease in thermodynamic driving force caused by physical cooling means that the oxidation pressure provided by the system cannot be maintained at the set threshold.

[0131] The solid black line in the figure, representing Example 2 (the simultaneous cooling and acid reduction group), remains within a stable high potential range. Under the same cooling conditions, the absolute redox potential of the system in Example 2 consistently fluctuates within a small range of 416.9 mV to 424.1 mV, without any potential drop, indicating that the chemical oxidation pressure of the system was not weakened by the cooling. In Example 2, citric acid was added dropwise during the synchronous period of linear temperature decrease, lowering the pH value of the system from 6.5 to 4.5. The smooth increase in hydrogen ion concentration thermodynamically enhanced the standard redox potential of tert-butyl hydroperoxide. The enhanced chemical oxidation potential from the acid reduction operation compensated for the weakening of physical diffusion and the decrease in reaction rate caused by the cooling. The dynamic hedging coupling mechanism of temperature and pH parameters eliminated the reaction activity decay caused by a single cooling variable, maintaining a constant absolute oxidation pressure within the reactor. This figure visually confirms the role of the dual-parameter coupling hedging mechanism of temperature and pH in maintaining a constant oxidation potential, providing a stable physicochemical environment for subsequent targeted oxidation screening.

[0132] Test Example 3:

[0133] At the end of the 48-hour constant stress phase 2, 100 seeds were randomly collected from the reaction vessels of Example 2, Comparative Example 4 and Comparative Example 5 respectively, and the seeds were rinsed with deionized water to remove the residual soaking solution on the surface.

[0134] Seeds from each group were longitudinally cut along the seed ridge and immersed in a 0.1% TTC (triphenyltetrazolium chloride) solution for staining in a 30°C incubator in the dark for 2 hours. The physiological activity of the seeds was determined based on the staining results: seeds with bright red embryos and cotyledons were considered viable, while those with light pink, white, or softened tissues were considered dead. Viable and dead seeds within the same group were collected separately.

[0135] Seed coats were removed from various seeds, and 0.5 g of embryo and endosperm tissue were accurately weighed. Pre-cooled 0.1 mol / L phosphate buffer was added, and the mixture was homogenized in an ice bath. The homogenate was centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was collected as the crude enzyme solution for testing.

[0136] The α-amylase activity in the extract was determined by the 3,5-dinitrosalicylic acid colorimetric method, with units of U / gFW; the malondialdehyde content was determined by the thiobarbituric acid (TBA) method at a specific wavelength, with units of nmol / gFW.

[0137] Table 3. Seed physiological and biochemical data at the end of the constant stress phase.

[0138] Treatment group and physiological state α-Amylase activity (U / gFW) Malondialdehyde (MDA) content (nmol / gFW) Example 2 (Surviving Part) 12.8 16.4 Example 2 (Death Part) 91.3 142.7 Comparative Example 4 (All Samples) 15.6 39.2 Comparative Example 5 (All Samples) 64.9 167.3

[0139] in conclusion:

[0140] Based on the data in Table 3, combined with Figure 4 (The horizontal axis of the figure is divided into four test groups: Example 2 (surviving part), Example 2 (dead part), Comparative Example 4 (all samples without gibberellin), and Comparative Example 5 (all samples under constant 15℃ high temperature). The graph uses a dual vertical axis system. The left primary axis and its corresponding dark gray bars represent α-amylase activity, reflecting the intensity of seed germination metabolic initiation; the right secondary axis and its corresponding light gray bars represent malondialdehyde content, reflecting the degree of damage to cell membrane lipid peroxidation in the seed. The absolute values ​​of the corresponding data are marked above the bars. This graph quantitatively shows the physiological and biochemical response characteristics of seeds under different induced stress mechanisms, such as survival, escape, and indiscriminate lethality.)

[0141] The results of Example 2 showed a clear polarization distribution between surviving and dead seeds. Dead seeds exhibited α-amylase activity of 91.3 U / gFW and malondialdehyde (MDA) content as high as 142.7 nmol / gFW; while surviving seeds showed only α-amylase activity of 12.8 U / gFW and MDA content maintained at a low level of 16.4 nmol / gFW. In the system containing gibberellin and tert-butyl peroxide, the receptors of non-dwarfing and non-cold-resistant inferior seeds were highly sensitive to gibberellin. After absorbing gibberellin, inferior seeds forcibly initiated amylase transcription and metabolic recovery mechanisms, leading to the degradation of endogenous abscisic acid and antioxidant enzyme systems. The vulnerable cells, after the metabolic barrier was removed, were completely exposed to the strong oxidative environment created by tert-butyl peroxide, triggering irreversible and severe membrane lipid peroxidation, ultimately resulting in death. High-quality cold-resistant dwarfing seeds remained insensitive to gibberellin and maintained a deep defensive dormant state, thus preventing the spread of peroxidative damage and surviving silently.

[0142] In Comparative Example 4, without the addition of gibberellin, the α-amylase activity of its extract remained at an extremely low level of 15.6 U / gFW, while the content of malondialdehyde, a cell membrane lipid peroxidation product, accumulated to 39.2 nmol / gFW, far below the lethal threshold. The lack of gibberellin as a suicide inducer prevented inferior seeds from undergoing erroneous metabolic awakening, allowing many non-dwarf seeds to evade oxidative killing by tert-butyl peroxide in a dormant state through intact native stress response mechanisms, thus losing their targeted elimination ability.

[0143] In Comparative Example 5, the malondialdehyde (MDA) content in all seeds reached a lethal level of 167.3 nmol / gFW during the reaction at a high temperature of 15°C. Without programmed cooling, the target seeds were unable to respond to the low temperature and enter and maintain a defensive deep dormancy state. The excessively high basal metabolic activity within the seeds, coupled with the undiminished high activation rate of tert-butyl hydroperoxide at 15°C, triggered intense and non-selective lipid peroxidation. This excessive oxidative storm exceeded the tolerance limits of all seeds, resulting in indiscriminate lethality of all seeds, including those with superior genes.

[0144] Test Example 4:

[0145] Seeds treated according to the various examples and comparative examples were sown in seedling trays containing standard seedling substrate and cultured in an artificial climate chamber at 25°C and 75% relative humidity. The total time from the start of the seed soaking treatment to the radicle breaking through 2 mm of the seed coat was recorded. The final seedling rate on the 45th day after sowing was calculated.

[0146] The surviving seedlings from each group were transferred to a standard greenhouse for further cultivation for 3 months. The greenhouse environment was controlled with a day / night temperature of 25℃ / 18℃ and a photoperiod of 14h / 10h.

[0147] Seedlings that have grown for 3 months were selected from each group, and the absolute length of the first 5 internodes of the main stem was measured using a vernier caliper with an accuracy of 0.02 mm. The mean internode length of each group of seedlings and the variance of the sample population were calculated.

[0148] Mature leaves from the same position on each group of seedlings were harvested, washed, dried, and chopped into small pieces. The leaf pieces were placed in a gradient cooling apparatus and subjected to a gradient cooling treatment from 4℃ to -20℃. The leaves were then removed, added to deionized water, and extracted by shaking at room temperature. The initial conductivity was measured. The extracted tissue was boiled and cooled to measure the final conductivity. The relative conductivity was calculated, and the logistic equation was used to fit the curve of relative conductivity versus treatment temperature to determine the median lethal temperature (LT) for each group of seedlings. 50 .

[0149] Table 4. Data on the measurement of core indicators for breeding of final products in each treatment system

[0150] Processing Group Total germination time (d) Final seedling survival rate (%) Average intersegment length (mm) Intersegment length variance <![CDATA[Lethal temperature 50% (LT 50 (°C)]]> Example 1 4.6 13.5 14.2 1.14 -14.3 Example 2 4.9 11.8 13.6 0.92 -15.8 Example 3 5.3 10.2 12.7 0.85 -16.4 Comparative Example 1 67.4 78.6 32.5 56.41 -4.5 Comparative Example 2 6.2 35.1 23.8 25.73 -7.2 Comparative Example 3 5.5 42.4 25.1 31.05 -6.6 Comparative Example 4 4.8 84.3 30.9 48.66 -4.8 Comparative Example 5 -- 0 -- -- --

[0151] Note: "--" indicates no data or cannot be measured.

[0152] in conclusion:

[0153] Based on the data in Table 4, combined with Figure 5 (Comparative Example 5, lacking seedlings, is not included; the height of the bars in the figure reflects the average internode length of the population, and the error bars at the top reflect the standard deviation calculated from the data variance.) Figure 6 (The vertical axis represents negative values ​​for temperature) for analysis.

[0154] The total time from seed treatment to germination in Examples 1 to 3 was between 4.6 and 5.3 days, compared to 67.4 days in Comparative Example 1 using traditional stratification treatment, thus shortening the dormancy breaking process. The final seedling rate in these examples was controlled within the range of 10.2% to 13.5%. Figure 5 In the example group, shorter columns and extremely narrow error bars corresponded to an average internode length distribution of 12.7 to 14.2 mm for surviving seedlings, with an internode length variance between 0.85 and 1.14. This clearly demonstrates the dwarfing phenotype and high purity characteristics of the plants. Figure 6 In the example group, the curve extends downwards significantly, representing the plant's ultimate tolerance to low temperatures. Its actual half-lethal temperature LT... 50 Reaching temperatures of -14.3℃ to -16.4℃, it exhibits cold resistance. The coupling of low seedling survival rate and high trait purity demonstrates that the treatment system created directional selection pressure, eliminating non-targeted seeds lacking cold resistance and dwarfing genes before sowing. Figure 6 The distribution clearly demonstrates the decisive advantage of the example system over the various control treatments in enhancing and purifying the cold resistance trait in the offspring.

[0155] Comparative Example 1, used as a conventional breeding control, had a longer total breeding time and a seedling survival rate of 78.6%. Figure 5 The extremely wide error bars in the comparative group indicate severe phenotypic segregation and confounding within the population; the internode length of its offspring reaches 32.5 mm with a variance of 56.41. LT 50 The temperature was -4.5℃. Conventional methods lack trait selection mechanisms, leading to segregation and mixing in the offspring population.

[0156] Comparative Example 2, without the addition of a polar shear agent, experienced gelation at low temperatures, hindering mass transfer. Some non-targeted seeds did not absorb sufficient inducing and killing agents, thus escaping the elimination process. Its seedling survival rate was 35.1%, and the variance in internode length increased to 25.73. LT 50 When the temperature was raised to -7.2℃, the resulting seedlings were in a mixed state and targeted purification was not achieved.

[0157] Comparative Example 3 did not undergo acid reduction treatment during the cooling process, and the oxidative driving force decreased with decreasing temperature. The lethal pressure failed to overcome the antioxidant and stress response mechanisms of inferior seeds, resulting in incomplete elimination. The seedling survival rate reached 42.4%, and the variance of internode length was similar to that of LT. 50 All indicators showed a non-homozygous state.

[0158] In Comparative Example 4, the removal of gibberellin resulted in no metabolic recovery signals being received by non-dwarfing and non-cold-resistant seeds, thus maintaining their dormancy defense barrier. Tert-butyl hydroperoxide did not cause substantial damage, and the seedling survival rate reached 84.3%. The variance in seedling internode length and cold resistance were consistent with those in Comparative Example 1 under natural conditions, indicating that the screening system was ineffective.

[0159] Comparative Example 5 was reacted at a constant temperature of 15℃. The seeds had active internal metabolism and high activation of tert-butyl hydrogen peroxide, resulting in indiscriminate oxidative damage. The seedling rate was 0, and no surviving plants were obtained.

Claims

1. A hybridization breeding method using Xinjiang wild apple to select cold-resistant and dwarfing rootstocks, characterized in that, Includes the following steps: Xinjiang wild apple was used as the female parent and conventional dwarfing rootstock M9 was used as the male parent for artificial hybridization. The hybrid fruits were collected, washed and the hybrid seeds were extracted. The hybrid seeds were then air-dried and shriveled and mechanically damaged seeds were removed. The hybrid seeds are placed into a reaction vessel equipped with a temperature control device and a stirring device, and a composite soaking solution is pumped in for constant temperature stirring reaction. The composite soaking solution contains citrate-disodium hydrogen phosphate buffer base solution, polyethylene glycol 6000, dimethyl sulfoxide, tert-butyl hydrogen peroxide and gibberellin. After the constant temperature stirring reaction, linear cooling is performed. During the synchronous period of the linear cooling temperature drop, citric acid aqueous solution is added dropwise to the reaction vessel at a uniform rate to adjust the pH value in the reaction vessel. After the linear cooling and the addition of the citric acid aqueous solution are stopped, the reaction is carried out at a constant temperature and constant pH value. The composite soaking solution was drained, and the hybrid seeds were rinsed with phosphate buffer solution. Then the hybrid seeds were transferred to sterile pure water for rehydration. The hybrid seeds, after being soaked and rehydrated, were buried in a sterile vermiculite substrate, placed in a completely dark environment for static buffering and recovery, and then transferred to an incubator for routine germination.

2. The hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples according to claim 1, characterized in that, The preparation process of the compound seed soaking solution includes: Prepare a citrate-disodium hydrogen phosphate buffer solution with a concentration of 0.05 mol / L to 0.15 mol / L by adding a 1 mol / L NaOH solution or HCl solution to adjust the initial pH of the citrate-disodium hydrogen phosphate buffer solution to 6.2 to 6.

8. Maintain the temperature of the citrate-disodium hydrogen phosphate buffer solution at 25°C to 30°C, and add polyethylene glycol PEG-6000 in batches while stirring at 100 rpm to 200 rpm, controlling the final mass fraction of polyethylene glycol PEG-6000 to be 15% to 25%, and stir until clear; Add the dimethyl sulfoxide (DMSO) and control the volume fraction of the DMSO to be 0.3% to 0.8%, and continue stirring for 30 minutes; After the mixture containing the added dimethyl sulfoxide (DMSO) is allowed to cool naturally to 20°C, the tert-butyl hydroperoxide (t-BHP) is added, with the final concentration of t-BHP controlled at 5 mmol / L to 10 mmol / L. Then, the gibberellin (GA3) is added, with the final concentration of GA3 controlled at 1.5 mg / L to 3.0 mg / L. The mixture is stirred at 50 rpm for 15 minutes under light-protected conditions.

3. The hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples according to claim 1, characterized in that, In the constant temperature stirring reaction, the solid-liquid mass ratio of the hybrid seed to the composite soaking solution is 1:8 to 1:

12. The temperature control device is set to maintain the temperature inside the reactor at 12°C to 18°C. The stirring device is turned on and the speed is set to 40 rpm to 60 rpm. The reaction continues for 10 to 15 hours.

4. The hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples according to claim 1, characterized in that, In the linear cooling process, the temperature inside the reactor is reduced from 12°C to 18°C ​​to 1°C to 4°C at a rate of 0.8°C / h to 1.5°C / h. During the pH adjustment, an aqueous solution of citric acid with a concentration of 0.5 mol / L to 1.0 mol / L is added dropwise at a uniform rate to linearly adjust the pH value in the reaction vessel from the initial 6.2 to 6.8 to 4.0 to 5.

0.

5. The hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples according to claim 4, characterized in that, During the reaction, which is carried out under constant temperature and pH, the temperature inside the reactor is maintained at 1°C to 4°C, the pH inside the reactor is controlled at 4.0 to 5.0, the stirring device is kept at a speed of 40 rpm to 60 rpm, and the reaction is carried out for 36 to 60 hours.

6. The hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples according to claim 1, characterized in that, In the rinsing process, the concentration of the phosphate buffer is 0.1 mol / L, the pH value is 7.0, and the solid-liquid ratio of the hybrid seeds to the phosphate buffer is 1:

5. The hybrid seeds were centrifuged and rinsed at a speed of 400 rpm to 600 rpm for 10 to 15 minutes each time, and the operation was repeated 3 to 5 times.

7. The hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples according to claim 1, characterized in that, The soaking and rehydration time is 3 to 5 hours.

8. The hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples according to claim 1, characterized in that, In the static buffer recovery process, the moisture content of the sterile vermiculite matrix is ​​55% to 65%, the temperature of the completely dark environment is controlled at 12°C to 18°C, and the static buffer recovery time is 4 to 7 days.

9. The hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples according to claim 1, characterized in that, In the conventional germination process, the temperature of the incubator is 22°C to 28°C, the relative humidity is 80% to 90%, and the light cycle is set to alternate between 16 hours of light and 8 hours of darkness per day.

10. The hybridization breeding method for selecting cold-resistant and dwarfing rootstocks from Xinjiang wild apples according to claim 1, characterized in that, In the air-drying process, the ambient temperature is 20°C to 25°C, and the hybrid seeds are air-dried until the absolute moisture content is 8% to 12%.

Images