Method for rapid screening of cold-resistant and early-flowering apple rootstocks from hybrid seedlings

Abstract

This invention discloses a method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings, comprising the following steps: 1. Scion collection: scions are collected from mother apple seedlings; 2. Hydroponics: the collected scions are placed in a container for hydroponic cultivation; 3. Observation and recording of the budding status of the hydroponically cultivated scions; 4. Screening for cold-resistant scions based on budding status; 5. Grafting and planting: grafting the grafted plant onto the screened cold-resistant scions, and planting the grafted scions in a nursery; 6. During the 2nd-3rd year after planting, the plant height and number of flower buds of the grafted scions are counted; 7. Combining the plant height and number of flower buds of the grafted scions, apple rootstocks with both cold resistance and early flowering characteristics are screened. This invention forms a two-stage technical closed loop of "rapid initial screening → precise verification," compressing the traditional breeding cycle by more than 60% and reducing screening costs by 70%.

Description

Technical Field

[0001] This invention belongs to the field of apple germplasm resource evaluation and utilization technology, specifically relating to a method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings. Background Technology

[0002] Apple dwarfing and high-density planting technology has become the mainstream direction of modern apple industry development due to its advantages such as saving land resources, increasing yield per unit area, facilitating mechanized management, and improving fruit quality. In this technology system, the selection of rootstock is particularly crucial. It must possess both stress resistance (such as cold resistance) and early flowering and fruiting characteristics to ensure the adaptability of fruit trees in complex environments and the rapid realization of economic benefits. Especially in the harsh winters of northern my country, the cold resistance of the rootstock directly determines the survival rate and overwintering safety of apple trees, while early flowering and fruiting characteristics can effectively shorten the vegetative growth period of young trees, promote the early entry of trees into the reproductive growth stage, inhibit excessive vegetative growth, and extend the fruiting cycle, thereby helping fruit farmers shorten the investment return cycle and improve the economic benefits of the industry.

[0003] Current evaluation systems for rootstock cold resistance and early flowering still face significant technical bottlenecks. In cold resistance evaluation, traditional methods mainly rely on artificial simulated low-temperature treatment or natural overwintering trials in the field, requiring observation of rootstock survival rates and frost damage over months or even years, resulting in long cycles and significant dependence on environmental conditions. Although the detection of physiological and biochemical indicators (such as conductivity and malondialdehyde content) can partially reflect cold resistance, the process is cumbersome, data repeatability is poor, and high-throughput screening is difficult. For example, although the invention patent CN111996276B proposes a technology for identifying the cold resistance of Hanfu apple hybrid progeny based on SNP molecular markers, its application is limited to Hanfu apple and its hybrid varieties, lacking universality.

[0004] In the field of early-flowering evaluation, existing technologies generally require long-term field observation of the flowering time and quantity of grafted rootstocks, combined with hormone regulation experiments or anatomical observations for comprehensive judgment. This not only consumes a lot of time and manpower but is also easily affected by cultivation management measures and climate fluctuations, resulting in insufficient stability of evaluation results. For example, the method for selecting early-flowering and early-fruiting apple rootstocks described in invention patent application publication number CN107568056A requires 4 years from hybridization breeding to regional trials; while the technical solution proposed in invention patent application publication number CN109526733A, although it involves selection through seedling grafting, tissue culture seedling cultivation, and rootstock-scion combination screening, still requires 9-10 years for the selection process. The above methods generally suffer from common problems such as lengthy evaluation cycles, high human and material costs, complex indicator systems, and low standardization, which seriously restrict the efficiency of selecting superior rootstock varieties and the process of industrialization and promotion.

[0005] Therefore, there is an urgent need to develop an efficient, accurate, and low-cost comprehensive evaluation technology for the cold resistance and early flowering of rootstocks, so as to overcome the limitations of the existing evaluation system and provide reliable technical support for the breeding of rootstocks for dwarf and dense apple cultivation. Summary of the Invention

[0006] The present invention adopts the following technical solution:

[0007] This invention provides a method for rapidly screening cold-resistant, early-flowering apple rootstocks from hybrid seedlings, comprising the following steps:

[0008] Step 1, scion collection: Collect scions from apple seedling mother plants;

[0009] Step 2, hydroponics: Place the collected scions in a container for hydroponic cultivation;

[0010] Step 3: Observe and record the budding status of the scions grown hydroponically;

[0011] Step 4: Select scions with cold resistance based on the germination status;

[0012] Step 5, grafting and planting: graft the grafted plant onto the selected cold-resistant scion, and plant the grafted scion in the nursery.

[0013] Step 6: During the 2nd to 3rd year after the grafted scion is planted, the plant height and number of flower buds of the grafted scion are counted.

[0014] Step 7: Based on the combined plant height and number of flower buds of the grafted scions, screen out apple rootstocks that have both cold resistance and early flowering characteristics.

[0015] Furthermore, the specific method for collecting scions in step 1 is as follows: the scions are collected one month before the apple seedling mother plant sprouts in February each year. The scions are selected from stem segments of more than 20 cm above ground from the apple seedling mother plant. The apple seedling mother plant meets the conditions of upright plants, robust development, and freedom from pests and diseases.

[0016] Furthermore, in step 2, hydroponics specifically involves dividing the collected scions into small portions and placing them in containers for hydroponic cultivation.

[0017] The environmental control conditions for hydroponic cultivation are as follows: day and night temperature of 15℃ / 10℃, ambient humidity of 40%-50%, light intensity of 1500-1800lx, daily light duration of 10 hours, water changed every 3 days, and each time the water is changed, a small portion of the base of the scion is cut off to expose the new stubble.

[0018] Further, in step 3, observing and recording the budding status of the hydroponically grown scion specifically involves investigating the budding status of the leaf buds of the scion daily after 7-8 days of hydroponic cultivation, continuously recording the number of sprouting buds, the number of opening buds, and the total number of buds, and calculating the budding rate according to the following formula (1):

[0019] Germination rate = A / B × 100%......(1),

[0020] Where A represents the number of budding buds plus the number of blooming buds, and B represents the total number of buds.

[0021] Furthermore, in step 4, the selection of cold-resistant scions based on the germination status specifically involves selecting scions with a germination time of less than 10 days and a germination rate of over 50% as cold-resistant scions.

[0022] Further, in step 5, the grafting and planting specifically involves using the single-bud side grafting method to graft the grafted plant onto the selected cold-resistant scion. The grafted scion is then transplanted to the nursery with a planting density of 15cm-20cm×40cm. After transplanting, the plant is directly planted. After watering, it is thoroughly watered every 2 to 3 days. After watering three times, it is transferred to conventional field management. The grafted plant is either Changhong or Changfu No. 2, which are difficult to flower.

[0023] Further, in step 7, the selection of apple rootstocks with both cold resistance and early flowering characteristics is based on the plant height and number of flower buds of the grafted scion in the third year, the number of flower buds in the second year, and the number of flower buds in the third year. The plant height and the number of flower buds are each divided into 5 levels and assigned 1-5 points respectively. The comprehensive score is calculated according to the following formula (2), and the scion with a comprehensive score ≥2.5 points is selected as a cold-resistant and early-flowering apple rootstock.

[0024] Overall score = ω1*0.2 + ω2*0.4 + ω3*0.4......(2),

[0025] Wherein, ω1 is the assigned score of the plant height of the grafted scion in the third year, ω2 is the assigned score of the number of flower buds of the grafted scion in the second year, and ω3 is the assigned score of the number of flower buds of the grafted scion in the third year.

[0026] Furthermore, the plant height range corresponding to the assigned score for the third year's plant height from high to low is 2.0-2.5 meters, 1.5-2.0 meters, 2.5-3.0 meters, below 1.5 meters, and above 3 meters.

[0027] Furthermore, the range of flower buds corresponding to the assigned score for the number of flower buds in the second year from high to low is: more than 15, 10-15, 5-10, 1-5, and 0.

[0028] Furthermore, the range of flower bud numbers corresponding to the assigned scores for the number of flower buds in the third year from high to low is: more than 40, 30-40, 20-30, 10-20, and less than 10.

[0029] Compared with the prior art, the superior effects of the present invention are as follows:

[0030] 1. The method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings described in this invention employs an innovative hydroponic germination test technique, which can complete the preliminary screening of cold resistance in just 7 to 10 days. Compared with traditional methods that rely on years of field overwintering observation, this invention significantly improves the screening efficiency by tens of times.

[0031] 2. The method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings described in this invention shortens the early-flowering evaluation cycle from 4-10 years in traditional methods to 2-3 years after grafting. Combined with a quantitative scoring model, it achieves accurate early judgment and significantly accelerates the breeding process.

[0032] 3. The method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings described in this invention deeply integrates the evaluation indicators of cold resistance (germination rate) and early flowering (number of flower buds / plant height). By using a standardized scoring model (comprehensive score ≥ 2.5 points), rootstocks with both characteristics are screened, breaking through the limitations of traditional single-trait screening and ensuring the comprehensive trait advantages of the rootstocks.

[0033] 4. The method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings described in this invention employs precise control of environmental parameters (day and night temperature and humidity, light intensity) during the hydroponic stage, combined with periodic pruning of the scion base, to ensure the repeatability and stability of the budding test. Hundreds of samples can be processed in a single batch, and the screening throughput far exceeds that of traditional methods.

[0034] 5. The method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings described in this invention uses Changhong / Changfu 2, which is difficult to flower, as the rootstock for grafting. By amplifying the differences in early-flowering characteristics of the scion through the flowering inhibition effect of the grafted plant, the phenotypic differentiation is significantly improved and the risk of misjudgment is reduced.

[0035] 6. The method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings described in this invention establishes a rapid and non-destructive physiological index detection method for cold resistance to replace complex biochemical detection, thereby reducing the technical threshold. At the same time, it constructs a multi-dimensional scoring system to classify and quantify plant height and flower bud number, and reflects the contribution of early flowering traits through weight allocation, thereby achieving data-driven scientific decision-making.

[0036] 7. The method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings described in this invention is not limited by season during the hydroponic stage and can be carried out throughout the year, avoiding the dependence of traditional field trials on climate conditions. Furthermore, after grafting and planting, dense planting management (15-20cm×40cm) is adopted, saving nursery land. Combined with an automated irrigation system, management costs are reduced, making it suitable for large-scale promotion and application.

[0037] 8. The method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings described in this invention can be adapted to the cold-resistant / early-flowering requirements of different ecological zones by adjusting hydroponic parameters (such as low-temperature gradient settings) and scoring thresholds. The technical framework can be extended to the screening of rootstocks for Rosaceae fruit trees such as pear and peach, and has broad application prospects.

[0038] 9. The method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings described in this invention quickly identifies cold-resistant germplasm through hydroponic germination rate, and achieves early prediction of early flowering by combining a plant height-flower bud dynamic scoring model after grafting. This forms a two-stage technical closed loop of "rapid initial screening → precise verification", which reduces the traditional breeding cycle by more than 60% and reduces screening costs by 70%, providing a disruptive solution for the targeted breeding of dwarfing apple rootstocks. Attached Figure Description

[0039] Figure 1 This is a flowchart illustrating the method for rapidly screening cold-resistant, early-flowering apple rootstocks from hybrid seedlings according to the present invention.

[0040] Figure 2 This is a schematic diagram of the leaf buds in step 3 of the method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings according to the present invention.

[0041] Figure 3 This is a schematic diagram of flower buds in step 6 of the method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings described in this invention.

[0042] Figure 4 This is a schematic diagram illustrating the principle of measuring plant height in step 6 of the method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings described in this invention.

[0043] Figure 5 This is a schematic diagram of the budding status of leaf buds after hydroponics in the method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings as described in this invention.

[0044] Figure 6 This is a schematic diagram illustrating the survey of flower buds in the scion after transplanting, as described in the method for rapidly screening cold-resistant and early-flowering apple rootstocks from hybrid seedlings in this invention. Detailed Implementation

[0045] To better understand the above-mentioned objectives, features and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0046] like Figures 1 to 6 As shown, this invention provides a method for rapidly screening cold-resistant, early-flowering apple rootstocks from hybrid seedlings.

[0047] like Figure 1 As shown, the method for rapidly screening cold-resistant, early-flowering apple rootstocks from hybrid seedlings includes the following steps:

[0048] Step 1, Scion Collection: Scions are collected one month before the apple seedling mother plants sprout in February each year. The scions are selected from stem segments of more than 20 cm above ground from the apple seedling mother plants. After collection, the scions are soaked in an 800-fold carbendazim solution for 10 minutes and rinsed three times with clean water to reduce the risk of microbial contamination. Select one-year-old lignified stem segments with a diameter of 3-5 mm and plump buds. When cutting, retain a length of 40-50 cm and retain 15-20 buds. The cut is tilted at 45° to increase the water absorption area. The apple seedling mother plants should meet the conditions of upright plants, vigorous development and no diseases or pests.

[0049] Step 2, hydroponics: Divide the collected scions into small portions and place them in containers for hydroponic cultivation. The environmental control conditions for hydroponic cultivation are as follows: the water quality is deionized water or distilled water, the day and night temperature is 15℃ / 10℃, the ambient humidity is 40%-50%, the light intensity is 1500-1800lx, and the light is simulated by fluorescent lamps with a photoperiod of 10 hours of light / 14 hours of darkness to simulate the light environment of spring. Change the water every 3 days. Each time the water is changed, cut off 0.5cm from the base of the scion to expose the new stubble. Use garden shears to cut at an angle to expand the water absorption surface.

[0050] Step 3, as follows Figure 5 As shown, the budding status of the leaf buds on the scions was investigated daily starting 7-8 days after the scions were hydroponically cultured, and recorded for a total of 20 days. The apple leaf buds were as follows... Figure 2 As shown, the number of sprouting buds, the number of opening buds, and the total number of buds were continuously recorded, and the germination rate was calculated according to the following formula (1):

[0051] Germination rate = (number of budding buds + number of blooming buds) / total number of buds × 100%......(1);

[0052] Step 4: Selecting cold-resistant scions based on budding status and cold resistance grading standards. Specifically, scions with a budding time of less than 10 days and a budding rate of more than 50% are selected as cold-resistant scions and included in the evaluation of early-flowering apple resources. The budding rate refers to the budding rate on the 10th day after the daily survey of the budding status of the leaf buds of the scion begins, and the budding time refers to the time from the start of the daily survey of the budding status of the leaf buds of the scion until the leaf buds begin to bud.

[0053] Studies have shown that during the dormant period, cold-resistant varieties convert starch in their branches into soluble sugars such as glucose and sucrose to lower the freezing point of cell sap and improve overwintering ability. After the low-temperature period ends, these substances can provide energy for bud break and promote early metabolism. Therefore, the bud break rate of different cold-resistant scions may be positively correlated with their cold resistance after meeting certain chilling requirements. There is also evidence that some cold-resistance genes in plants synergistically regulate the expression of bud break-related genes, such as ICE1 and MYB15.

[0054] Step 5, grafting and planting: use the single-bud side grafting method to graft the grafted plant onto the selected cold-resistant scion. The grafted plant varieties are Changhong or Changfu No. 2, which are difficult to flower. Transplant the grafted scion to the nursery with a planting density of 15cm-20cm×40cm. After transplanting, plant directly. After watering, water thoroughly every 2 to 3 days. After watering 3 times, transfer to regular field management.

[0055] Step 6, as follows Figure 6 As shown, during the 2nd-3rd year after grafting, the plant height in the 3rd year, the number of flower buds in the 2nd year, and the number of flower buds in the 3rd year were recorded. Apple flower buds are shown in the figure. Figure 3 As shown, the principle of plant height measurement is as follows: Figure 4 As shown;

[0056] Step 7: Based on the scion's height in the third year, the number of flower buds in the second year, and the number of flower buds in the third year, grade and assign values ​​to each. Plant height and the number of flower buds are each divided into 5 grades, assigned values ​​from 1 to 5 points respectively. The plant height in the third year, from highest to lowest score, corresponds to the following ranges: 2.0-2.5 meters, 1.5-2.0 meters, 2.5-3.0 meters, below 1.5 meters, and above 3 meters. The number of flower buds in the second year, from highest to lowest score, corresponds to the following ranges: more than 15, 10-15, 5-10, 1-5, and 0. The number of flower buds in the third year, from highest to lowest score, corresponds to the following ranges: more than 40, 30-40, 20-30, 10-20, and less than 10.

[0057] Calculate the comprehensive score according to the following formula (2), and select grafted scions with a comprehensive score ≥ 2.5 as cold-resistant and early-flowering apple rootstocks;

[0058] Overall score = ω1*0.2 + ω2*0.4 + ω3*0.4......(2),

[0059] Wherein, ω1 is the assigned score of the plant height of the grafted scion in the third year, ω2 is the assigned score of the number of flower buds of the grafted scion in the second year, and ω3 is the assigned score of the number of flower buds of the grafted scion in the third year.

[0060] It should be noted that the goal of this invention is to select apple rootstocks with cold resistance and early flowering and fruiting characteristics suitable for apple dwarfing and high-density planting techniques. In step 7, the core selection criteria are easy shaping (quickly reaching the target tree height of about 3.0 meters) and easy flowering (promoting early flowering and fruiting of apple trees). The weight of each indicator will be different. In the experiment, it was found that the difference in tree height in the second year was relatively small, and it was not easy to classify and assign values. Therefore, the tree height in the second year was not considered.

[0061] Traditional methods for assessing cold resistance require simulating low-temperature environments and evaluating electrical conductivity. First, this method requires a large amount of material, at least 21 one-year-old branches with consistent growth. Second, it necessitates low-temperature treatment equipment, such as a high-low temperature alternating chamber, which results in high costs. Finally, measuring electrical conductivity is time-consuming and labor-intensive, and the accuracy of the results is often unsatisfactory. In contrast, the method proposed in this invention does not require a large number of branches or special equipment; the screening process can be completed simply through hydroponic observation.

[0062] This invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims.

Claims

1. A method for rapidly screening cold-resistant, early-flowering apple rootstocks from hybrid seedlings, characterized in that, Includes the following steps: Step 1, Scion Collection: Collect scions from apple seedling mother plants. Scions are collected one month before the apple seedling mother plants sprout in February each year. The scions are selected from stem segments of more than 20 cm above ground from the apple seedling mother plants. After collection, the scions are soaked in an 800-fold carbendazim solution for 10 minutes and rinsed three times with clean water to reduce the risk of microbial contamination. One-year-old lignified stem segments with a diameter of 3-5 mm and full buds are selected. When cutting, the length is retained to 40-50 cm and 15-20 buds are retained. The cut is tilted at 45° to increase the water absorption area. The apple seedling mother plants meet the conditions of upright plants, vigorous development and no diseases and pests. Step 2, hydroponics: Place the collected scions in a container for hydroponic cultivation. The environmental control conditions for hydroponic cultivation are as follows: day and night temperature of 15℃ / 10℃, ambient humidity of 40%-50%, light intensity of 1500-1800lx, daily light duration of 10 hours, water change every 3 days, and each time the water is changed, a small part of the base of the scion is cut off to expose the new stubble. Step 3: Observe and record the budding status of the scions grown hydroponically: After 7-8 days of hydroponic cultivation of the scions, the budding status of the leaf buds on the scions was investigated daily. The number of budding buds, the number of opening buds, and the total number of buds were continuously recorded, and the budding rate was calculated according to the following formula (1): Germination rate = A / B × 100% (1), Where A is the number of sprouting buds + the number of blooming buds, and B is the total number of buds; Step 4: Select scions with cold resistance based on germination status; Scions with a germination time of less than 10 days and a germination rate of over 50% are selected as cold-resistant scions; Step 5, grafting and planting: graft the grafted plant onto the selected cold-resistant scion, and plant the grafted scion in the nursery. Step 6: During the 2nd to 3rd year after the grafted scions are planted, count the plant height and number of flower buds of the grafted scions. Step 7: Based on the combined plant height and number of flower buds of the grafted scion, screen out apple rootstocks that have both cold resistance and early flowering characteristics. The scion is graded and assigned a value according to the plant height in the third year, the number of flower buds in the second year, and the number of flower buds in the third year after grafting. The plant height and the number of flower buds are each divided into 5 grades and assigned a value of 1-5 points respectively. The comprehensive score is calculated according to the following formula (2). The scion with a comprehensive score ≥2.5 points is selected as a cold-resistant and early-flowering apple rootstock. Overall score = ω1×0.2+ω2×0.4+ω3×0.4 (2), Wherein, ω1 is the assigned score of the plant height of the grafted scion in the third year, ω2 is the assigned score of the number of flower buds of the grafted scion in the second year, and ω3 is the assigned score of the number of flower buds of the grafted scion in the third year.

2. The method for rapidly screening cold-resistant, early-flowering apple rootstocks from hybrid seedlings according to claim 1, characterized in that, In step 5, grafting and planting specifically involves using the single-bud side grafting method to graft the grafted plant onto the selected cold-resistant scion. The grafted scion is then transplanted to the nursery at a planting density of 15-20cm × 40cm. After transplanting, the plant is directly planted. After watering, the plant is thoroughly watered every 2 to 3 days. After watering three times, the plant is transferred to regular field management. The grafted plant is either Changhong or Changfu No. 2, which are difficult to flower.

3. The method for rapidly screening cold-resistant, early-flowering apple rootstocks from hybrid seedlings according to claim 1, characterized in that, The plant height scores assigned in the third year, from highest to lowest, correspond to the following plant height ranges: 2.0-2.5 meters, 1.5-2.0 meters, 2.5-3.0 meters, below 1.5 meters, and above 3 meters.

4. The method for rapidly screening cold-resistant, early-flowering apple rootstocks from hybrid seedlings according to claim 1, characterized in that, The numerical values ​​for the number of flower buds in the second year, from highest to lowest, correspond to the following ranges: more than 15, 10-15, 5-10, 1-5, and 0.

5. The method for rapidly screening cold-resistant, early-flowering apple rootstocks from hybrid seedlings according to claim 1, characterized in that, The numerical values ​​for the number of flower buds in the third year, from highest to lowest, correspond to the following ranges: more than 40, 30-40, 20-30, 10-20, and less than 10.

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