Apple rootstock space mutation breeding method

Abstract

The invention discloses an apple rootstock space mutation breeding method which comprises the following steps: selecting full apple rootstock seeds, drying in the shade, and storing at 4 DEG C; the seeds are loaded on a return spacecraft and are in orbit for 7-180 days; after returning, grading and laminating according to hundred-grain weight: laminating less than or equal to 1.387 g for 46 days; 1.388 g to 2.746 g of the product is laminated for 53 d to 56 d; more than or equal to 2.747 g lamination is carried out for 56 days; the temperature is 2-4 DEG C, and the humidity is 60%; sowing single seeds in a nutrition pot after 50% of white buds appear, establishing a single-plant electronic file after seedling emergence, and recording seedling height, seedling thickness, leaf area, leaf form and images; in the last ten days of July, pruning branch sections with 3-4 buds, leaving one leaf at the top end, soaking in rooting liquid for 20-30 minutes, and cutting in vermiculite bowls with the humidity of more than or equal to 90%, the arched shed height of 2 m and the temperature of less than or equal to 30 DEG C; according to the apple rootstock space mutation breeding method, the breeding period is shortened, the mutant screening efficiency is improved, and the cutting rooting rate is increased.

Description

Technical Field

[0001] This invention relates to the field of fruit tree breeding, and in particular to a method for space-induced mutation breeding of apple rootstock, which is based on space-induced mutation, and includes a supporting hierarchical stratification, rapid screening of electronic archival phenotypes, rapid propagation by softwood cuttings / tissue culture, a special leaf area measuring ruler, and recursive mutation. Background Technology

[0002] Fruit trees, as perennial plants, have long juvenile periods and highly heterogeneous genes. Conventional breeding methods are greatly limited by factors such as low mutation probability and long selection time, resulting in significant constraints on the selection of new varieties. Since the 1960s, my country has conducted extensive work on fruit tree mutation breeding, including chemical mutagenesis and radiation-induced mutagenesis. Breeding objectives have mainly focused on dwarfing mutations, shortening the juvenile period, altering the maturity period, improving quality, and enhancing disease resistance. In addition, mutation breeding has played a crucial role in addressing specific breeding objectives, such as seedless breeding of fruit trees. Currently, morphological identification is the most intuitive, feasible, and convincing method in mutation breeding. This experiment investigated, compared, and analyzed the field botanical characteristics of *Malus spectabilis* variegated rootstock in a space-induced mutation breeding nursery for apple, screened individual plants with mutant characteristics, provided materials for the identification of beneficial mutations, and summarized field screening techniques for apple space-induced mutations.

[0003] Conventional breeding of apple rootstocks has a long cycle (10–15 years), low mutation probability, and difficulty in breaking the linkage of undesirable traits. Space mutagenesis can significantly increase the mutation frequency, but it faces three major bottlenecks in perennial fruit trees: 1. Early screening of mutants is inefficient, and traditional leaf area measurement methods are prone to large errors and are time-consuming; 2. Asexual propagation is slow, tissue culture is costly, and the rooting rate of softwood cuttings is low; 3. Lack of a systematic closed-loop technology system of "carrying-layering-screening-propagation-verification-recursive mutation". Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for space-induced mutation breeding of apple rootstock, which facilitates a shorter breeding cycle, improves the efficiency of mutant screening, and increases the rooting rate of cuttings.

[0005] The present invention achieves its objective by employing the following technical solution: A method for space-induced mutation breeding of apple rootstocks, including (1) Select plump apple rootstock seeds, dry them in the shade, and store them at 4℃; (2) The seeds were carried on a recoverable spacecraft and remained in orbit for 7–180 days; (3) After returning, stratify and stack according to 100-grain weight: ≤1.387 g, layering time 46 days; 1.388–2.746 g, layering time 53–56 days; ≥2.747 g, layering time 56 days; temperature 2–4 ℃, humidity 60%; (4) Sow 50% of the seedlings in nutrient pots after they have sprouted. After emergence, establish an electronic file for each seedling to record seedling height, seedling diameter, leaf area, leaf morphology and images. (5) In late July, cut off branches with 3-4 buds, leaving 1 leaf at the top, soak them in rooting solution for 20-30 minutes, and insert them into vermiculite pots with humidity ≥90%, greenhouse height 2 m, and temperature ≤30 ℃; (6) Phenotypic mutants were screened based on electronic records. The screening indicators were leaf area increase rate ≥15%, plant height increase rate ≥20%, and stem diameter increase rate ≥10%. (7) Rapid propagation of mutants by tissue culture: using tender shoots as explants, callus was induced by MS + 6-BA 2 mg / L + NAA 0.2 mg / L, buds were induced by subculture medium MS + 6-BA 1 mg / L + IBA 0.5 mg / L, and rooting medium 1 / 2 MS + IBA 1 mg / L. Complete plants were obtained after 30 days. (8) Establish a field breeding nursery: 3 m × 1 m of mutation mother plants and 1 m × 0.5 m of cutting test nursery; (9) In autumn, using the control plant as a benchmark, the drought resistance, cold resistance, salt and alkali resistance and dwarfing ability of the mutant plant were tested; (10) The seeds of superior mother plants were carried a second time and recursive mutagenesis screening was carried out; (11) After passing the grafting compatibility test and 2–3 years of multi-location regional trials, apply for variety approval.

[0006] As a further limitation of this technical solution, the ratio of the number of seeds carried in space to the number of control seeds on the ground is 3:1 or 4:1.

[0007] As a further limitation of this technical solution, the stratification medium is fine river sand with a water content of 60%; when it is necessary to delay the emergence of white to unify the sowing period, the humidity is reduced to 10% in stages and then restored to 60%.

[0008] As a further limitation of this technical solution, the sowing substrate is unsterilized vermiculite, which increases the germination rate by 10–25% compared to sterilized substrate.

[0009] As a further limitation of this technical solution, the electronic record system uses a mobile terminal APP, which connects to the leaf area measuring ruler via Bluetooth to upload seedling trait data and images to the cloud database in real time.

[0010] As a further limitation of this technical solution, a) a transparent grid plate, engraved with 1 cm × 1 cm thick-lined squares and 0.5 cm × 0.5 cm thin-lined squares; b) Set graduations with an accuracy of 0.1 cm on the horizontal and vertical center lines; c) Measurement formula: S=(Sa×1 cm) 2 )+(Sb×0.25 cm 2 Sa represents 1 cm of 100% coverage. 2 The number of squares, Sb is ≥50% coverage of 0.25 cm. 2 Number of squares; d) Compared with the YMJ-B handheld leaf area meter, the correlation coefficient is ≥0.983.

[0011] As a further limitation of this technical solution, the material is 2 mm transparent acrylic, which can be rolled up for storage, and the measurement range is 0–200 cm. 2 .

[0012] As a further limitation of this technical solution, the softwood cuttings are taken from secondary branches that sprout after the current year's branches are cut in late August, which increases the rooting rate by 2.4–4.6 times compared to primary branches.

[0013] As a further limitation of this technical solution, adding 0.1% activated carbon to the rooting medium for rapid tissue culture propagation increases the rooting rate to over 95%.

[0014] As a further limitation of this technical solution, drought resistance was tested using a potted water control method, and the survival rate was determined when the soil moisture content dropped to 40% of field capacity; cold resistance was tested using an artificial climate chamber at −25 ℃ for 6 h and then the electrolyte leakage rate was determined; salt and alkali resistance was tested using 200 mmol / L NaCl solution for 30 days and then the plant height inhibition rate was determined; dwarfing was tested using the plant height / stem diameter ratio as an indicator, with a smaller ratio indicating stronger dwarfing.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are: The breeding cycle has been significantly shortened through a closed-loop system of "space-borne, hierarchical stratification, rapid screening, efficient propagation, and recursive mutagenesis," reducing the traditional 10-15 year breeding cycle to 5-8 years and saving more than 40% of the time cost.

[0016] The efficiency of mutant screening is improved by 5–10 times. A dedicated leaf area measuring ruler (correlation coefficient ≥0.983, single plant measurement ≤30 s) is linked with the mobile terminal APP and cloud electronic archives to achieve high-throughput, digital phenotypic screening in the seedling stage, significantly reducing the rate of missed and incorrect selection.

[0017] The combination of space-induced mutagenesis and 2–3 recursive mutagenesis can enhance both mutation frequency and mutation spectrum, thereby significantly broadening the mutation spectrum, breaking the linkage of undesirable traits, and increasing the probability of obtaining rootstocks with excellent comprehensive traits.

[0018] Through systematic evaluation, the drought survival rate of the selected variety "Hangzhen No. 1" was increased by 45% and the plant height was reduced by 20%; the plant height inhibition rate of "Hangzhen No. 2" was only 18% under 200 mmol / L NaCl, and the dwarfing coefficient was reduced to 85, achieving a three-in-one rootstock improvement of stress resistance, dwarfing and compatibility.

[0019] With high regional adaptability and industrial promotion value, it has been tested simultaneously in three major ecological zones: the Loess Plateau, the Bohai Bay, and the cold highlands of Southwest China. It has shown stable performance in all three zones, with a grafting survival rate of ≥85% and an average yield increase of 8-18%. It can be directly applied to the renovation of old orchards and the construction of new orchards in my country's main production areas.

[0020] The integration of low-cost materials and simple facilities, such as non-sterile vermiculite sowing substrate, rollable transparent acrylic measuring ruler, and disposable plastic cup tissue culture containers, allows grassroots research units and seedling enterprises to quickly replicate this technology system without expensive equipment.

[0021] In summary, this invention, with space-induced mutation as its core and supported by key technologies such as hierarchical stratification, digital screening, efficient propagation, and recursive mutagenesis, solves long-standing problems such as long breeding cycles for apple rootstocks, difficulty in early screening of mutants, and low efficiency of asexual propagation. It provides the apple industry with new rootstock varieties with short cycles, superior traits, and wide adaptability, along with their supporting technology system, resulting in significant economic, social, and ecological benefits. Attached Figure Description

[0022] Figure 1 Table showing the differences in botanical characteristics between mutant and control plants of Malus spectabilis.

[0023] Figure 2 A statistical table of leaf morphological characteristics of mutant and control plants of Malus spectabilis.

[0024] Figure 3 Statistical analysis table of botanical characteristics of mutant and control plants of Malus spectabilis.

[0025] Figure 4 A statistical table comparing the leaf cleavage degree of different species in induced mutations and control groups of Begonia variegata.

[0026] Figure 5 The morphology of the control plant is shown for the variegated crabapple.

[0027] Figure 6The plant morphology of a mutant Begonia variegata.

[0028] Figure 7 This is a statistical table of the botanical characteristics of field mutant strains.

[0029] Figure 8 Statistical table comparing leaf morphology of mutants screened in the field for variegated crabapple.

[0030] Figure 9 This includes the seed variety, collection time, and storage conditions.

[0031] Figure 10 A statistical table of seed stratification time for different apple rootstocks.

[0032] Figure 11 Statistical table of seed stratification time and conditions for different apple rootstocks and varieties subjected to space-induced mutation.

[0033] Figure 12 The effect of different treatments on the germination rate of the same rootstock in the same substrate.

[0034] Figure 13 The structural principle and actual measurement diagram of a self-made leaf area measuring ruler.

[0035] Figure 14 The structural principle and actual measurement diagram of a custom-made leaf area measuring ruler.

[0036] Figure 15 A map for quickly measuring the 'base area'.

[0037] Figure 16 The graph shows the relationship between the measured values ​​of the leaf area measuring instrument and the measured values ​​of the YMJ-B leaf area meter. Detailed Implementation

[0038] The following detailed description of a specific embodiment of the present invention is provided in conjunction with the accompanying drawings. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiment.

[0039] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0040] The terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to mechanical connections or electrical connections; they can refer to direct connections or indirect connections through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art will understand the specific meaning of these terms in this invention based on the specific circumstances.

[0041] The following description of exemplary embodiments refers to the accompanying drawings. The same reference numerals in different figures denote the same or similar elements. The following detailed description does not limit the invention. Rather, the scope of the invention is defined by the appended claims. For simplicity, the following embodiments describe the terminology and structure of the system; however, the embodiments described below are not limited to this system but can be applied to any other applicable system.

[0042] like Figures 1-16 As shown, the present invention includes (1) selecting plump apple rootstock seeds, drying them in the shade, and storing them at 4 ℃; (2) The seeds were carried on a recoverable spacecraft and remained in orbit for 7–180 days; (3) After returning, stratify and stack according to 100-grain weight: ≤1.387 g, layering time 46 days; 1.388–2.746 g, layering time 53–56 days; ≥2.747 g, layering time 56 days; temperature 2–4 ℃, humidity 60%; (4) Sow 50% of the seedlings in nutrient pots after they have sprouted. After emergence, establish an electronic file for each seedling to record seedling height, seedling diameter, leaf area, leaf morphology and images. (5) In late July, cut off branches with 3-4 buds, leaving 1 leaf at the top, soak them in rooting solution for 20-30 minutes, and insert them into vermiculite pots with humidity ≥90%, greenhouse height 2 m, and temperature ≤30 ℃; (6) Phenotypic mutants were screened based on electronic records. The screening indicators were leaf area increase rate ≥15%, plant height increase rate ≥20%, and stem diameter increase rate ≥10%. (7) Rapid propagation of mutants by tissue culture: using tender shoots as explants, callus was induced by MS + 6-BA 2 mg / L + NAA 0.2 mg / L, buds were induced by subculture medium MS + 6-BA 1 mg / L + IBA 0.5 mg / L, and rooting medium 1 / 2 MS + IBA 1 mg / L. Complete plants were obtained after 30 days. (8) Establish a field breeding nursery: 3 m × 1 m of mutation mother plants and 1 m × 0.5 m of cutting test nursery; (9) In autumn, using the control plant as a baseline, the drought resistance, cold resistance, salt and alkali resistance and dwarfing ability of the mutant plant were tested; (10) The seeds of superior mother plants were carried a second time and recursive mutagenesis screening was carried out; (11) After passing the grafting compatibility test and 2–3 years of multi-location regional trials, apply for variety approval.

[0043] The ratio of the number of space-borne seeds to ground-based control seeds is 3:1 or 4:1.

[0044] The stratification medium is fine river sand with a water content of 60%; when it is necessary to delay the emergence of white to unify the sowing period, the humidity is reduced to 10% in stages and then restored to 60%.

[0045] The sowing substrate is unsterilized vermiculite, which increases the germination rate by 10–25% compared to sterilized substrate.

[0046] The electronic record system uses a mobile terminal APP, which connects to the leaf area measuring ruler via Bluetooth to upload seedling trait data and images to the cloud database in real time.

[0047] In practice, the softwood cuttings are made from secondary branches that sprout after the current year's branches are cut in late August, which increases the rooting rate by 2.4–4.6 times compared to primary branches.

[0048] The tissue culture rapid propagation method mentioned above, by adding 0.1% activated carbon to the rooting medium, increases the rooting rate to over 95%.

[0049] The drought resistance test used a potted water control method, and the survival rate was measured when the soil moisture content dropped to 40% of the field capacity. The cold resistance test used an artificial climate chamber to freeze at -25 ℃ for 6 h and then measured the electrolyte leakage rate. The salt and alkali resistance test used 200 mmol / L NaCl solution for irrigation for 30 days and then measured the plant height inhibition rate. The dwarfing test used the plant height / stem diameter ratio as an indicator, and the smaller the ratio, the stronger the dwarfing effect.

[0050] The scions used in the grafting compatibility test are any one of Fuji, Gala, or Jonagold, and the grafting survival rate is ≥85%.

[0051] This application also provides a recursive mutagenesis system in which seeds from single plants with excellent comprehensive traits in the previous breeding nursery are carried again, with the cycle repeated 2–3 times to superimpose the mutation effect.

[0052] The regional trials were conducted simultaneously in three ecological zones: the Loess Plateau, the Bohai Bay, and the Southwest Cold Highlands, with three replicate sites in each zone and ≥50 plants per site.

[0053] This application also provides an apple rootstock variety, "Hangzhen No. 1," which was bred from Malus spectabilis through in-orbit mutagenesis for 7 days in 2020 and selected using the method described in this application. Its characteristics are: a) Leaf area increased by 116% compared to the original species; b) Plant height reduced by 20%; c) Survival rate increased by 45% under drought stress; d) Grafting success rate with Fuji apples: 92%.

[0054] This application also provides a dedicated leaf area measuring ruler, including a) A transparent grid board, engraved with 1 cm × 1 cm thick-lined squares and 0.5 cm × 0.5 cm thin-lined squares; b) Set graduations with an accuracy of 0.1 cm on the horizontal and vertical center lines; c) Measurement formula: S=(Sa×1 cm) 2 )+(Sb×0.25 cm 2 Sa represents 1 cm of 100% coverage. 2 The number of squares, Sb is ≥50% coverage of 0.25 cm. 2 Number of squares; d) Compared with the YMJ-B handheld leaf area meter, the correlation coefficient is ≥0.983.

[0055] It is made of 2 mm transparent acrylic, can be rolled up for storage, and has a measuring range of 0–200 cm. 2 .

[0056] This application also provides an apple rootstock variety, "Hangzhen No. 2," which was bred from Malus baccata through a 20-day simulated mutagenesis induced by a high-altitude balloon and selected using the method described in claims 1-13, characterized in that: a) Under 200 mmol / L NaCl, the plant height inhibition rate was only 18%; b) After two recursive mutagenesis, the dwarfing coefficient (plant height / stem diameter) decreased to 85.

[0057] In practice, Table 1 shows the differences in botanical characteristics between the mutant and control plants of *Malus variegata*. The average leaf length and width of the mutant and control plants did not show significant differences (p>0.05), meaning that the average leaf length and width were consistent and showed no significant differences. However, the average leaf area of ​​both mutant and control plants showed significant differences (p<0.05). Specifically, the mutant plants showed a 0.01a significance for average leaf area (t=-8.666, p=0.000). Furthermore, the average leaf area of ​​the control plants (12.11) was significantly lower than that of the mutant plants (17.83). This means that there were significant differences in average leaf area between the mutant and control plants of *Malus spectabilis*. The plant length (cm) of both mutant and control plants was significantly different (p<0.05), with the mutant plants showing a 0.05a significance level for plant length (cm) (t=-2.530, p=0.013). Furthermore, the average value of the control plants (67.62) was significantly lower than the average value of the mutant plants (76.00). All treatments showed significant differences in branch diameter (cm) (p<0.05), indicating that there were differences in plant diameter (cm) between the mutant and control plants of *Malus spectabilis*. The mutant strains showed a 0.01a significance for plant diameter (cm) (t=-3.380, p=0.001). Further comparison revealed that the average value of the control strains (0.40) was significantly lower than the average value of the mutant strains (0.46). In summary, all treatments showed significant differences in plant diameter (cm). However, no significant differences were observed in average internode length (cm) among the different treatments (p>0.05), indicating that all treatments showed consistency in average internode length (cm) with no significant differences.

[0058] Table 2 shows the morphological characteristics of leaves in mutant and control plants of *Malus variegata*. In the control samples, 99% of the leaves were horizontal and 1% were drooping; 100% of mature leaves were dark green; 100% of the leaf margins had sharp serrations; 99% were flat and 1% were rolled back; 44% were completely split and 56% were partially split, with 3% overlapping. In the mutant samples, 1.47% of the leaves were upright; 0.59% of the mature leaves were yellowish-green and 0.29% were light green; 1.47% were involute; and 2.65% were slightly split.

[0059] Table 3, the statistical analysis of botanical characteristics of the mutant and control plants of *Malus spectabilis*, and Table 4, the comparative statistics of leaf lobes of the mutant and control plants of *Malus spectabilis*, show that the leaf length of the mutant and control plants is 3.0–7.17 cm, the leaf width is 2.63–6.47 cm, the leaf area is 5.2–26.83 cm², the plant height is 7.3–115 cm, the plant diameter is 0.1–0.63 cm, and the internode length is 0.8–3.5 cm. The leaves of the control plants and most treatments are horizontal, dark green, with sharp serrations on the leaf margins, and are smooth. The lobes are 3 deeply lobed, 3 half-lobed, 5 deeply lobed, and 5 half-lobed. The leaves are ovate to broadly ovate, with oblong-ovate to oblong lobes, and the petioles have narrow wings and are ovate-lanceolate. Figure 5 The morphological diagram of the control plants for the variegated begonia shows that the control plants are thin and weak with little taper, and are very easy to fall over when sprayed with water.

[0060] Table 5, a statistical table of the botanical characteristics of the field mutants, shows that the average leaf length, average leaf area, and average plant diameter of mutants 015, 131, 197, 225, 239, 282, and 285 were all greater than the average values ​​of the control. The leaf area of ​​mutant 225 (26.83 cm²) was more than twice that of the control (12.41 cm²). Table 6 shows that the leaf lobes of the seven mutants were significantly smaller than those of the control. The leaves were ovate to ovate-orbicular, with an acute apex and a rounded to broadly cuneate base. The petioles were winged, ovate-lanceolate, and had an acute apex. The leaves of mutant 197 showed a drooping, dorsally rolled morphology, while those of mutant 015 were upright. Figure 6 The morphological images of the mutant Begonia variegatedii plants show that the mutant plants are upright and sturdy, with significantly increased leaf area and plant diameter, and strong resistance to lodging. Based on comprehensive analysis, plants 015, 131, 197, 225, 239, 282, and 285 were identified as mutant plants and will undergo further observation, identification, and screening for beneficial mutagenesis.

[0061] 1. Analysis and comparison of the botanical characteristics between space-induced mutant and control plants of *Malus variegata* revealed that the mutant and control plants showed consistency in average leaf length, leaf width, and average internode length, with no significant differences; however, the average leaf area, plant length, and plant diameter showed significant differences of 0.01 a, 0.05 a, and 0.01 a, respectively. The plant height, plant diameter, and leaf area of ​​space-induced *Malus variegata* seedlings can be used as screening indicators for field mutants in comparative analysis.

[0062] 2. In the field, *Malus spectabilis* exhibits a slender, weak growth habit with a small taper. The plant height ranges from 7.3 to 115 cm, the diameter from 0.1 to 0.63 cm, and the internode length from 0.8 to 3.5 cm. The leaves are divided into 3 deeply lobed, 3 half-lobed, 5 deeply lobed, or 5 half-lobed lobes. The leaves are ovate to broadly ovate, 2.97 to 7.17 cm long, 2.63 to 6.47 cm wide, and have an area of ​​5.2 to 26.83 cm². The base is rounded to broadly cuneate, with irregularly serrated margins. The lobes are oblong-ovate to oblong-elliptic. The petioles have narrow wings, are ovate-lanceolate, and have an acute apex. The plant exhibits poor resistance to lodging.

[0063] 3. In the field control samples of *Malus spectabilis*, the overall leaf orientation was horizontal, mature leaves were dark green, leaf margins had sharp serrations, and the leaves were flat and evenly divided into fully and partially lobed sections. In the mutagenic samples, the leaves were mostly horizontal or drooping; mature leaves were dark green; leaf margins had sharp serrations; the leaves were either flat or rolled back; and the leaves were partially and fully lobed, with some plants showing micro-lobes. Leaf characteristics are of practical and effective value as a screening indicator for identifying mutant plants in the field through space-induced mutagenesis of apples.

[0064] 4. The seven mutant plants selected from the field (Nos. 015, 131, 197, 225, 239, 282, and 285) had average leaf length, leaf area, and plant diameter greater than the control average. They were also upright, robust, and had small leaf lobes, and their lodging resistance was enhanced. They can be used as beneficial mutagenesis materials for further identification and screening.

[0065] As another specific embodiment of this application: This study investigated the stratification and seedling emergence of apple seeds induced by space mutation. The experimental materials included four apple rootstock seeds and two apple varieties induced by space mutation in my country in 2020 and 2021, as well as ground-based control seeds (see Table 7 for seed varieties, collection time, and storage conditions). Seeds were stratified in sand in batches, and germination and emergence rates were tested. The seeds induced by space mutation in 2020 were *Malus hualaiensis*, *Malus variegata*, and *Malus baccata*; the seeds induced by space mutation in 2021 were *Tianwang No. 1*, *E'ai No. 2*, and *Pingyi Sweet Tea*.

[0066] 1.2.1 Stratification time required for bud break under different rootstocks and varieties Soak the seeds carried on the ground and the control seeds reserved on the ground in clean water for 24 hours. Then mix them with clean fine river sand at a ratio of 1:5. The sand should be moist enough to clump together when squeezed but crumble when released. Place the mixture in a glass bottle with a plastic cap that has ventilation holes and put it in a refrigerator at 2°C for stratification. Keep the sand moist throughout the stratification process. When 20% of the seeds show signs of sprouting, calculate the number of stratification days and then sow and raise seedlings.

[0067] On November 26, 2020, the seeds of Malus spectabilis, Malus hualaiensis, Malus spectabilis and Malus baccata carried in 2020, as well as the ground-reserved control seeds, were subjected to low-temperature stratification in a refrigerator. On December 27, 2022, the seeds of Tianwang No. 1, E'ai No. 2 and Pingyi Sweet Tea carried in 2021 were subjected to low-temperature stratification in a refrigerator.

[0068] 1.2.2 Effects of different treatments on seed germination rate using the same substrate Vermiculite was selected as the sowing substrate for stratification of seeds, and four treatments were set up. Treatment 1: Vermiculite autoclaving + rooting powder; Treatment 2: Vermiculite autoclaving; Treatment 3: Vermiculite without sterilization + rooting powder; Treatment 4: Vermiculite without sterilization.

[0069] 1.3 Data Statistical Analysis Seed germination (emergence) rate = number of germinated (emerged) seeds / number of tested seeds × 100%. Data were statistically analyzed using Excel.

[0070] 2 Results and Analysis 2.1 Effects of different apple rootstocks and varieties on stratification time Table 8 shows the statistical results of stratification time for seeds of different apple rootstocks. The results indicate significant differences in the stratification time required for seeds to develop white kernels. On November 26, 2020, the stratification time for *Malus spectabilis* was the shortest, only 35 days; *Malus macrocarpa* had the longest, at 60 days; and *Malus hualaiensis* had 53 days. The stratification results showed that smaller seeds required shorter stratification times. The stratification time for *Malus spectabilis*, *Malus hualaiensis*, and *Malus macrocarpa* increased from lowest to longest based on seed weight.

[0071] Table 9. Statistical Table of Stratification Time and Conditions for Different Apple Air-Induced Mutation Rootstocks and Varieties. Following the 4.5-point segmented probability grading method, the 100-seed weight and stratification days for the three treatments were segmented using (X±0.5246S), where X represents the mean and S represents the standard deviation. The range of 100-seed weight and stratification time for apple seeds under relative temperature and humidity conditions was determined. Under sand storage conditions of approximately 60% humidity and refrigerator temperature of 2℃, seeds with a 100-seed weight below 1.387g had a maximum stratification time of 46 days; seeds with a 100-seed weight between 1.388 and 2.746g had a stratification time between 53 and 56.0 days; and seeds with a 100-seed weight above 2.747g had a minimum stratification time of 56 days.

[0072] On December 27, 2022, the stratified seeds of Pingyi Sweet Tea, E'ai No. 2, and Tianwang No. 1 were stratified. The moisture content of the stratified sand was gradually reduced from 60% to 10% at the beginning. After 62 days, the sand moisture content was increased back to about 60%. On March 31, 2023, the seeds of all three treatments simultaneously showed signs of germination. Table 3 shows that reducing the moisture content of the stratified sand can prolong the germination time of the stratified seeds. The stratification time for Pingyi Sweet Tea seeds (1.02g per 100 seeds) was 94 days, which was 59 days longer than that for the normal treatment of 0.70g per 100 seeds of Malus spectabilis and 34 days longer than that for Malus macrocarpa (3.28g per 100 seeds). The seeds of Pingyi Sweet Tea (1.02g per 100 seeds) and E'ai No. 2 (3.60g per 100 seeds) showed signs of germination simultaneously. The experiment proves that by reducing the moisture content of the sand used for seed stratification, the germination period of the seeds can be delayed, the interlayer time between different seeds can be shortened, and the goal of simultaneous stratification and sowing of different varieties of seeds can be achieved.

[0073] 2.2 Effects of different sowing substrate treatments on seed germination rate Table 10 shows the effect of different treatments of the same substrate on the germination rate of the same rootstock. The germination rates of *Malus spectabilis* seed-carrying substrates treated with high-temperature sterilization of vermiculite plus rooting powder were 63.77% and 50%, respectively; the germination rates of vermiculite sterilization alone were 60% and 54.16%; the germination rates of vermiculite without sterilization plus rooting powder were 77.5% and 83.3%; and the germination rates of vermiculite without sterilization were 87.5% and 68.75%. The results indicate that the germination rate of sterilized substrates was actually lower than that of unsterilized substrates. The germination rates of mutagenic seeds and ground-reserved seeds were not affected by the sowing substrate, with average germination rates of 66.41% and 64.80%, respectively.

[0074] 3 Results and Discussion 1. Apple rootstocks and varietal seeds need to be exposed to certain humidity, low temperature and time conditions to break seed dormancy and achieve seed germination.

[0075] 2. The required low temperature for apple rootstock seeds increases from small to large. Under conditions of refrigerator temperature of 2℃ and sand storage humidity of about 60%, apple seeds with a 100-seed weight of less than 1.387g require a maximum of 46 days of stratification; seeds with a 100-seed weight between 1.388 and 2.746g require 53 to 56 days of stratification; and seeds with a 100-seed weight of more than 2.747g require a minimum of 56 days of stratification.

[0076] 3. The time required for apple seed stratification can be extended by reducing the humidity of the stratification sand (60%~10%), thus reducing the difference in stratification time between seeds of different sizes and achieving the goal of stratifying and sowing seeds of different sizes at the same time.

[0077] 4. Apple rootstock seeds: Using vermiculite as the sowing substrate, there is no need to treat the vermiculite by high pressure sterilization or add rooting powder. Direct sowing can achieve the corresponding germination rate.

[0078] As another specific embodiment of this application, we have developed a simple and non-destructive tool for measuring plant leaf area. The morphology and area of ​​plant leaves are important indicators for studying plant physiology, biochemistry, genetics, breeding, and crop cultivation. Establishing a simple and accurate method for measuring leaf area and developing an easy-to-use measuring tool are of great significance for carrying out agricultural variety selection, yield and quality prediction, research on efficient cultivation techniques, and rational fertilization.

[0079] Traditional methods for measuring leaf area include the grid method, leaf-shaped paper weighing method, leaf sample weighing method, and regression equation method. The grid method involves laying the leaf flat on grid paper, drawing its outline, calculating the number of grids, and determining the leaf area. The leaf-shaped paper weighing method involves copying or tracing the leaf shape onto uniformly thick paper, cutting out the leaf shape, weighing it, and calculating the leaf area based on the weight of the whole sheet of paper and the cut leaf-shaped paper. The leaf sample weighing method involves weighing leaves with known areas, determining the leaf area-to-weight ratio, and then weighing the sample to calculate the leaf area. The regression equation method involves measuring the area and length / width parameters of a certain number of similar leaves, fitting a regression coefficient for a class of leaves using the known parameters, and then calculating the area based on the leaf length and width. This method requires establishing the regression coefficient first, and the results are more inaccurate for irregular leaves. All of these methods require in vitro measurement or taking a portion of leaves to establish the equation, and the measurement methods are complex and the sample size is limited. The main research instruments used in production are planimeters and leaf area meters. A planimeter calculates the area of ​​a leaf by moving the instrument's needle along the leaf's outline and taking a reading on a counting disc or measuring wheel. While accurate, this method is complex and has limited sample size. It's often used as a standard to check the deviations of other measurement methods, but impractical for large-scale scientific research. A leaf area meter, theoretically capable of measuring the area of ​​leaves of any shape, is commonly available in portable field non-destructive measurement and indoor benchtop in vitro measurement methods. Measurement methods include scanning and multi-parameter analysis with software. These are expensive and precision instruments. Non-destructive measurement is less accurate due to factors such as measurement orientation, leaf morphology, and operator technique. In recent years, many scholars have used digital cameras to acquire digital images of leaves, using software analysis and data modeling to calculate the area. Digital image processing can achieve non-destructive measurement, but image acquisition is complex, requiring each leaf image to be taken at equal distances, making operation difficult, measurement reliability poor, and time-consuming. Furthermore, when measuring uneven leaves, the photographed leaf area differs from the actual leaf area. Furthermore, the use of computer software or computer modeling requires operators to possess advanced computer skills and data analysis abilities, which significantly limits its widespread adoption. Therefore, developing a portable, easy-to-use, reliable, highly accurate, and low-cost handheld tool for measuring the leaf area of ​​living organisms is of great importance.

[0080] 1. Detection principle and structural design of measuring tools 1.1 Detection Principle The detection principle is to use the standard area of ​​the grid to divide the leaf into squares of known area, and by counting the number of squares, the area of ​​the leaf being measured can be measured simply and quickly.

[0081] 1.2 Structural Design This tool is a transparent plate with graduated squares. During measurement, the transparent plate is placed over the leaf being measured, and the squares on the plate are imprinted on the leaf, dividing it into small squares of known area. By counting the number of squares on the leaf, the area of ​​the leaf can be calculated. This tool can be made at home or by a professional tool manufacturer, with a length and width exceeding the maximum length and width of the leaf being measured, depending on the size and shape of the leaves of different tree species. It can be used by the user or provided to scientific and technical personnel.

[0082] The measuring tool's detection principle and actual measurement diagram are as follows: Figure 13 Structural principle and experimental diagram of a self-made leaf area measuring ruler Figure 14 The structural principle and actual measurement diagram of a custom-made leaf area measuring ruler.

[0083] 1.3 Scale settings.

[0084] To facilitate operation and reading, a basic grid of 1×1cm is printed on the transparent plate, with an area of ​​1cm², highlighted by dark, thin lines. Within the basic grid, a 0.5×0.5cm grid, with an area of ​​0.25cm², is marked with light, thin lines. A 0.1cm graduation is set on the horizontal center line of the vertical line of the basic grid for measuring the length and width of the blade.

[0085] 2. Measurement Method 2.1 Measurement of length and width.

[0086] Place the measuring ruler over the blade being measured, aligning the blade's length and width with the horizontal and vertical scales of the measuring ruler, and directly read the blade's length and width with an accuracy of 0.1 cm.

[0087] 2.2 Leaf area measurement.

[0088] 2.2.1 Precise Measurement. Place the measuring ruler over the leaf being measured, aligning the leaf veins with a vertical grid line. Count the number of basic squares that are 100% covered and the number of 0.5×0.5cm squares that are 50% covered. Multiply the number of basic squares by 1 cm² and add the number of 0.5×0.5cm squares that are 50% covered by multiplying by 0.25 cm². This sum is the leaf area value of the measured leaf, calculated using the formula: S = (Sa×1cm²) + (Sb×0.25cm²) (where Sa is the number of basic squares 100% covered and Sb is the number of 0.5×0.5cm squares 50% covered). 0.5×0.5cm squares with less than 50% coverage are not counted.

[0089] 2.2.2 Rapid Measurement. When the sample size is large, select the smaller leaf from the same batch of measured leaves and mark the area of ​​the squares completely covered by this leaf on the measuring ruler. This area is used as the "base area" of the measured leaves in the same batch. When measuring other leaves, use a precise measurement method to count the area of ​​the squares outside the "base area". The area of ​​the measured leaf is the sum of the "base area" and the area of ​​the squares outside the base area.

[0090] 3. Measurement accuracy verification The project selected 75 leaves of *Malus spectabilis* and measured their area using a leaf area measuring ruler developed in this project, following a precise measurement method. The leaf area was then measured again using a YMJ-B handheld leaf area meter manufactured by Jinan Prunte Biotechnology Co., Ltd. Correlation analysis was performed on the two sets of measurements. Figure 15 Quickly measure the 'base area' marking diagram Figure 16 The graph showing the relationship between the measured values ​​of the leaf area measuring ruler and the measured values ​​of the YMJ-B leaf area meter indicates that the correlation coefficient between the measured values ​​of the leaf area measuring ruler and the measured values ​​of the YMJ-B meter reaches 0.983.

[0091] Conclusions: 1. The leaf area measuring ruler developed in this project, compared with the Prandtl YMJ-B high-precision leaf area meter, is unaffected by leaf size, shape, hardness, etc., and technicians can carry it to the field or onto trees for measurement at any time. The coefficient of determination of the measured value can reach 0.983 compared with the high-precision instrument, providing a new and convenient measuring tool for plant leaf area measurement.

[0092] 2. The leaf area measuring ruler developed in this project combines the traditional leaf area grid measurement method with the function of a ruler. Depending on the size of the leaf being measured, it can be made by technicians themselves or by professional institutions into different sizes. This tool is simple to manufacture, has low environmental requirements, and can be used for measurements anytime, anywhere in the field or laboratory. It overcomes the complexities and destructive nature of traditional testing methods, as well as the high cost and operational instability of advanced instruments, reducing the skill requirements for operators and possessing strong practicality and operability.

[0093] Throughout this specification, the terms "an embodiment" or "an embodiment" mean that a specific feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the disclosed subject matter. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment. Furthermore, any suitable manner may be adopted to incorporate a specific feature, structure, or characteristic in one or more embodiments. It should be understood that this specification is not intended to limit the invention. Rather, exemplary embodiments are intended to cover alternatives, modifications, and equivalents that are included within the spirit and scope of the invention as defined by the appended claims. Furthermore, numerous specific details are set forth in the detailed description of exemplary embodiments to provide a comprehensive understanding of the claimed invention. However, those skilled in the art will understand that various embodiments may also be practiced without these specific details.

[0094] Although features and elements of these exemplary embodiments have been described in particular combination in the embodiments, each feature and element may be used alone without the other features and elements of the embodiments, or in combination with or without the other features and elements disclosed herein.

[0095] This written description uses examples, including the best mode, to disclose the invention and enables any person skilled in the art to practice the invention, including making and utilizing any apparatus or system and performing any combined methods. The patentable scope of the invention is defined by the claims and may include other examples as would be apparent to those skilled in the art. Such other examples are considered to be included within the scope of the claims if they have structural elements that are not different from the verbal language of the claims, or if they include structural elements equivalent to those described in the verbal language of the claims.

Claims

1. A method for space-induced mutation breeding of apple rootstocks, characterized in that, include: (1) Select plump apple rootstock seeds, dry them in the shade, and store them at 4℃; (2) The seeds were carried on a recoverable spacecraft and remained in orbit for 7–180 days; (3) After returning, stratify and stack according to 100-grain weight: ≤1.387 g, layering time 46 days; 1.388–2.746 g, layering time 53–56 days; ≥2.747 g, layering time 56 days; temperature 2–4 ℃, humidity 60%; (4) Sow 50% of the seedlings in nutrient pots after they have sprouted. After emergence, establish an electronic file for each seedling to record seedling height, seedling diameter, leaf area, leaf morphology and images. (5) In late July, cut off branches with 3-4 buds, leaving 1 leaf at the top, soak them in rooting solution for 20-30 minutes, and insert them into vermiculite pots with humidity ≥90%, greenhouse height 2 m, and temperature ≤30 ℃; (6) Phenotypic mutants were screened based on electronic records. The screening indicators were leaf area increase rate ≥15%, plant height increase rate ≥20%, and stem diameter increase rate ≥10%. (7) Rapid propagation of mutants by tissue culture: using tender shoots as explants, callus was induced by MS + 6-BA 2 mg / L + NAA 0.2 mg / L, shoots were induced by subculture medium MS + 6-BA 1 mg / L + IBA 0.5 mg / L, and rooting medium 1 / 2 MS + IBA 1 mg / L. Complete plants were obtained after 30 days. (8) Establish a field breeding nursery: 3 m × 1 m of mutation mother plants and 1 m × 0.5 m of cutting test nursery; (9) In autumn, using the control plant as a benchmark, the drought resistance, cold resistance, salt and alkali resistance and dwarfing ability of the mutant plant were tested; (10) The seeds of superior mother plants were carried a second time and recursive mutagenesis screening was carried out; (11) After passing the grafting compatibility test and 2–3 years of multi-location regional trials, apply for variety approval.

2. The method for space-induced mutation breeding of apple rootstock according to claim 1, characterized in that: The ratio of the number of seeds carried in space to the number of control seeds on the ground is 3:1 or 4:

1.

3. The method for space-induced mutation breeding of apple rootstock according to claim 1, characterized in that: The stratification medium is fine river sand with a water content of 60%; when it is necessary to delay the emergence of white to unify the sowing period, the humidity is reduced to 10% in stages and then restored to 60%.

4. The method for space-induced mutation breeding of apple rootstock according to claim 1, characterized in that: The sowing substrate is unsterilized vermiculite, which increases the germination rate by 10–25% compared to sterilized substrate.

5. The method for space-induced mutation breeding of apple rootstock according to claim 4, characterized in that: The electronic record system uses a mobile terminal APP, which connects to the leaf area measuring ruler via Bluetooth, and uploads seedling trait data and images to the cloud database in real time.

6. A dedicated leaf area measuring ruler for use in the method of claim 1, characterized in that: a) A transparent grid board, engraved with 1 cm × 1 cm thick-lined squares and 0.5 cm × 0.5 cm thin-lined squares; b) Set graduations with an accuracy of 0.1 cm on the horizontal and vertical center lines; c) Measurement formula: S=(Sa×1 cm²)+(Sb×0.25 cm²), where Sa is the number of 1 cm² squares with 100% coverage, and Sb is the number of 0.25 cm² squares with ≥50% coverage; d) Compared with the YMJ-B handheld leaf area meter, the correlation coefficient is ≥0.

983.

7. The method for space-induced mutation breeding of apple rootstock according to claim 6, characterized in that: Made of 2 mm transparent acrylic, it can be rolled up for storage and has a measurement range of 0–200 cm².

8. The method for space-induced mutation breeding of apple rootstock according to claim 5, characterized in that: The softwood cuttings were taken in late August using secondary branches that sprouted after the current year's growth was cut off, which increased the rooting rate by 2.4–4.6 times compared to primary branches.

9. The method for space-induced mutation breeding of apple rootstock according to claim 8, characterized in that: The tissue culture rapid propagation is achieved by adding [a certain ingredient] to the rooting medium. Adding 0.1% activated carbon increases the rooting rate to over 95%.

10. The method for space-induced mutation breeding of apple rootstock according to claim 9, characterized in that: The drought resistance test used the pot water control method, and the survival rate was measured when the soil moisture content dropped to 40% of the field capacity. The cold resistance test used an artificial climate chamber to freeze at -25 ℃ for 6 h and then measured the electrolyte leakage rate. The salt and alkali resistance test used 200 mmol / L NaCl solution for irrigation for 30 days and then measured the plant height inhibition rate. The dwarfing test used the plant height / stem diameter ratio as an indicator, and the smaller the ratio, the stronger the dwarfing effect.

Images