Caenorhabditis elegans cross breeding method based on flowering phase difference regulation and control

Abstract

The invention discloses an acer elegans cross breeding method based on flowering phase difference regulation and control, and belongs to the technical field of garden plant breeding. The method comprises the following steps: selecting an acer elegans variety of which the natural early flowering phase difference is greater than or equal to 10 days and the genetic distance is greater than or equal to 0.5 as a cross parent, carrying out low-temperature refrigeration and shading blue light supplementing treatment on a female parent to delay the flowering phase, and carrying out prolonged illumination, red light supplementing and hormone and nutrient solution spraying on a male parent to advance the flowering phase; male parent pollen is collected through a vacuum negative pressure adsorption device and stored in a closed container, a sucrose solution is sprayed to female parent stigmas before pollination, and repeated pollination is conducted through a directional nozzle at 9-11 o'clock every day; carrying out stratification treatment and temperature-difference temperature-control germination on the hybrid seeds, and carrying out light-temperature regulation and control seedling culture after sowing; the molecular marker is used for verifying the hybridization authenticity and target traits, phenotype determination is carried out on three-year-old seedlings to complete breeding, the hybridization obstacle caused by asynchronous flowering phases of the caenorhabditis elegans varieties is solved through the flowering phase regulation and control technology, and the hybridization success rate is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of biological breeding, and in particular to a method for hybrid breeding of Acer elegans based on differential regulation of flowering periods. Background Art

[0002] The elegant maple (Acer palmatum) is a key ornamental tree species in the Aceraceae family. Its graceful form, varied leaf color, and excellent adaptability make it a popular choice for landscaping applications. With the rapid development of urban greening and landscape construction, the market demand for new varieties of Acer palmatum with excellent ornamental qualities and strong adaptability is growing. Hybridization is an important method for obtaining new varieties. By hybridizing different varieties, the beneficial traits of the parent trees can be combined to produce new varieties with vibrant leaf color, robust growth, and strong resistance to stress.

[0003] In the field of plant hybrid breeding, synchronization of the flowering periods of the parents is a key prerequisite for successful hybridization. However, there are generally large differences in flowering periods between different varieties of Acer truncatum. The difference in the initial flowering period between some varieties can even reach more than 30 days, which seriously hinders the development of hybrid breeding. At present, there are many research reports on plant flowering period regulation technologies at home and abroad. For example, CN115281073A discloses a hybrid breeding method for regulating the flowering period of lilac. The method uses a temperature control device to regulate the flowering period of early-flowering lilac varieties and / or late-flowering lilac varieties, so that the flowering periods of varieties with large differences in early and late flowering periods meet, thereby improving the success rate of hybrid breeding. However, this method is mainly aimed at plants of the genus Syringa, and its regulation parameters and technical measures are difficult to be directly applied to Acer truncatum.

[0004] In terms of pollination technology, traditional artificial pollination methods suffer from low efficiency and low success rates. CN114831018A discloses a method for sexual hybridization of Astragalus membranaceus, which improves pollination success rates by selecting flowers in a specific open state for pollination. CN113080052A introduces a hybridization breeding method for a new tea variety, including pollen collection and artificial pollination. However, these methods fail to address the issues of pollen viability and pollination efficiency. This is particularly true for species with small, abundant flowers, such as Acer elegans, where traditional pollination methods are time-consuming, labor-intensive, and ineffective.

[0005] In terms of screening hybrid offspring, CN118020634A proposed a breeding method for efficiently cultivating new varieties of Cymbidium orchids, combining morphological markers for strain screening; CN108243946B introduced a method for artificial hybrid breeding of water lilies, which screened suitable parents by culturing early pollen in stigma mucus; however, these methods lack a systematic screening system for woody plants, especially Acer elegans, making it difficult to effectively identify and breed hybrid offspring with excellent ornamental and stress resistance traits.

[0006] In summary, existing technologies face the following challenges: First, significant differences in flowering periods exist between different Acer truncatum varieties, leading to a lack of effective technologies for synchronized flowering; second, traditional pollination methods are inefficient and have low success rates, particularly for species like Acer truncatum, which have small, abundant floral organs; and third, a lack of systematic and effective methods for screening hybrid offspring makes it difficult to obtain hybrids with superior traits. These issues have severely hampered the development of Acer truncatum hybrid breeding and the development of new varieties. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a hybrid breeding method for Acer truncatum based on the regulation of flowering period differences, in view of the problems that large differences in flowering periods between different Acer truncatum varieties lead to difficulties in hybrid breeding, traditional pollination methods are inefficient and have low success rates, and there is a lack of systematic and effective methods for screening hybrid offspring, which makes it difficult to obtain hybrid varieties with excellent traits.

[0008] The technical solution adopted by the present invention to solve the above technical problems is to provide a method for hybrid breeding of Acer truncatum based on differential regulation of flowering period, comprising the following steps: S1. Parent Selection and Flowering Synchronization: Select Acer elegans varieties with a natural flowering time difference of 10 days or more and a genetic distance of 0.5 as hybrid parents. The one with earlier flowering time will serve as the female parent, and the one with later flowering time will serve as the male parent. The female parent will be treated to delay flowering, including low-temperature refrigeration and shading with blue light supplementation. The male parent will be treated to advance flowering, including extended daylight hours, red light supplementation, and application of hormones and nutrient solutions. S2. Vacuum-assisted artificial pollination: Pollen from the male parent is collected using a vacuum suction device and stored in a sealed container. Before pollination, the female parent's stigma is sprayed with a sucrose solution. Repeated pollination is performed using a directional nozzle between 9:00 and 11:00 AM daily. Once the female parent is fully pollinated and fruiting, mature hybrid seeds are harvested. S3. Progeny screening: The hybrid seeds obtained in step S2 are subjected to stratification and differential temperature control for germination. After sowing, light and temperature-controlled seedling cultivation is carried out. SSR and SNP molecular markers are used to verify the authenticity of the hybrid and the target traits. Phenotypic determination is performed on three-year-old seedlings to complete the breeding.

[0009] Compared with the existing technology (related technology), the hybrid breeding method of Acer elegans based on differential regulation of flowering period in the present application has the following advantages: the present invention adopts a "two-way flowering period regulation strategy" to delay the flowering period of the early-flowering female parent by low-temperature refrigeration and shading-blue light supplementation on the one hand, and advance the flowering period of the late-flowering male parent by synchronously promoting flowering with red light, hormones and nutrient solution on the other hand, so that the parent varieties with a natural flowering period difference of ≥10 days can achieve artificial synchronous flowering, solving the technical problem of "different flowering periods cannot be hybridized" that has long restricted Acer elegans breeding; further, mature pollen is accurately collected by negative pressure adsorption, combined with low-temperature and low-humidity storage and sucrose-induced viscosity increase to ensure pollen activity, and pollination is repeated in a critical period using a directional nozzle, which greatly improves the pollination efficiency and fertilization rate, which is significantly better than the efficiency of traditional tweezers and brush operations; further, the present invention integrates molecular-level SSR / SNP markers with field phenotypic determination, Double verification of hybrid authenticity and efficient screening of excellent individuals avoids the problems of misselection and missed selection in traditional reliance on appearance judgment, shortens the breeding cycle, and improves the success rate of breeding. Through the "temperature and humidity gradient + light and temperature difference control" method to optimize stratification germination and seedling conditions, the problems of uneven seedling emergence and large growth differences in Acer elegans sowing are solved, providing a stable and consistent population basis for subsequent screening. The final offspring of the present invention are evaluated by comprehensive indicators such as leaf color, cold resistance, growth and photosynthesis. The screened plants perform better than their parents in terms of ornamental value, stress resistance and growth potential, achieving a synergistic effect of trait aggregation and genetic improvement. In summary, the present invention systematically integrates the key links of flowering period regulation, pollination method, offspring cultivation and screening to form a complete, efficient and scientific Acer elegans hybrid breeding method system, which significantly improves the breeding success rate and new variety selection efficiency, and has important theoretical innovation value and industrial promotion prospects.

[0010] In one possible embodiment, the female parent is an early-flowering Acer truncatum variety "Coral Pavilion", "Red Shoes", "Spring Dawn" or "Morning Dew", and the male parent is a late-flowering variety "Red Feather Clothes", "Purple Clouds", "Golden Autumn" or "Muxue".

[0011] Compared with the existing technology, the above technical solution eliminates reproductive isolation barriers between varieties with the same flowering period. By identifying early-flowering and late-flowering resources and fixing the combination with staggered flowering periods as the maternal / paternal structure, it is beneficial to the efficiency and directionality of subsequent flowering period regulation operations. In addition, the setting of genetic distance ≥ 0.5 helps to improve the phenotypic separation of hybrid offspring, providing a genetic basis for trait recombination and high-quality strain screening. Through structured parent selection, the probability of recombination of target traits and the richness of variation in hybridization are significantly improved, reducing the risks of population convergence and trait fixation brought about by close hybridization.

[0012] In one possible embodiment, in step S1, the conditions for delaying the flowering period of the mother plant are as follows: 1-year-old branches with a diameter of ≥0.8 cm are refrigerated at 0-4°C and a humidity of 60-70% for 35 days and then moved into a greenhouse, and shaded to ≤8 hours of light per day, while supplemented with blue light of a wavelength of 450±10 nm and a light intensity of 50 μmol / m 2 / s.

[0013] Compared with the existing technology, the above-mentioned technical solution can effectively delay the bud germination time of the early-flowering mother plant by simulating the natural winter low-temperature dormancy period and regulating the artificial photoperiod and light quality, thereby achieving artificial flowering synchronization with the father plant. Without destroying the activity of the branches and the flowering ability, the flowering time of the mother plant can be effectively delayed by about 30-40 days to match the late-flowering father plant.

[0014] In one possible embodiment, in step S1, the conditions for delaying the flowering period of the male parent are as follows: 30 days before the flower bud differentiation of the male parent, red light with a wavelength of 660±15nm is supplemented daily, and the proportion of red light is ≥70%, the illumination time is extended to 14-16 hours, and 80ppm gibberellin and flower-promoting nutrient solution are sprayed simultaneously, and the treatment is performed once a week for 3 consecutive weeks.

[0015] Compared with the existing technology, the above technical solution uses 660±15nm red light, which belongs to the far-red light band, and can effectively activate the phytochrome signaling pathway in plants and promote flower bud formation; extending the light time to simulate long-day conditions is beneficial to inducing long-day plants (Acer elegans) to bloom early; gibberellins have a significant effect of breaking dormancy and promoting the growth of floral organs; further, the flower-promoting nutrient solution provides sufficient macro- and trace element support for reproductive growth, improving the quality of plant flower buds and flowering vitality.

[0016] In a possible embodiment, in step S1, the pH value of the flower-promoting nutrient solution is 5.8-6.2, the conductivity is ≤2.0 mS / cm, and the weekly root irrigation amount of the flower-promoting nutrient solution is 500 mL / plant; in terms of mass ratio, the nutrient solution includes: 0.1% potassium dihydrogen phosphate, 0.05% boric acid and 0.02% zinc sulfate.

[0017] Compared with the prior art, the above technical solution is adopted. In the specially compounded nutrient solution of the present invention, potassium dihydrogen phosphate provides phosphorus and potassium elements, which can enhance plant carbon metabolism, promote flower bud differentiation and pollen activity; boric acid participates in cell division and pollen tube elongation, significantly improving fertilization efficiency; zinc sulfate, as a cofactor of multiple enzymes, can regulate endogenous hormone balance and promote reproductive growth. Furthermore, by adjusting the formula of the above nutrient solution, the nutritional status and hormone sensitivity of plants in the flowering stage are significantly improved, and the flower bud differentiation efficiency and the quality of floral organ formation are improved; the pH value is further controlled to 5.8-6.2 and the conductivity is ≤2.0mS / cm, which optimizes the rhizosphere absorption environment and avoids nutrient backflow and ion poisoning; the above embodiment ensures a nutrient accumulation effect rather than excessive stimulation by controlling the application amount and frequency, thereby improving the reproductive adaptability of plants.

[0018] In one possible embodiment, in step S2, the conditions for the vacuum negative pressure adsorption device to collect the father parent pollen are: the adsorption pressure of the vacuum negative pressure adsorption device is -0.05±0.01MPa, the adsorption head diameter of the vacuum negative pressure adsorption device is 1±0.5mm, and the pollen activity loss rate is ≤5% during the process of collecting the father parent pollen by the vacuum negative pressure adsorption device.

[0019] Compared with the existing technology, the above-mentioned technical solution can overcome the problems of low efficiency, high pollution and serious pollen damage in traditional manual pollination operations. Through precisely controlled negative pressure suction (-0.05±0.01MPa), mature pollen can be quickly adsorbed into a sealed collection chamber without destroying the anther structure; the adsorption head has a precise size (1±0.5mm) to adapt to the small flower vase of the Acer truncatum, improving the collection accuracy and safety; low-pressure operation minimizes mechanical damage and oxidation exposure, avoids enzyme inactivation and water loss, thereby significantly improving the success rate of hybrid pollination and the stability of repeated pollination, shortening the breeding cycle, and improving the controllability and standardization of the entire Acer truncatum hybrid breeding system.

[0020] In a possible embodiment, in step S2, the condition for spraying the sucrose solution is: spraying 15% sucrose solution on the stigma of the female parent before pollination, with a spraying amount of 10 μL / flower.

[0021] Compared with the existing technology, the above technical solution can enhance the wettability of the stigma and improve the pollen adhesion rate by spraying an appropriate amount of 15% sucrose solution; at the same time, it provides a rapidly absorbable carbon source, activates the germination and elongation of pollen tubes, and promotes rapid recognition and affinity reaction between pollen and stigma; and the quantitative application amount control of 10μL can avoid osmotic stress and dilution effects, ensuring the stability of the stigma fertilization environment.

[0022] In a possible embodiment, in step S2, the pollen storage conditions are: the collected pollen is stored in a sealed container at a temperature of 4° C. and a humidity of ≤30% for a storage time of ≤15 days.

[0023] Compared with the existing technology, the above technical solution can significantly slow down the pollen metabolic activity by storing the pollen at a low temperature of 4°C, inhibiting respiration and endogenous enzyme activity, delaying aging, and further controlling the low humidity environment of ≤30% to prevent the pollen from absorbing moisture and becoming moldy or spontaneously germinating, thereby maintaining structural stability; the sealed container isolates the oxygen and foreign bacteria in the outside air from invading, avoiding oxidation and contamination. The above implementation plan broadens the pollen utilization window, improves the flexibility and success rate of artificial pollination, ensures the feasibility and high output of the hybridization plan, and promotes the steady improvement of the hybrid breeding efficiency of Acer truncatum.

[0024] In one possible embodiment, step S31 includes: mixing the hybrid seeds with moist vermiculite in a volume ratio of 1:3, stratifying the seeds under conditions of a gradient temperature of 0-25°C and a humidity of 80% for 60 days before sowing, and germinating and raising seedlings by controlling the temperature difference between day and night, with the conditions being 25°C during the day and 15°C at night, and the treatment cycle being 30 days.

[0025] Compared with the existing technology, the above technical solution uses moist vermiculite as a porous medium to evenly provide moisture and oxygen, promote the activation of seed hydrolases, and effectively break dormancy; the gradient temperature change of 0-25℃ simulates the natural environment in spring and promotes the development of radicles; 80% humidity maintains a high humidity environment that is conducive to seed coat softening and enzyme activation; and further control of the day and night temperature difference (25℃ during the day / 15℃ at night) forms physiological stimulation, induces changes in endogenous hormones (such as increased gibberellins), and promotes uniform germination.

[0026] In one possible embodiment, in step S32, the phenotypic determination includes the following items: (1) Leaf color: The leaf color in spring is RHS color chart N66A, and changes to RHS 34A in autumn, and the autumn color lasts for ≥45 days; (2) Growth traits: annual growth ≥ 20 cm, coefficient of variation of seedling height uniformity ≤ 15%; (3) Cold resistance: after 24 hours of low temperature stress treatment at -15℃, the survival rate is ≥90%, and the electrolyte leakage rate of branches is ≤25%; (4) Physiological characteristics: Leaf photosynthetic rate ≥ 18 μmol CO2 / m 2 / s, the measurement condition was PAR 800 μmol / m 2 / s, pore density ≤120 / mm 2 .

[0027] Compared with the existing technology, the adoption of the above technical scheme and the use of the internationally accepted RHS color card standard can achieve objective quantification of leaf color visual indicators, which is conducive to the stable evaluation of color genetic characteristics; the annual growth amount is combined with the uniformity variation coefficient to reflect the individual growth potential and population consistency; the above-mentioned cold resistance reflects the stability of the cell membrane through the electrolyte exudation rate, which is an objective indicator of stress tolerance at the cellular level. Further, the photosynthetic rate and stomatal density comprehensively evaluate the photosynthetic potential and water use efficiency, reflecting its physiological adaptability; through the above-mentioned determination, the present invention further ensures the scientificity, systematicness and success rate of the breeding of new varieties of Acer truncatum. DETAILED DESCRIPTION

[0028] First, those skilled in the art should understand that these embodiments are merely used to explain the technical principles of the embodiments of the present application and are not intended to limit the scope of protection of the embodiments of the present application. Those skilled in the art may adjust them as needed to suit specific application scenarios.

[0029] In the description of the embodiments of this application, it should be noted that, unless otherwise specified or limited, the terms "connected" and "connection" should be understood in a broad sense. 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. Those skilled in the art will understand the specific meanings of the above terms in the embodiments of this application based on the specific circumstances.

[0030] The present invention provides a method for hybrid breeding of Acer elegans based on differential regulation of flowering period, comprising the following steps: S1. Parent Selection and Flowering Synchronization: Select Acer elegans varieties with a natural flowering time difference of 10 days or more and a genetic distance of 0.5 as hybrid parents. The one with earlier flowering time will serve as the female parent, and the one with later flowering time will serve as the male parent. The female parent will be treated to delay flowering, including low-temperature refrigeration and shading with blue light supplementation. The male parent will be treated to advance flowering, including extended daylight hours, red light supplementation, and application of hormones and nutrient solutions. S2. Vacuum-assisted artificial pollination: Pollen from the male parent is collected using a vacuum suction device and stored in a sealed container. Before pollination, the female parent's stigma is sprayed with a sucrose solution. Repeated pollination is performed using a directional nozzle between 9:00 and 11:00 AM daily. Once the female parent is fully pollinated and fruiting, mature hybrid seeds are harvested. S3. Progeny screening: The hybrid seeds obtained in step S2 are subjected to stratification and differential temperature control for germination. After sowing, light and temperature-controlled seedling cultivation is carried out. SSR and SNP molecular markers are used to verify the authenticity of the hybrid and the target traits. Phenotypic determination is performed on three-year-old seedlings to complete the breeding.

[0031] As a preferred solution, in step S1, the female parent is an early-flowering Acer truncatum variety "Coral Pavilion", "Red Dancing Shoes", "Spring Dawn" or "Morning Dew", and the male parent is a late-flowering variety "Red Feather Clothes", "Purple Clouds", "Golden Autumn" or "Muxue".

[0032] As a preferred solution, in step S1, the conditions for delaying the flowering period of the mother plant are as follows: 1-year-old branches with a diameter of ≥0.8 cm are refrigerated at 0-4°C and a humidity of 60-70% for 35 days and then moved into a greenhouse, and shaded to ≤8 hours of light per day, while supplemented with blue light of 450±10nm wavelength and a light intensity of 50μmol / m 2 / s.

[0033] As a preferred solution, in step S1, the conditions for delaying the flowering period of the male parent are as follows: 30 days before the flower bud differentiation of the male parent, red light with a wavelength of 660±15nm is supplemented daily, and the proportion of red light is ≥70%, the illumination time is extended to 14-16 hours, and 80ppm gibberellin and flower-promoting nutrient solution are sprayed simultaneously, and the treatment is performed once a week for 3 consecutive weeks.

[0034] As a preferred solution, in step S1, the pH value of the flower-promoting nutrient solution is 5.8-6.2, the conductivity is ≤2.0mS / cm, and the weekly root irrigation amount of the flower-promoting nutrient solution is 500mL / plant; in terms of mass ratio, the nutrient solution includes: 0.1% potassium dihydrogen phosphate, 0.05% boric acid and 0.02% zinc sulfate.

[0035] As a preferred solution, in step S2, the conditions for the vacuum negative pressure adsorption device to collect the pollen of the father parent are: the adsorption pressure of the vacuum negative pressure adsorption device is -0.05±0.01MPa, the adsorption head diameter of the vacuum negative pressure adsorption device is 1±0.5mm, and the pollen activity loss rate is ≤5% during the process of collecting the pollen of the father parent by the vacuum negative pressure adsorption device.

[0036] As a preferred solution, in step S2, the condition for spraying the sucrose solution is: spraying 15% sucrose solution on the stigma of the female parent before pollination, with a spraying amount of 10 μL / flower.

[0037] As a preferred solution, in step S2, the pollen storage conditions are: the collected pollen is stored in a sealed container at a temperature of 4°C and a humidity of ≤30% for a storage time of ≤15 days.

[0038] As a preferred solution, step S31 includes: mixing the hybrid seeds with moist vermiculite in a volume ratio of 1:3, stratifying the seeds under conditions of a gradient temperature of 0-25°C and a humidity of 80% for 60 days before sowing, and germinating and raising seedlings by controlling the temperature difference between day and night, with the conditions being 25°C during the day and 15°C at night, and the treatment cycle being 30 days.

[0039] As a preferred solution, in step S32, the phenotypic determination includes the following items: (1) Leaf color: The leaf color in spring is RHS color chart N66A, and changes to RHS 34A in autumn, and the autumn color lasts for ≥45 days; (2) Growth traits: annual growth ≥ 20 cm, coefficient of variation of seedling height uniformity ≤ 15%; (3) Cold resistance: after 24 hours of low temperature stress treatment at -15℃, the survival rate is ≥90%, and the electrolyte leakage rate of branches is ≤25%; (4) Physiological characteristics: Leaf photosynthetic rate ≥ 18 μmol CO2 / m 2 / s, the measurement condition was PAR 800 μmol / m 2 / s, pore density ≤120 / mm 2 .

[0040] The hybrid breeding method for Acer elegans based on differential flowering period regulation provided by the present invention introduces scientific parameter control and technical collaboration in multiple key links, significantly improving breeding efficiency and success rate: First, in the parent selection stage, by combining the two standards of "difference in initial flowering period ≥10 days" and "genetic distance ≥0.5", it is ensured that the hybrid combination has the feasibility of adjustable flowering period misalignment on the basis of genetic diversity; further, the delayed flowering period of the maternal parent and the advanced flowering period of the paternal parent are respectively treated by low-temperature refrigeration + shading blue light and red light supplementation + hormone nutrient solution. These physical, light and chemical measures form a synergistic effect, significantly improving the success rate of flowering period synchronization, and breaking through the key technical bottleneck that has long restricted the hybrid pairing of Acer elegans varieties; Secondly, the pollination process utilizes a combination of vacuum-pressure precision pollen collection, repeated pollination with a directional nozzle, and pre-pollination stigma activation with a sucrose solution. This ensures both pollen viability and stigma fertilization efficiency, addressing the low efficiency of traditional pollination due to the small size and large number of flowers of the Acer elegans. Furthermore, pollen storage conditions (low temperature, low humidity, and airtight seal) are matched with pollen collection parameters (pressure and tip specifications), further reducing pollen viability loss to ≤5%.

[0041] During the progeny screening phase, stratification and light-temperature seedling cultivation, combined with the diurnal temperature differential to accelerate germination, promote rapid radicle dormancy, shorten the germination cycle, and improve uniformity. Subsequently, SSR / SNP molecular markers combined with a four-dimensional phenotypic evaluation system (ornamental traits, growth potential, stress resistance, and physiological indicators) are used for collaborative screening, effectively improving the accuracy of identifying high-quality individual plants and breeding efficiency.

[0042] In summary, the present invention has constructed a hybrid breeding system for Acer truncatum with a complete system, clear technical path, and coordinated optimization of each link by setting parameter combinations with scientific logic and synergistic mechanisms in each link of parent selection, flowering period regulation, pollination and screening. It has significantly improved the hybridization success rate, breeding efficiency and the stability of excellent traits of offspring, and provided a technical path with promotional value for breeding innovation of garden plants, especially woody ornamental tree species.

[0043] This invention, through synchronized flowering control technology, successfully achieved hybridization between Acer elegans varieties with naturally occurring flowering period differences of 10 days or more, resolving the hybridization difficulties associated with asynchronous flowering in traditional breeding. Compared with traditional methods, this flowering control technology can reduce flowering period differences of more than 30 days to less than 5 days, significantly improving hybridization feasibility. Furthermore, vacuum-assisted artificial pollination technology reduces pollen viability loss to ≤5%, significantly increasing pollination efficiency and success rates. Compared with traditional artificial pollination, this method increased the pollination success rate by more than 40% and the seed set rate by 35%, greatly improving the efficiency of hybrid breeding. Through a systematic progeny screening method, hybrid offspring with excellent traits were obtained, including excellent leaf color traits (leaf color is RHS color chart N66A in spring, changes to RHS 34A in autumn, and the autumn color lasts ≥45 days), good growth traits (annual growth ≥20cm, coefficient of variation of seedling height uniformity ≤15%), excellent cold resistance traits (survival rate ≥90% after 24 hours of low temperature stress treatment at -15℃, branch electrolyte exudation rate ≤25%) and good physiological traits (leaf photosynthetic rate ≥18μmol CO2 / m² / s, stomatal density ≤120 / mm²). The combined performance of the above traits is better than that of the parents and existing varieties, providing new germplasm resources for the improvement of Acer truncatum varieties.

[0044] The following provides examples including specific data to further expand the above-mentioned technical solutions of the present invention: Example 1: This embodiment provides a method for hybrid breeding of Acer elegans based on differential regulation of flowering period, comprising the following steps: S1: Parent selection and flowering synchronization regulation; Select Acer truncatum varieties with a natural flowering start difference of ≥10 days and a genetic distance ≥0.5 as hybrid parents, with the one that starts flowering earlier serving as the female parent and the one that starts flowering later serving as the male parent. In this example, the early-flowering Acer truncatum variety "Coral Court" was selected as the female parent, and the late-flowering variety "Red Feather Clothes" was selected as the male parent. Through previous observations, the natural flowering start of "Coral Court" was in early March, and the natural flowering start of "Red Feather Clothes" was in mid-April. The difference in the natural flowering start of the two varieties reached 40 days. Molecular marker analysis showed that the genetic distance between the two varieties was 0.68, which met the parent selection criteria.

[0045] The mother plant "Coral Court" was treated to delay the flowering period. The specific operation was as follows: select one-year-old branches with a diameter of ≥0.8cm and refrigerate them in a cold storage room at 0-4℃ and 60-70% humidity for 35 days. During the cold storage period, check the condition of the branches once a week to ensure that they do not dry out or mold. After the cold storage, move the branches into a greenhouse environment and control the greenhouse temperature at 18-22℃. In the greenhouse, the mother plant was shaded so that the daily light time was ≤8 hours, and blue light with a wavelength of 450±10nm and a light intensity of 50μmol / m 2 / s. The blue light treatment used an LED light source, with a daily illumination time of 8 hours, from 8 a.m. to 4 p.m. This series of treatments delayed the flowering period of the mother plant, "Coral Court," to mid-April.

[0046] The male parent, "Hong Yu Yi," was treated to advance flowering. Specifically, treatment began 30 days before flower bud differentiation (mid-February). Red light with a wavelength of 660±15nm was supplemented daily, with the red light accounting for ≥70% of the plant's light intensity, and the light duration was extended to 14-16 hours. Professional horticultural LED lights were used for red light treatment, with continuous illumination from 6:00 AM to 10:00 PM, for a total light duration of 16 hours. Simultaneously, the male parent was sprayed with an 80ppm gibberellin solution and a flower-promoting nutrient solution once a week for three consecutive weeks. The gibberellin solution was sprayed at a rate of 100mL per plant, evenly applied to the branches. The pH of the flower-promoting nutrient solution was controlled between 5.8 and 6.2, with an electrical conductivity of ≤2.0mS / cm. Root irrigation was provided at a rate of 500mL per plant per week. The flower-promoting nutrient solution consisted of 0.1% potassium dihydrogen phosphate, 0.05% boric acid, and 0.02% zinc sulfate (by weight). Through this series of treatments, the flowering period of the male parent "Red Feather Clothes" was advanced to mid-April;

[0047] After the above-mentioned flowering period regulation treatment, the flowering periods of the female parent "Coral Pavilion" and the male parent "Red Feather Clothes" were synchronized, creating conditions for subsequent hybridization and pollination.

[0048] S2: vacuum-assisted artificial pollination; When the pollen of the male parent, "Red Feather Clothes," matures, it is collected using a vacuum negative pressure adsorption device. The adsorption pressure of the vacuum negative pressure adsorption device is set to -0.05±0.01MPa, and the adsorption head diameter is 1±0.5mm. During the collection process, the adsorption head is gently touched to the surface of the anther, and the negative pressure draws the mature pollen into the collection tube. Using this method to collect pollen, the pollen activity loss rate is ≤5%, ensuring the success rate of subsequent pollination.

[0049] Store the collected pollen in a sealed container at a temperature of 4°C and a humidity of ≤30% for no more than 15 days. Use a sealed glass bottle with a desiccant to store the pollen. Check the pollen vitality regularly to ensure that the pollen vitality is not less than 90% of the initial vitality during pollination.

[0050] Before pollination, spray the stigmas of the female plant, "Coral Court," with a 15% sucrose solution at a rate of 10 μL per flower. This spraying helps increase stigma stickiness and pollen germination. Pollination should be performed between 9:00 and 11:00 a.m. daily, when temperature and humidity conditions are optimal for pollen germination. Use a directional nozzle to evenly spray pollen onto the female plant's stigma. Pollinate each flower for three consecutive days to maximize pollination success.

[0051] After pollination is complete, mark the pollinated flowers and record the pollination date. Harvest the mature hybrid seeds after pollination and fruiting of the female parent, which have gradually matured and changed color. Seeds are harvested approximately 120 days after pollination, when they are fully mature and have a brown seed coat. After washing and drying, the harvested seeds are stored at 4°C in preparation for subsequent seedling cultivation.

[0052] S3: progeny screening; S31: performing stratification treatment and temperature difference control germination on the hybrid seeds obtained in step S2, and carrying out light and temperature control seedling cultivation after sowing; The specific procedure is to mix hybrid seeds with moist vermiculite in a volume ratio of 1:3 and stratify them for 60 days at a gradient temperature of 0-25°C and 80% humidity. Stratification is done in layers, with the bottom layer being moist vermiculite, the middle layer being the seed-vermiculite mixture, and the top layer being moist vermiculite. During the treatment period, humidity levels are checked weekly and water is added as needed to keep the vermiculite moist but not waterlogged.

[0053] After 60 days of stratification, the seeds were sown in seedling trays using a mixture of peat, vermiculite, and perlite (5:3:2 by volume). After sowing, the seeds were germinated and raised under a controlled day / night temperature regime of 25°C during the day and 15°C at night for 30 days. During this period, the substrate was kept moist and the light intensity was controlled at 200-300 μmol / m² / s for 12 hours per day.

[0054] S32: Use SSR and SNP molecular markers to verify hybrid authenticity and target traits.

[0055] DNA was extracted from seedling leaves and amplified by PCR using 10 pairs of polymorphic SSR primers and 20 SNP marker loci, followed by sequencing analysis to verify the authenticity of the hybrid offspring. Successful hybrids were confirmed by comparing the molecular marker profiles of the hybrid offspring with those of the parents. Furthermore, molecular markers associated with target traits were used to preliminarily screen the hybrid offspring for superior traits.

[0056] The hybrid offspring were cultivated and managed in the field for 3 years. When the seedlings grew to 3 years old, phenotypic tests were performed, including the following items: (1) Leaf color traits: The leaf color in spring and autumn was measured using the RHS color chart. Individuals whose leaf color in spring was RHS color chart N66A, which changed to RHS 34A in autumn, and whose autumn color lasted for ≥45 days were selected. The autumn color duration was determined by regularly observing and recording the number of days from the beginning of leaf color change to leaf fall.

[0057] (2) Growth traits: Measure annual growth and select individuals with annual growth ≥ 20 cm and a seedling height uniformity coefficient of variation ≤ 15%. Annual growth is determined by measuring the length of branches grown in the current year, and the seedling height uniformity coefficient of variation is calculated through statistical analysis;

[0058] (3) Cold resistance: The cut branches were placed in a low-temperature environment of -15℃ for 24 hours, and the survival rate and electrolyte exudation rate were measured. Individuals with a survival rate ≥90% and a branch electrolyte exudation rate ≤25% after low-temperature stress treatment were selected. The survival rate was determined by observing the proportion of branches that recovered to normal growth, and the electrolyte exudation rate was measured using a conductivity meter;

[0059] (4) Physiological characteristics: The photosynthetic rate of leaves was measured using a photosynthesis meter under the condition of PAR 800 μmol / m² / s. The photosynthetic rate of leaves was selected to be ≥18 μmol CO2 / m 2 / s, pore density ≤120 / mm 2 The stomatal density was determined by microscopic observation and counting;

[0060] Through the above-mentioned phenotypic determination, new hybrid varieties with excellent comprehensive traits are finally screened out, completing the breeding process.

[0061] Example 2 This embodiment provides a method for hybrid breeding of Acer elegans based on differential regulation of flowering period, comprising the following steps: S1: Parent selection and flowering synchronization regulation; Select Acer truncatum varieties with a natural flowering start difference of ≥10 days and a genetic distance ≥0.5 as hybrid parents, with the one that starts flowering earlier as the female parent and the one that starts flowering later as the male parent. In this embodiment, the early-flowering Acer truncatum variety "Red Dance Shoes" was selected as the female parent, and the late-flowering variety "Purple Clouds" was selected as the male parent. Through previous observations, the natural flowering start of "Red Dance Shoes" was mid-March, and the natural flowering start of "Purple Clouds" was late April. The difference in the natural flowering start of the two varieties reached 35 days. Through molecular marker analysis, the genetic distance between the two varieties was 0.62, which met the parent selection conditions.

[0062] The mother plant "Red Shoes" was treated to delay the flowering period. The specific operation was as follows: one-year-old branches with a diameter of ≥0.8 cm were selected and refrigerated in a cold storage room at 2°C and 65% humidity for 35 days. During the cold storage period, the branches were checked once a week to ensure that they did not dry out or mold. After the cold storage, the branches were moved into a greenhouse environment and the greenhouse temperature was controlled at 20°C. In the greenhouse, the mother plant was shaded to ensure that the daily light time was 8 hours, and blue light with a wavelength of 450±10nm and a light intensity of 50μmol / m 2 / s. The blue light treatment used an LED light source, with a daily illumination time of 8 hours, from 8 a.m. to 4 p.m. This series of treatments delayed the flowering period of the mother plant, "Red Shoes," to late April.

[0063] The male parent, Zixia, was treated to advance flowering. Specifically, treatment began 30 days before flower bud differentiation (late February). Red light with a wavelength of 660±15nm was supplemented daily, accounting for 75% of the total light duration, and the light duration was extended to 15 hours. Professional horticultural LED lights were used for red light treatment, with continuous illumination from 6:00 AM to 9:00 PM, for a total light duration of 15 hours. Simultaneously, the male parent was sprayed with an 80ppm gibberellin solution and a flower-promoting nutrient solution once a week for three consecutive weeks. The gibberellin solution was sprayed at a rate of 100mL per plant, evenly applied to the branches. The flower-promoting nutrient solution maintained a pH of 6.0 and a conductivity of 1.8mS / cm, and the root irrigation rate was 500mL per plant per week. The flower-promoting nutrient solution consisted of 0.1% potassium dihydrogen phosphate, 0.05% boric acid, and 0.02% zinc sulfate (by weight). Through this series of treatments, the flowering period of the male parent "Zixia" was advanced to late April;

[0064] After the above-mentioned flowering period regulation treatment, the flowering periods of the female parent "Red Dance Shoes" and the male parent "Purple Clouds" were synchronized, creating conditions for subsequent hybridization and pollination.

[0065] S2: vacuum-assisted artificial pollination; When the pollen from the male parent, Zixia, matures, it is collected using a vacuum suction device. The suction pressure of the vacuum suction device is set to -0.04 MPa, and the suction tip has a diameter of 1 mm. During collection, the suction tip is gently placed in contact with the anther surface, and negative pressure draws the mature pollen into the collection tube. This method reduces pollen viability to 4%, ensuring a successful subsequent pollination.

[0066] Store the collected pollen in a sealed container at a temperature of 4°C and a humidity of 25% for no more than 10 days. Use a sealed glass bottle with a desiccant to store the pollen. Check the pollen viability regularly to ensure that it is no less than 92% of its initial viability during pollination.

[0067] Before pollination, spray the stigmas of the female plant "Red Shoes" with a 15% sucrose solution at a rate of 10 μL per flower. This spraying helps increase the stickiness of the stigmas and the germination rate of pollen. Pollination should be performed at 10:00 AM daily, when the temperature and humidity conditions are optimal for pollen germination. Use a directional nozzle to evenly spray pollen onto the stigmas of the female plant. Pollinate each flower for three consecutive days to increase pollination success.

[0068] After pollination is complete, mark the pollinated flowers and record the pollination date. Harvest the mature hybrid seeds after pollination and fruiting of the female parent, which have gradually matured and changed color. Seeds are harvested approximately 125 days after pollination, when they are fully mature and have a brown seed coat. After washing and drying, the harvested seeds are stored at 4°C in preparation for subsequent seedling cultivation.

[0069] S3: progeny screening; S31: performing stratification treatment and temperature difference control germination on the hybrid seeds obtained in step S2, and carrying out light and temperature control seedling cultivation after sowing; The specific procedure is to mix hybrid seeds with moist vermiculite in a volume ratio of 1:3 and stratify them for 60 days at a gradient temperature of 0-25°C and 80% humidity. Stratification is done in layers, with the bottom layer being moist vermiculite, the middle layer being the seed-vermiculite mixture, and the top layer being moist vermiculite. During the treatment period, humidity levels are checked weekly and water is added as needed to keep the vermiculite moist but not waterlogged.

[0070] After 60 days of stratification, the seeds were sown in seedling trays using a mixture of peat, vermiculite, and perlite (5:3:2 by volume). After sowing, the seeds were germinated and raised under the control of the day and night temperature difference, with the conditions being 25°C during the day and 15°C at night, for a treatment period of 30 days. During the seedling raising period, the substrate was kept moist and the light intensity was controlled at 250 μmol / m 2 / s, and the lighting time is 12 hours / day.

[0071] S32: Use SSR and SNP molecular markers to verify hybrid authenticity and target traits.

[0072] DNA was extracted from seedling leaves and amplified by PCR using 12 pairs of polymorphic SSR primers and 25 SNP marker loci, followed by sequencing analysis to verify the authenticity of the hybrid offspring. Successful hybrids were confirmed by comparing the molecular marker profiles of the hybrid offspring with those of the parents. Furthermore, molecular markers associated with target traits were used to preliminarily screen hybrid offspring for superior traits.

[0073] The hybrid offspring were cultivated and managed in the field for 3 years. When the seedlings grew to 3 years old, phenotypic tests were performed, including the following items: (1) Leaf color traits: The leaf color in spring and autumn was measured using the RHS color chart. Individuals whose leaf color in spring was RHS color chart N66A, which changed to RHS 34A in autumn, and whose autumn color lasted for 48 days were selected. The autumn color duration was determined by regularly observing and recording the number of days from the beginning of leaf color change to leaf fall.

[0074] (2) Growth traits: Measure annual growth and select individuals with an annual growth of 22 cm and a coefficient of variation of seedling height uniformity of 13%. Annual growth is determined by measuring the length of branches grown in the current year, and the coefficient of variation of seedling height uniformity is calculated through statistical analysis;

[0075] (3) Cold resistance: The cut branches were placed in a low-temperature environment of -15°C for 24 hours, and the survival rate and electrolyte exudation rate were measured. Individuals with a survival rate of 92% and a branch electrolyte exudation rate of 23% after low-temperature stress treatment were selected. The survival rate was determined by observing the proportion of branches that recovered to normal growth, and the electrolyte exudation rate was measured using a conductivity meter;

[0076] (4) Physiological characteristics: The photosynthetic rate of leaves was measured using a photosynthesis meter under the conditions of PAR 800 μmol / m² / s. The photosynthetic rate of leaves was selected to be 19 μmol CO2 / m 2 / s, pore density is 115 / mm 2 The stomatal density was determined by microscopic observation and counting;

[0077] Through the above-mentioned phenotypic determination, new hybrid varieties with excellent comprehensive traits are finally screened out, completing the breeding process.

[0078] Example 3 This embodiment provides a method for hybrid breeding of Acer elegans based on differential regulation of flowering period, comprising the following steps: S1: Parent selection and flowering synchronization regulation; Select Acer truncatum varieties with a natural flowering start difference of ≥10 days and a genetic distance ≥0.5 as hybrid parents, with the one that starts flowering earlier serving as the female parent and the one that starts flowering later serving as the male parent. In this example, the early-flowering Acer truncatum variety "Chunxiao" was selected as the female parent, and the late-flowering variety "Jinqiu" was selected as the male parent. Previous observations showed that the natural flowering start of "Chunxiao" is in early March, while the natural flowering start of "Jinqiu" is in mid-April, with a difference of 45 days between the two varieties. Molecular marker analysis also showed a genetic distance of 0.71 between the two varieties, meeting the parent selection criteria.

[0079] The mother plant "Chun Xiao" was treated to delay the flowering period. The specific operation was as follows: one-year-old branches with a diameter of 1.0 cm were selected and refrigerated in a cold storage room at 0°C and 70% humidity for 35 days. During the cold storage period, the branches were checked once a week to ensure that they did not dry out or mold. After the cold storage, the branches were moved to a greenhouse environment and the greenhouse temperature was controlled at 19°C. In the greenhouse, the mother plant was shaded to ensure that the daily light time was 7 hours, and blue light with a wavelength of 450±10nm and a light intensity of 50μmol / m 2 / s. The blue light treatment used an LED light source, with a daily illumination time of 7 hours, from 9:00 AM to 4:00 PM. Through this series of treatments, the flowering period of the mother plant "Chun Xiao" was delayed to mid-April.

[0080] The male parent, "Golden Autumn," was treated to advance flowering. Specifically, treatment began 30 days before flower bud differentiation (mid-February). Red light with a wavelength of 660±15nm was supplemented daily, accounting for 80% of the plant's total light duration, and the light duration was extended to 16 hours. Professional horticultural LED lights were used for red light treatment, with continuous illumination from 6:00 AM to 10:00 PM, for a total light duration of 16 hours. Simultaneously, the male parent was sprayed with an 80ppm gibberellin solution and a flower-promoting nutrient solution once a week for three consecutive weeks. The gibberellin solution was sprayed at a rate of 100mL per plant, evenly applied to the branches. The flower-promoting nutrient solution maintained a pH of 5.8 and a conductivity of 1.5mS / cm, and the root irrigation rate was 500mL per plant per week. The flower-promoting nutrient solution consisted of 0.1% potassium dihydrogen phosphate, 0.05% boric acid, and 0.02% zinc sulfate (by weight). Through this series of treatments, the flowering period of the male parent "Golden Autumn" was advanced to mid-April;

[0081] After the above-mentioned flowering period regulation treatment, the flowering periods of the female parent "Chunxiao" and the male parent "Jinqiu" were synchronized, creating conditions for subsequent hybridization and pollination.

[0082] S2: vacuum-assisted artificial pollination; When the pollen of the male parent, "Golden Autumn," matures, it is collected using a vacuum suction device. The suction pressure of the vacuum suction device is set to -0.06 MPa, and the suction tip has a diameter of 0.8 mm. During collection, the suction tip is gently placed in contact with the anther surface, and the negative pressure draws the mature pollen into the collection tube. This method reduces pollen viability to 3%, ensuring a successful subsequent pollination.

[0083] Store the collected pollen in a sealed container at a temperature of 4°C and a humidity of 20% for no more than 12 days. Use a sealed glass bottle with a desiccant to store the pollen. Check the pollen viability regularly to ensure that it is no less than 95% of its initial viability during pollination.

[0084] Before pollination, spray the stigmas of the female plant "Chunxiao" with a 15% sucrose solution at a rate of 10 μL per flower. This spraying helps increase stigma stickiness and pollen germination. Pollination should be performed at 9:00 AM daily, when temperature and humidity conditions are optimal for pollen germination. Use a directional nozzle to evenly spray pollen onto the stigma of the female plant. Pollinate each flower for three consecutive days to increase pollination success.

[0085] After pollination is complete, mark the pollinated flowers and record the pollination date. Harvest the mature hybrid seeds after pollination and fruiting of the female parent, which have gradually matured and changed color. Seeds are harvested approximately 130 days after pollination, when they are fully mature and have a brown seed coat. After washing and drying, the harvested seeds are stored at 4°C in preparation for subsequent seedling cultivation.

[0086] S3: progeny screening; S31: performing stratification treatment and temperature difference control germination on the hybrid seeds obtained in step S2, and carrying out light and temperature control seedling cultivation after sowing; The specific procedure is to mix hybrid seeds with moist vermiculite in a volume ratio of 1:3 and stratify them for 60 days at a gradient temperature of 0-25°C and 80% humidity. Stratification is done in layers, with the bottom layer being moist vermiculite, the middle layer being the seed-vermiculite mixture, and the top layer being moist vermiculite. During the treatment period, humidity levels are checked weekly and water is added as needed to keep the vermiculite moist but not waterlogged.

[0087] After 60 days of stratification, the seeds were sown in seedling trays using a mixture of peat, vermiculite, and perlite (5:3:2 by volume). After sowing, the seeds were germinated and raised under the control of the day and night temperature difference, with the conditions being 25°C during the day and 15°C at night, for a treatment period of 30 days. During the seedling raising period, the substrate was kept moist and the light intensity was controlled at 280 μmol / m 2 / s, the light duration is 12 hours / day;

[0088] S32: Use SSR and SNP molecular markers to verify hybrid authenticity and target traits.

[0089] DNA was extracted from seedling leaves and amplified by PCR using 15 pairs of polymorphic SSR primers and 30 SNP marker loci, followed by sequencing analysis to verify the authenticity of the hybrid offspring. Successful hybrids were confirmed by comparing the molecular marker profiles of the hybrid offspring with those of the parents. Furthermore, molecular markers associated with target traits were used to preliminarily screen the hybrid offspring for superior traits.

[0090] The hybrid offspring were cultivated and managed in the field for 3 years. When the seedlings grew to 3 years old, phenotypic tests were performed, including the following items: (1) Leaf color traits: The leaf color of spring and autumn was measured using the RHS color chart. Individuals whose leaf color in spring was RHS color chart N66A, which changed to RHS 34A in autumn, and whose autumn color lasted for 52 days were selected. The autumn color duration was determined by regularly observing and recording the number of days from the beginning of leaf color change to leaf fall.

[0091] (2) Growth traits: Measure annual growth and select individuals with an annual growth of 25 cm and a coefficient of variation of seedling height uniformity of 10%. Annual growth is determined by measuring the length of branches grown in the current year, and the coefficient of variation of seedling height uniformity is calculated through statistical analysis.

[0092] (3) Cold resistance: The cut branches were placed in a low-temperature environment of -15℃ for 24 hours, and the survival rate and electrolyte exudation rate were measured. Individuals with a survival rate of 95% and a branch electrolyte exudation rate of 20% after low-temperature stress treatment were selected. The survival rate was determined by observing the proportion of branches that recovered normal growth, and the electrolyte exudation rate was measured using a conductivity meter.

[0093] (4) Physiological characteristics: The photosynthetic rate of leaves was measured using a photosynthesis meter under the conditions of PAR 800 μmol / m² / s. The photosynthetic rate of leaves was selected to be 21 μmol CO2 / m 2 / s, pore density is 105 / mm 2 The stomatal density was determined by counting the stomata under a microscope.

[0094] Through the above-mentioned phenotypic determination, new hybrid varieties with excellent comprehensive traits are finally screened out, completing the breeding process.

[0095] Example 4 This embodiment provides a method for hybrid breeding of Acer elegans based on differential regulation of flowering period, comprising the following steps: S1: Parent selection and flowering synchronization regulation Select Acer truncatum varieties with a natural flowering period difference of ≥10 days and a genetic distance of ≥0.5 as hybrid parents, among which the one that starts flowering earlier is the female parent, and the one that starts flowering later is the male parent. In this embodiment, the early-flowering Acer truncatum variety "Chenlu" is selected as the female parent, and the late-flowering variety "Muxue" is selected as the male parent. Through previous observations, the natural flowering period of "Chenlu" is March 5, and the natural flowering period of "Muxue" is March 15. The difference in the natural flowering period of the two is 10 days, and through molecular marker analysis, the genetic distance between the two varieties is 0.58, which meets the parent selection conditions;

[0096] Treatment for delayed flowering of female parent: Select one-year-old branches with a diameter of ≥0.8cm and refrigerate them in a cold storage room at 0-4℃ and 65% humidity for 15 days. During the cold storage period, check the condition of the branches once a week to ensure that they do not dry out or mold. After the cold storage, move the branches into a greenhouse environment and control the greenhouse temperature at 18-22℃. In the greenhouse, shade the mother plant so that the daily light time is ≤8 hours, and supplement with blue light with a wavelength of 450±10nm and a light intensity of 50μmol / m 2 / s. The blue light treatment used an LED light source, with a daily illumination time of 8 hours, from 8:00 AM to 4:00 PM. This series of treatments delayed the flowering period of the mother plant "Morning Dew" from March 5 to March 12 (a delay of 7 days).

[0097] Treatment of male parent’s flowering period in advance: The treatment started 20 days before the flower bud differentiation of the male parent (i.e. February 25). Red light with a wavelength of 660±15nm (red light accounts for 70%) was supplemented daily, and the lighting time was extended to 14 hours. Professional horticultural LED lights were used for red light treatment, and continuous irradiation was performed from 6 am to 8 pm. At the same time, the male parent was sprayed with 60ppm gibberellin solution (lower than 80ppm in Example 1, adapted to smaller regulation needs) and flower-promoting nutrient solution every week, and the treatment was carried out once a week for 2 consecutive weeks. The pH value of the flower-promoting nutrient solution was controlled between 5.8-6.2, the conductivity was ≤2.0mS / cm, the weekly root irrigation volume was 300mL / plant (reduced dosage), and the composition of the flower-promoting nutrient solution was: 0.1% potassium dihydrogen phosphate, 0.05% boric acid and 0.02% zinc sulfate (mass ratio). Through this series of treatments, the flowering period of the male parent "Muxue" was advanced from March 15 to March 13 (2 days in advance);

[0098] Synchronization results: The first flowering date after the female parent's regulation was March 12; the first flowering date after the male parent's regulation was March 13; the difference in artificial flowering period was 1 day (far ≤ 5 days, meeting the pollination requirements).

[0099] S2: Vacuum-assisted artificial pollination When the pollen of the male parent, "Muxue," matures, it is collected using a vacuum negative pressure adsorption device. The adsorption pressure of the vacuum negative pressure adsorption device is set to -0.05±0.01MPa, and the adsorption head diameter is 1±0.5mm. During the collection process, the adsorption head is gently touched to the surface of the anther, and the negative pressure draws the mature pollen into the collection tube. Using this method to collect pollen, the pollen activity loss rate is ≤5%, ensuring the success rate of subsequent pollination.

[0100] Store the collected pollen in a sealed container at 4°C and ≤30% humidity for less than 7 days (shorten the storage period to accommodate the smaller control window). Use sealed glass bottles with desiccant to store pollen. Check pollen viability regularly to ensure that pollen viability is no less than 90% of its initial viability during pollination.

[0101] Before pollination, spray the stigmas of the "Morning Dew" female parent with a 15% sucrose solution at a rate of 10 μL per flower. Pollination should be performed between 9:00 and 11:00 daily. Use a directional nozzle to evenly spray pollen onto the female parent's stigma. Pollinate each flower for two consecutive days (reducing the number of repetitions). After pollination, mark the pollinated flowers and record the pollination date. Harvest mature hybrid seeds approximately 120 days after pollination, when the seeds are fully mature and have a brown seed coat. After washing and air-drying, store the harvested seeds at 4°C.

[0102] S3: Offspring screening S31: Stratification and temperature-controlled germination Hybrid seeds were mixed with moist vermiculite in a volume ratio of 1:3 and stratified for 40 days at a gradient temperature of 0-25°C and 80% humidity. The stratification process used a layered method, with moist vermiculite as the bottom layer, the seed-vermiculite mixture in the middle layer, and moist vermiculite as the top layer. During the treatment period, the humidity was checked weekly and water was added as needed to keep the vermiculite moist but not waterlogged.

[0103] After stratification, the seeds were sown in seedling trays using a mixture of peat, vermiculite, and perlite (volume ratio 5:3:2). After sowing, the seeds were germinated and seedlings were grown under the control of the day and night temperature difference, with the temperature at 25°C during the day and 15°C at night for 30 days. During the seedling cultivation period, the substrate was kept moist and the light intensity was controlled at 200-300 μmol / m 2 / s, and the lighting time is 12 hours / day.

[0104] S32: Molecular marker validation and phenotyping DNA was extracted from seedling leaves and amplified by PCR using eight pairs of polymorphic SSR primers and 15 SNP marker loci, followed by sequencing analysis to verify the authenticity of the hybrid offspring. Successful hybrids were confirmed by comparing the molecular marker profiles of the hybrid offspring with those of the parents. Furthermore, molecular markers associated with target traits were used to preliminarily screen hybrid offspring for superior traits.

[0105] The hybrid offspring were cultivated and managed in the field for 3 years. When the seedlings grew to 3 years old, phenotypic tests were performed, including the following items: Leaf color traits: The leaf color in spring is RHS color card N66A, which changes to RHS 34A in autumn, and the autumn color lasts for 50 days (better than the 45 days of the male parent "Muxue").

[0106] Growth traits: Annual growth is 18cm (reaching 90% of the ≥20cm standard, retaining potential individuals), and the coefficient of variation of seedling height uniformity is 12%.

[0107] Cold resistance: After 24 hours of low temperature stress treatment at -15℃, the survival rate was 88% and the electrolyte exudation rate of the branches was 22%.

[0108] Through the above-mentioned phenotypic determination, new hybrid varieties with excellent comprehensive traits are finally screened out, completing the breeding process.

[0109] Physiological characteristics: Leaf photosynthetic rate is 19μmol CO2 / m 2 / s, pore density is 110 / mm 2 .

[0110] It should be noted that Example 1, Example 2, Example 3, and Example 4 are all a method for hybrid breeding of Acer truncatum based on differential regulation of flowering period.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

[0112] In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "in the present embodiment", "specific example", or "some examples" means that the specific features, mechanisms, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, mechanisms, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

[0113] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A method for hybrid breeding of Acer elegans based on differential regulation of flowering period, characterized in that: The following steps are involved: S1. Parent selection and flowering synchronization regulation: Select Acer elegans varieties with a natural flowering time difference of ≥10 days and a genetic distance of ≥0.5 as hybrid parents, with the one with earlier flowering as the female parent and the one with later flowering as the male parent; The female parent is subjected to a treatment for delaying the flowering period, including low-temperature refrigeration and shading and supplementing blue light; the male parent is subjected to a treatment for advancing the flowering period, including extending the light period, supplementing red light, and spraying hormones and nutrient solutions; S2. Vacuum-assisted pollination: Use a vacuum negative pressure adsorption device to collect pollen from the male parent and store it in a sealed container. Spray a sucrose solution on the female parent's stigma before pollination. Repeat pollination is performed using a directional nozzle between 9:00 and 11:00 daily. Once the female parent is fully pollinated and fruiting, harvest the mature hybrid seeds. S3. Progeny screening: S31: performing stratification treatment and temperature-differential temperature control germination on the hybrid seeds obtained in step S2, and carrying out light and temperature-controlled seedling cultivation after sowing; S32: Use SSR and SNP molecular markers to verify the authenticity of hybridization and target traits; perform phenotypic determination on 3-year-old seedlings to complete breeding.

2. The method for hybrid breeding of Acer elegans based on differential regulation of flowering period according to claim 1, characterized in that: In step S1, the female parent is an early-flowering Acer truncatum variety "Coral Court", "Red Shoes", "Spring Dawn" or "Morning Dew", and the male parent is a late-flowering variety "Red Feather Clothes", "Purple Clouds", "Golden Autumn" or "Muxue".

3. The hybrid breeding method of Acer elegans based on differential regulation of flowering period according to claim 1, characterized in that: In step S1, the conditions for delaying the flowering period of the mother plant are as follows: 1-year-old branches with a diameter of ≥0.8 cm are refrigerated at 0-4°C and a humidity of 60-70% for 35 days and then moved into a greenhouse, and shaded to a light intensity of ≤8 hours per day, while supplemented with blue light of 450±10nm wavelength and a light intensity of 50μmol / m 2 / s.

4. The method for hybrid breeding of Acer elegans based on differential regulation of flowering period according to claim 1, characterized in that: In step S1, the conditions for delaying the flowering period of the male parent are as follows: 30 days before the flower bud differentiation of the male parent, red light with a wavelength of 660±15nm is supplemented daily, and the proportion of red light is ≥70%, the illumination time is extended to 14-16 hours, and 80ppm gibberellin and flower-promoting nutrient solution are sprayed simultaneously, and the treatment is carried out once a week for 3 consecutive weeks.

5. The hybrid breeding method of Acer elegans based on differential regulation of flowering period according to claim 4, characterized in that: In step S1, the pH value of the flower-promoting nutrient solution is 5.8-6.2, the conductivity is ≤2.0 mS / cm, and the weekly root irrigation amount of the flower-promoting nutrient solution is 500 mL / plant; Calculated by mass ratio, the nutrient solution includes: 0.1% potassium dihydrogen phosphate, 0.05% boric acid and 0.02% zinc sulfate.

6. The method for hybrid breeding of Acer elegans based on differential regulation of flowering period according to claim 1, characterized in that: In step S2, the conditions for the vacuum negative pressure adsorption device to collect the pollen of the father parent are: the adsorption pressure of the vacuum negative pressure adsorption device is -0.05±0.01MPa, the adsorption head diameter of the vacuum negative pressure adsorption device is 1±0.5mm, and the pollen activity loss rate is ≤5% during the process of collecting the pollen of the father parent by the vacuum negative pressure adsorption device.

7. The method for hybrid breeding of Acer elegans based on differential regulation of flowering period according to claim 1, characterized in that: In step S2, the conditions for spraying the sucrose solution are: spraying 15% sucrose solution on the stigma of the female parent before pollination, with a spraying amount of 10 μL / flower.

8. The method for hybrid breeding of Acer elegans based on differential regulation of flowering period according to claim 1, characterized in that: In step S2, the pollen storage conditions are as follows: the collected pollen is stored in a sealed container at a temperature of 4° C. and a humidity of ≤30% for a storage time of ≤15 days.

9. The method for hybrid breeding of Acer elegans based on differential regulation of flowering period according to claim 1, characterized in that: The step S31 comprises: mixing the hybrid seeds with moist vermiculite in a volume ratio of 1:3, stratifying the seeds under conditions of a gradient temperature of 0-25°C and a humidity of 80% for 60 days before sowing, and germinating and raising seedlings by controlling the temperature difference between day and night, with the conditions being 25°C during the day and 15°C at night, and the treatment cycle being 30 days.

10. The method for hybrid breeding of Acer elegans based on differential regulation of flowering period according to claim 1, characterized in that: In step S32, the phenotypic determination includes the following items: (1) Leaf color: The leaf color in spring is RHS color chart N66A, and changes to RHS 34A in autumn, and the autumn color lasts for ≥45 days; (2) Growth traits: annual growth ≥ 20 cm, coefficient of variation of seedling height uniformity ≤ 15%; (3) Cold resistance: survival rate ≥90% after 24 hours of low temperature stress treatment at -15℃, and electrolyte leakage rate of branches ≤25%; (4) Physiological characteristics: Leaf photosynthetic rate ≥ 18 μmol CO2 / m 2 / s, the measurement condition was PAR 800μmol / m 2 / s, pore density ≤ 120 / mm 2 .