Method for improving fruiting of male sterile lagerstroemia speciosa 'Yunnan'

Abstract

The method comprises the following steps: selecting a male sterile lagerstroemia speciosa 'Yunnan' plant which grows robustly and is free from plant diseases and insect pests as a female parent, and screening stigmas with strong receptivity through a benzidine-hydrogen peroxide method; spraying a composite solution containing chitosan, trehalose and gibberellin to enhance pollen adhesion and germination; male fertile male parent pollen is collected and subjected to low-temperature activation and sucrose-boric acid suspension treatment, and the pollen vitality can be improved by 80% or above; bagging protection is carried out after pollination is continuously carried out for 3 days, seed dormancy is synergistically broken through the vitality agent solution, and light and nitrogen regulation is carried out in the seedling stage. According to the technology, the technical bottleneck that male sterile lagerstroemia speciosa 'Yunnan' hardly bears fruits is overcome, the fruit setting rate is remarkably increased to 38.5%, the germination rate is increased to 13.2%, powerful support is provided for genetic improvement and variety innovation of the male sterile lagerstroemia speciosa 'Yunnan', and new hope and possibility are brought to the field of ornamental plant breeding.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for improving the fruit setting of male sterile Lagerstroemia speciosa 'Yunshang'.

Background Art

[0002] Lagerstroemia speciosa plays an important role in the field of landscape gardening, and its variety improvement and breeding techniques have always been the focus of research.

[0003] Among them, Lagerstroemia indica 'Yunchang' is a new double-flowered variety of Lagerstroemia speciosa discovered by Beijing Forestry University and Guangxi Forestry Research Institute through the selection of natural double-flowered mutants. It obtained the national new plant variety right in December 2020 (variety right number: 20200154).

[0004] Lagerstroemia indica 'Yunchang' is an arbor. The plant growth habit is semi-erect, the trunk bark is gray, and the four edges of the small branches are not obvious. The leaves are large, oval, dark green, hairless on both sides, the leaf margin has no undulation, and there are 9-13 pairs of lateral veins. The terminal panicle is axillary, and the flower axis, pedicel and outside of the calyx are all covered with a small amount of purplish-red bran-like dense felt hairs; the flower bud is spherical, purple, the suture line protrudes, and the top has a protrusion; the calyx is long, the lobes are triangular, reflexed, hairless inside, with obvious appendages in the shape of scales; the flower is double-flowered, the flower is violet (RHS N81B), there are 5-6 petals, 2.5-3.5 cm long, the petal margin is obviously wrinkled, there is a thick purple-red petal claw, about 0.5 cm long; there are hundreds of stamens, all anthers are petaloid, about 2.5 cm long, the petal claws are filamentous, about 1 cm long; the style is atrophied, about 5 mm long; the flowering period is from June to August. The ovary is spherical and atrophied, with a diameter of about 0.7 cm, the ovary wall is thick and hairless; it hardly bears fruit.

[0005] This variety is suitable for planting in tropical and south subtropical regions such as Fujian, Guangdong, and Guangxi in China for urban greening and courtyard cultivation, etc. Due to the male sterility of the plant, it is advisable to use grafting or cutting methods for propagation.

[0006] The existing Lagerstroemia speciosa cross-breeding technology faces multiple bottlenecks. The most significant one is the male sterility problem. As a male sterile variety, Lagerstroemia indica 'Yunchang' has a very low fruit setting rate, which directly limits its application in cross-breeding. Traditional cross-breeding methods rely on natural pollination, but male sterility leads to the inability to form effective pollen, and thus the normal fertilization process cannot be carried out. In addition, existing propagation methods such as grafting or cutting, although they can maintain the excellent characteristics of the variety, cannot fundamentally solve the problem of low fruit setting rate. Therefore, exploring a method that can improve the fruit setting rate of male sterile Lagerstroemia indica 'Yunchang' is of great significance for promoting the development of Lagerstroemia speciosa cross-breeding technology.

[0007] The prior art has deficiencies in aspects such as pollen viability detection, precise determination of stigma receptivity, and standardization of post-pollination protection measures, resulting in large fluctuations in hybridization success rates and low breeding efficiency. Although the "Distant Hybridization Breeding Method for Lagerstroemia excelsa" with the patent publication number CN118542233A has promoted the development of Lagerstroemia hybridization breeding to a certain extent, there are still problems of low seed setting rates in practical applications. This method solves the problem that the pollen tube cannot elongate normally in the style after pollination between Lagerstroemia indica and Lagerstroemia excelsa and cannot complete fertilization by applying a specific stigma coating agent before the stigma of the female parent secretes mucus. However, it has deficiencies in pollen viability detection, precise determination of stigma receptivity, and standardization of post-pollination protection measures, resulting in large fluctuations in hybridization success rates and low breeding efficiency, making it difficult for the seed setting rate after hybridization to reach the expected level.

[0008] In summary, there are many limitations in the propagation and breeding technologies of Lagerstroemia speciosa and male-sterile Lagerstroemia speciosa, which severely restrict the popularization and application of excellent varieties of male-sterile Lagerstroemia speciosa. Therefore, carrying out research on innovative breeding technologies for Lagerstroemia speciosa, especially exploring efficient and reliable hybridization breeding technologies for male-sterile Lagerstroemia speciosa, has great theoretical significance and practical application value for solving the current problems and meeting the market demand for male-sterile Lagerstroemia speciosa.

[0009] The disclosure of the above background technical content is only used to assist in understanding the inventive concept and technical solution of the present invention, and it does not necessarily belong to the prior art of this patent application. Without clear evidence indicating that the above content was publicly available on the filing date of this patent application, the above background technology should not be used to evaluate the novelty and inventiveness of this application.

Summary of the Invention

[0010] The object of the present invention is to propose a method for improving the seed setting of male-sterile Lagerstroemia speciosa 'Yunshang', aiming to stimulate the development of female organs and improve the seed setting rate through artificial assisted pollination combined with treatment with specific plant growth regulators.

[0011] To this end, the present invention adopts the following technical solutions:

[0012] A method for improving the seed setting of male-sterile Lagerstroemia speciosa 'Yunshang' includes the following steps:

[0013] (1) Selecting the female parent: Selecting a male-sterile Lagerstroemia speciosa 'Yunshang' plant with strong growth and no diseases and pests as the female parent;

[0014] (2) Detection of stigma receptivity: Using the benzidine-hydrogen peroxide method to detect the stigma receptivity of the stigma of the initially blooming female parent, and selecting the stigma with strong receptivity for subsequent pollination;

[0015] (3) Pollen collection and pretreatment: Collect the pollen of male-fertile Lagerstroemia indica with excellent breeding target traits. Collect the pollen at 8:00 - 12:00 in the morning on the day when the male flowers bloom. Immediately place the pollen at 4°C for low-temperature activation and store it for 24 hours. Subsequently, use a 10% sucrose + 50 mg / L boric acid solution for suspension treatment and detect the viability;

[0016] (4) Select the male parent: Select male-fertile Lagerstroemia indica that has passed the viability detection in step (3) and whose viability needs to reach or exceed 80% as the male parent;

[0017] (5) Hybrid pollination: When the petals of the female parent are fully unfolded and the stigma of the pistil with strong receptivity after the stigma receptivity detection in step (2) reaches full maturity, start the pollination operation;

[0018] (6) Sowing and strengthening in the seedling stage: After harvesting the mature fruits, remove the pericarp to obtain F1-generation seeds and weigh their thousand-grain weight; then, treat the seeds with a vigor agent solution; finally, sow the treated seeds in peat soil with a water content maintained at 80% - 90%; Implement an alternating treatment of 16 hours of light / 8 hours of darkness in the seedling stage, cycle daily for 30 days; The nitrogen supply fluctuates by ±30% according to the standard amount and is adjusted every 10 days to enhance stress resistance.

[0019] Further, the specific steps of the stigma receptivity detection in step (2) are as follows: Drop the prepared 0.5% benzidine-hydrogen peroxide solution on a grooved slide, collect the stigma of the newly opened flower of the female parent and immerse it in the solution. After 5 minutes, observe under a microscope. If the reaction solution turns blue and a large number of bubbles are generated, it indicates that the stigma has strong receptivity; if only a small number of bubbles are generated, the receptivity of the stigma is weak; if no bubbles are generated, it indicates that the stigma does not have receptivity.

[0020] Further, the specific steps of the viability detection in step (3) are as follows: Take a clean slide, use a dropper to suck a small amount of the prepared triphenyltetrazolium chloride solution and drop one drop in the center of the slide; Use a pollen brush to dip a small amount from the treated pollen and sprinkle it into the drop of triphenyltetrazolium chloride solution on the slide, then cover the cover glass; Place the prepared slide in a petri dish, incubate at 35 - 38°C for 15 - 30 minutes, and then use a microscope for microscopic examination. Count the pollen stained red or pink as viable pollen.

[0021] Further, the concentration of the triphenyltetrazolium chloride solution used in the viability detection in step (3) is 0.5 - 1.0%.

[0022] Further, for the selection of the male parent in step (4), one of Lagerstroemia speciosa (light red), Lagerstroemia speciosa (purple), Lagerstroemia indica 'Red Rocket', Lagerstroemia indica 'Velvet', Lagerstroemia balansae, Lagerstroemia tomentosa, and Lagerstroemia siamica can be selected.

[0023] Furthermore, the specific steps of the pollination operation in step (5) are as follows: When the petals of the female parent are fully unfolded and the pistil stigmas are fully mature after the stigma receptivity test in step (2), start the pollination operation; conduct precise pollination for 3 consecutive days. Before pollination, spray a chitosan composite solution on the stigmas of the female parent to form a bioadhesive film. Dip a sterilized soft brush into the suspended male parent pollen, and the dipping amount is that the pollen is evenly attached to the tip of the brush hair. Then evenly apply the male parent pollen on the stigmas of the female parent pistils; the chitosan composite solution contains 0.04 - 0.06% chitosan, 0.008 - 0.012% trehalose, and 0.008 - 0.012% gibberellin solution.

[0024] Furthermore, the specific time for the three - day pollination in step (5) is carried out during the period with the strongest stigma receptivity from 10:00 - 12:00 every morning in the first three days after the female parent blossoms; the pollination environment is controlled at a temperature of 28 ± 2°C, sunny days, and a humidity ≤ 75%.

[0025] Furthermore, during the whole process of the pollination operation in step (5), regularly check whether the plants have pests and diseases. Set up yellow sticky traps and blue insect traps around in advance to monitor and trap pests. Once pests and diseases are found, select appropriate biological control or low - toxicity chemical control methods according to the specific types of pests and diseases.

[0026] Furthermore, for the flowers after pollination in step (5), after each pollination, a waterproof sulfuric acid paper bag with a titanium dioxide nano - coating on the inner layer needs to be put on. The size of the paper bag can completely cover the flower and part of the flower stalk; when the ovary begins to swell, remove the waterproof sulfuric acid paper bag and replace it with a nylon mesh bag with a light transmittance ≥ 80% and a pore diameter of 0.8 mm.

[0027] Furthermore, the specific steps of treating the seeds with a vigor agent solution in step (6) are as follows: Prepare a vigor agent solution by adding 1 μM brassinolide to a 48 - 52 mg / L gibberellin solution, soak the seeds in the vigor agent solution for 11 - 13 h, and then rinse them with water 3 - 5 times.

[0028] The technical principles and beneficial effects of each step of the present invention are as follows:

[0029] I. Technical principles

[0030] 1. Selection of female parent: The male-sterile Lagerstroemia speciosa 'Yunshang' is selected as the female parent. Since it cannot produce functional pollen on its own, it can avoid self-pollination, thus ensuring that the genetic source of the hybrid offspring mainly comes from the male parent and realizing gene recombination and the introduction of excellent traits. Selecting a plant with strong growth and no pests and diseases as the female parent is because such a female parent has good physiological status and growth ability, can provide sufficient nutrition and a suitable growth environment for the development of the hybrid offspring, and is conducive to improving the success rate of cross-breeding and the quality of the offspring.

[0031] 2. Detection of stigma receptivity: The benzidine-hydrogen peroxide method is based on the chemical reaction between stigma secretions and benzidine-hydrogen peroxide solution. When the stigma has strong receptivity, its secretions can quickly react with the solution to produce a large number of bubbles and be accompanied by blue color. This is because the stigma secretions contain active substances such as peroxidase, which catalyze the decomposition of hydrogen peroxide to produce oxygen, forming bubbles, and at the same time undergo a color reaction with benzidine to present blue color. Through this method, the ability of the stigma to receive pollen and complete fertilization can be quickly and simply evaluated. Conducting stigma receptivity detection can not only reflect the metabolic activity of stigma cells, but also determine the number of receptors on the stigma surface, enzyme activity, and the physiological status of cells. For a stigma that produces a large number of bubbles and is accompanied by blue color, its cell metabolism is vigorous, the number of receptors is large and the activity is high, which can better recognize and bind pollen, promote pollen germination and pollen tube growth, and provide more favorable conditions for the fertilization process. This grading detection method can more accurately select stigmas with strong receptivity and ensure the pollination effect.

[0032] 3. Pollen collection and pretreatment: In the cross-breeding work of male-sterile Lagerstroemia speciosa, pollen viability detection is a crucial step. Pollen is collected from 8:00 to 12:00 in the morning on the day when male flowers are in bloom. At this time, the pollen maturity is relatively appropriate. Immediately after pollen collection, the pollen is placed at 4°C for low-temperature activation and stored for 24 hours because the low-temperature environment can slow down the physiological metabolism rate of pollen, maintain the viability of pollen, and prepare for the subsequent pollination process. The pollen is suspended in a 10% sucrose + 50 mg / L boric acid solution. Sucrose provides carbon source and energy for pollen, maintaining the physiological activity of pollen; boric acid has a promoting effect on pollen germination, promoting the germination and growth of pollen tubes. This pretreatment method can significantly improve the viability of pollen. High-quality pollen has a high germination rate and can more effectively complete the fertilization process, thus improving the pollination success rate and seed setting rate. Part of the pollen is subjected to viability detection. The principle of the triphenyltetrazolium chloride (TTC) staining method used for detection is to utilize the characteristics of TTC. TTC is a colorless oxidized dye. When it comes into contact with viable pollen cells, the dehydrogenase in the living cells will catalyze the dehydrogenation of the substrates in the cells, and the generated hydrogen will reduce the colorless TTC to red, insoluble triphenylformazan. However, due to the cessation or weakening of the metabolic activities of non-viable pollen cells, the dehydrogenase loses its activity and cannot reduce TTC, so it will not be stained or will be stained very lightly. By observing the staining situation of the pollen, the viability of the pollen can be judged, and then the proportion of viable pollen can be counted to evaluate the quality and fertilization ability of the pollen.

[0033] 4. Selection of male parent: Select male-fertile Lagerstroemia as the male parent with excellent breeding target traits (such as flower color, flower shape, flowering period, etc.). It is based on the principle of cross-breeding. Through the gene recombination of the male parent and the female parent, the excellent traits of the male parent are transmitted to the hybrid offspring.

[0034] 5. Hybrid Pollination: Before pollination, the stigma of the female parent is sprayed with a chitosan composite solution. Chitosan can form a protective film to fix pollen, inhibit microbial contamination, and reduce the stigma infection rate. Trehalose helps maintain cell stability. Gibberellin can promote the elongation of heterologous pollen tubes, increase the fertilization rate and fruit set rate. The three work together. The chitosan protective film reduces external interference and allows gibberellin to play a better role; trehalose enhances the sensitivity of cells to gibberellin, and gibberellin enables the pollen tube to quickly utilize the fixation advantage of chitosan and accelerate fertilization. When the stigma is mature, the pollen of the male parent is applied. The pollen germinates into a pollen tube smoothly with the help of the composite solution to complete fertilization and produce hybrid seeds. The specific time for pollination for three days is from 10:00 to 12:00 every morning in the first three days after the female parent blooms when the stigma receptivity is the strongest; the pollination environment is controlled at a temperature of 28±2°C, sunny and humidity ≤75%, because avoiding the high-temperature period at noon is beneficial to the germination and growth of pollen tubes, can maintain the vitality of the stigma and pollen, and too high or too low humidity will have an adverse impact on pollination; pollination is carried out once on the same stigma for three consecutive days to increase the chance of contact between pollen and stigma and improve the fruit set rate of pollination. Setting yellow sticky traps and blue insect traps to monitor and trap pests and adopting biological control or low-toxic chemical control methods are to prevent pests and diseases from damaging the pollination process and flowers and ensure the smooth progress of hybrid pollination. Covering with a waterproof sulfuric acid paper bag with a titanium dioxide nanocoating on the inner layer is to prevent pollen contamination and water loss and create a relatively stable microenvironment for the development of pollinated flowers and ovaries. The titanium dioxide nanocoating catalyzes the decomposition of ethylene under light, which can delay stigma senescence and extend the effective pollination period; when the ovary swells, it is replaced with a nylon mesh bag with a light transmittance ≥80% and a pore diameter of 0.8 mm, which can not only ensure normal gas exchange and growth space for the fruit but also prevent the fruit from being damaged by external mechanical damage and pests and diseases. These pollination protection measures comprehensively increase the fruit set rate to 38.5%.

[0035] 6. Enhancement during sowing and seedling stage: After harvesting mature fruits, remove the pericarp to obtain F1 generation seeds and weigh the 1000-seed weight, which can help understand the plumpness and quality of the seeds and provide reference for subsequent sowing rate and management. Immerse the seeds in the vigor agent solution for 11 - 13 h. The vigor agent solution is prepared by adding brassinolide to gibberellin solution, taking advantage of the synergistic effect of gibberellin and brassinolide (BR). Gibberellin can regulate the physiological and metabolic processes inside the seeds, such as promoting the synthesis of hydrolytic enzymes like α-amylase, accelerating the decomposition of stored substances in the seeds, and providing energy and material basis for seed germination; brassinolide further breaks the deep dormancy of seeds by activating the cell elongation pathway and acting synergistically with gibberellin. Rinsing the seeds with water is to remove the residual vigor agent solution on the seed surface and avoid its adverse effects on seed germination and seedling growth. Sow the treated seeds in peat soil with appropriate water content (80% - 90%). Peat soil has good water retention and air permeability, providing a good germination and growth substrate for the seeds. Implement an alternating treatment of 16 h of light / 8 h of darkness during the seedling stage, cycling daily for 30 d, simulating the natural light cycle, regulating the photosynthesis and other physiological processes of the seedlings, and promoting the normal growth and development of the seedlings. The nitrogen supply fluctuates by ±30% according to the standard amount and is adjusted every 10 days, enabling the seedlings to be exercised under different nitrogen levels, enhancing their adaptability to environmental changes, and improving stress resistance.

[0036] II. Beneficial effects

[0037] 1. Improve the hybridization purity and success rate: Select the male-sterile Lagerstroemia speciosa 'Yunshang' as the female parent to completely prevent self-pollination from the source, making the genes of the hybrid offspring mainly derived from the male parent, which effectively guarantees the high purity of hybridization. Use the benzidine-hydrogen peroxide method for accurate stigma receptivity detection, and at the same time, cooperate with strict pollen collection and pretreatment processes (such as low-temperature activation, suspension in a specific solution, and viability detection, etc.) to screen for high-viability pollen. Coupled with reasonable pollination operations, including precisely pollinating at the strongest stigma receptivity period for 3 consecutive days, spraying chitosan composite solution before pollination, strict pest and disease control, and scientific bagging protection and other coordinated steps, significantly improve the fruit setting rate of hybridization and greatly increase the probability of obtaining hybrid offspring with target excellent traits.

[0038] 2. Achieve trait improvement and innovation: According to breeding requirements, flexibly select male-fertile Lagerstroemia as the male parent with different excellent breeding target traits, such as the flower color of Lagerstroemia speciosa (light red) and the plant type and flower color of Lagerstroemia indica 'Red Rocket', etc. Through hybridization, introduce these excellent traits into the female parent to achieve the improvement and innovation of Lagerstroemia speciosa varieties in aspects such as flower color and plant type, meeting diverse ornamental and application needs.

[0039] 3. Standardized operation and strong controllability: Each step of the entire technical solution has detailed and clear operation methods and technical parameters, such as the concentration of 0.5% benzidine-hydrogen peroxide solution in the stigma receptivity test; the specific amount of pollen dipped in the pollination operation, the pollination frequency for 3 consecutive days, etc. The entire hybrid breeding process is standardized and easy to master, and each link is highly controllable, which can effectively reduce the risk of failure caused by improper operation and improve the stability and repeatability of breeding work.

[0040] 4. Comprehensive guarantee of breeding quality: From the careful selection of the mother plant, the scientific collection and strict testing of pollen, the precise implementation of pollination operations, the proper handling and sowing of seeds, to the intensive management of the seedling stage, each step cooperates with each other and is closely linked to form a complete technical system to fully guarantee the quality of hybrid breeding. Through the meticulous control of each link, the role of each factor can be maximized to ensure that the new large-flowered crape myrtle varieties finally cultivated have excellent performance in growth potential, ornamental traits, etc., and have high market value and application prospects.

Brief Description of the Drawings

[0041] Figure 1 This is a picture of the hybrid F1 fruit obtained in Example 1 of the present invention;

[0042] Figure 2 This is a diagram of a mature fruit of the hybrid F1 generation of Example 1 of the present invention;

[0043] Figure 3 This is a hybrid F1 seedling diagram of Example 1 of the present invention;

[0044] Figure 4 This is a hybrid F1 seedling diagram of Example 3 of the present invention. [Specific implementation method]

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0047] In an embodiment of the present invention, a method for improving the fruit set of male sterile Lagerstroemia indica 'Yunshang' is adopted, comprising the following steps:

[0048] (1) Selection of female parent: Select a male-sterile Lagerstroemia speciosa 'Yunshang' plant with strong growth and no pests or diseases as the female parent;

[0049] (2) Detection of stigma receptivity: Use the benzidine-hydrogen peroxide method to detect the stigma receptivity of the stigma of the initially blooming female parent, and select the stigma with strong receptivity to participate in subsequent pollination;

[0050] (3) Pollen collection and pretreatment: Collect the pollen of male-fertile Lagerstroemia with excellent breeding target traits. Collect the pollen at 8:00-12:00 in the morning on the day when the male flowers bloom. Immediately place the pollen at 4°C for low-temperature activation and store it for 24 hours. Then, suspend and treat it with a 10% sucrose and 50 mg / L boric acid solution and detect its viability;

[0051] (4) Selection of male parent: Select a male-fertile Lagerstroemia that has passed the viability test in step (3) and whose viability needs to reach or exceed 80% as the male parent;

[0052] (5) Cross-pollination: When the petals of the female parent are fully unfolded and the stigma of the pistil that is stronger after the stigma receptivity test in step (2) reaches full maturity, start the pollination operation;

[0053] (6) Sowing and strengthening in the seedling stage: After harvesting the mature fruits, remove the pericarp to obtain F1 generation seeds and weigh their 1000-grain weight; then, treat the seeds with a vigor agent solution; finally, sow the treated seeds in peat soil with a water content maintained at 80%-90%; implement an alternating treatment of 16 hours of light / 8 hours of darkness in the seedling stage, cycle daily for 30 days; the nitrogen supply fluctuates by ±30% according to the standard amount and is adjusted every 10 days to enhance stress resistance.

[0054] The specific steps of the stigma receptivity detection in step (2) are as follows: Drop the prepared 0.5% benzidine-hydrogen peroxide solution on a grooved slide, collect the stigma of the initially blooming female parent and immerse it in the solution. After 5 minutes, observe under a microscope. If the reaction solution turns blue and a large number of bubbles are generated, it indicates that the stigma has strong receptivity; if only a small number of bubbles are generated, the receptivity of the stigma is weak; if no bubbles are generated, it indicates that the stigma does not have receptivity.

[0055] The specific steps of the viability detection in step (3) are as follows: Take a clean slide, use a dropper to suck a small amount of the prepared triphenyl tetrazolium chloride solution and drop one drop in the center of the slide; use a pollen brush to dip a small amount of the treated pollen and sprinkle it into the drop of triphenyl tetrazolium chloride solution on the slide, then cover the cover slip; place the prepared slide in a petri dish, incubate it at 35-38°C for 15-30 minutes, and then use a microscope for microscopic examination. Count the pollen stained red or pink as viable pollen.

[0056] The concentration of the triphenyltetrazolium chloride solution used in the viability detection of step (3) is 0.5 - 1.0%.

[0057] For the selection of the male parent in step (4), one of Lagerstroemia speciosa (light red), Lagerstroemia speciosa (purple), Lagerstroemia indica 'Red Rocket', Lagerstroemia indica 'Velvet', Lagerstroemia balansae, Lagerstroemia tomentosa, and Lagerstroemia siamica can be selected.

[0058] The specific steps of the pollination operation in step (5) are as follows: When the petals of the female parent are fully unfolded and the pistil stigmas with strong receptivity after the stigma receptivity detection in step (2) reach full maturity, start the pollination operation; conduct precise pollination for 3 consecutive days. Before pollination, spray the chitosan composite solution on the stigma of the female parent to form a biological adhesion film. Dip a sterilized soft brush into the suspended male parent pollen, and the dipping amount is that the tip of the brush is evenly attached with pollen. Then evenly apply the male parent pollen on the pistil stigma of the female parent; the chitosan composite solution contains 0.04 - 0.06% chitosan, 0.008 - 0.012% trehalose, and 0.008 - 0.012% gibberellin solution.

[0059] The specific time for the three - day pollination in step (5) is carried out during the period with the strongest stigma receptivity from 10:00 - 12:00 every morning in the first three days after the female parent blossoms; the pollination environment is controlled at a temperature of 28 ± 2°C, sunny, and humidity ≤ 75%.

[0060] During the whole process of the pollination operation in step (5), regularly check whether the plants have pests and diseases. Set up yellow sticky traps and blue insect traps around in advance to monitor and trap pests. Once pests and diseases are found, select appropriate biological control or low - toxicity chemical control methods according to the specific types of pests and diseases.

[0061] For the flowers after pollination in step (5), after each pollination, a waterproof sulfuric acid paper bag with a titanium dioxide nano - coating on the inner layer needs to be put on. The size of the paper bag can completely cover the flower and part of the flower stalk; when the ovary begins to swell, remove the waterproof sulfuric acid paper bag and replace it with a nylon mesh bag with a light transmittance ≥ 80% and a pore diameter of 0.8 mm.

[0062] The specific steps of treating the seeds with the vigor agent solution in step (6) are as follows: Prepare a vigor agent solution by adding 1 μM brassinolide to a 48 - 52 mg / L gibberellin solution, soak the seeds in the vigor agent solution for 11 - 13 h, and then rinse them with water 3 - 5 times.

[0063] The following is further illustrated by more specific examples.

[0064] Example 1

[0065] A method for improving the seed setting of male - sterile Lagerstroemia speciosa 'Yunshang' includes the following steps:

[0066] (1) Select the female parent: Select a male-sterile Lagerstroemia speciosa 'Yunshang' plant with strong growth and no pests and diseases as the female parent.

[0067] (2) Detection of stigma receptivity: Use the benzidine-hydrogen peroxide method to detect the stigma receptivity of the stigma of the initially blooming female parent. Drop the prepared 0.5% benzidine-hydrogen peroxide solution on a grooved slide, collect the stigma of the initially blooming female parent and immerse it in the solution. After 5 minutes, observe under a microscope. If the reaction solution turns blue and a large number of bubbles are generated, it indicates that the stigma has strong receptivity; if only a small number of bubbles are generated, the receptivity of the stigma is weak; if no bubbles are generated, it indicates that the stigma does not have receptivity. (3) Pollen collection and pretreatment: Collect the pollen of male-fertile Lagerstroemia indica with excellent breeding target traits. Collect the pollen at 10 am on the day when the male flowers are in bloom, immediately place the pollen at 4°C for low-temperature activation and store it for 24 hours; then suspend the pollen after low-temperature activation with a 10% sucrose + 50 mg / L boric acid solution. Finally, select a part of the treated pollen for viability detection; take a clean slide, use a dropper to suck a small amount of the prepared triphenyltetrazolium chloride solution and drop a drop in the center of the slide; use a pollen brush to dip a small amount from the treated pollen and sprinkle it into the drop of triphenyltetrazolium chloride solution on the slide, then cover the cover slip; place the prepared slide in a petri dish, incubate it at 38°C for 22 minutes and then examine it under a microscope. Count the pollen stained red or pink as viable pollen. The concentration of the triphenyltetrazolium chloride solution used is 0.5%.

[0068] (4) Select the male parent: Select a male-fertile Lagerstroemia speciosa (light red) with a viability of 85% after the viability detection in step (3) as the male parent.

[0069] (5) Hybrid pollination: When the petals of the female parent are fully unfolded and the pistil stigmas are strongly receptive and fully mature after the stigma receptivity detection in step (2), the pollination operation begins; before pollination, the stigmas of the female parent are sprayed with a chitosan composite solution to form a biological adhesion film, where the chitosan composite solution contains 0.05% chitosan, 0.01% trehalose, and 0.01% gibberellin solution; a sterilized soft brush is used to dip into the suspended pollen of the male parent, and the dipping amount is that the tip of the brush is evenly attached with pollen, and the pollen of the male parent is evenly smeared on the stigmas of the female parent pistils. Ensure that the specific time for pollination for three days is at 10:00 am every day in the first three days after the female parent blooms. Pollinate the same stigma once on sunny days when the temperature is 26 - 30°C and the humidity is below 75%, and continue for three days. During the entire pollination operation process, regularly check whether the plants have pests and diseases, and set up yellow sticky traps and blue insect traps around in advance to monitor and trap pests. Once pests and diseases are found, select appropriate biological control or low-toxic chemical control methods according to the specific types of pests and diseases. For the pollinated flowers, after each pollination, they need to be covered with a waterproof sulfuric acid paper bag with a titanium dioxide nano-coating on the inner layer, and the size of the paper bag can completely cover the flower and part of the flower stalk; when the ovary begins to swell, remove the waterproof sulfuric acid paper bag and replace it with a nylon mesh bag with a light transmittance ≥80% and a pore size of 0.8 mm.

[0070] (6) Sowing and strengthening in the seedling stage: After harvesting the mature fruits, remove the fruit skins to obtain F1 generation seeds, and weigh their thousand-grain weight; then, soak the seeds in a vigor agent solution prepared from 50 mg / L gibberellin and 1 μM brassinolide for 12 h, and then rinse with water 4 times; finally, sow the treated seeds in peat soil with a water content maintained at 85%. Implement an alternating treatment of 16 h of light / 8 h of darkness in the seedling stage, cycle daily for 30 d; the nitrogen supply fluctuates by ±30% according to the standard amount and is adjusted every 10 days to enhance stress resistance. The figure of the F1 generation fruit obtained is shown in Figure 1 ; The figure of the mature fruit of the F1 generation is shown in Figure 2 ; The figure of the hybrid F1 generation seedlings is shown in Figure 3 .

[0071] Example 2

[0072] It is basically the same as Example 1, except that Lagerstroemia speciosa (purple) is selected as the male parent.

[0073] Example 3

[0074] It is basically the same as Example 1, except that Lagerstroemia indica 'Red Rocket' is selected as the male parent. The figure of the hybrid F1 generation seedlings is shown in Figure 4 .

[0075] Example 4

[0076] It is basically the same as Example 1, except that Lagerstroemia indica 'Velvet' is selected as the male parent.

[0077] Example 5

[0078] It is basically the same as Example 1, except that Lagerstroemia balansae is selected as the male parent.

[0079] Example 6

[0080] It is basically the same as Example 1, except that Lagerstroemia tomentosa is selected as the male parent.

[0081] Example 7

[0082] It is basically the same as Example 1, except that Lagerstroemia siamica is selected as the male parent.

[0083] Comparative Example 1

[0084] It is basically the same as Example 1, except that during pollen collection and pretreatment in step (3), the pollen is not activated at low temperature and the pollen is not suspended in a 10% sucrose + 50 mg / L boric acid solution.

[0085] Comparative Example 2

[0086] It is basically the same as Example 1, except that in step (5) of cross-pollination, the stigma of the female parent is not sprayed with the chitosan composite solution before pollination.

[0087] Comparative Example 3

[0088] It is basically the same as Example 1, except that in step (6) of sowing, the seeds are soaked in a 50 mg / L gibberellin solution without adding brassinolide.

[0089] Comparative Example 4

[0090] It is basically the same as Example 1, except that during pollen collection and pretreatment in step (3), the pollen is not activated at low temperature and the pollen is not suspended in a 10% sucrose + 50 mg / L boric acid solution; in step (5) of cross-pollination, the stigma of the female parent is not sprayed with the chitosan composite solution before pollination. In step (6) of sowing, the seeds are soaked in a 50 mg / L gibberellin solution without adding brassinolide.

[0091] Single-factor experiment of chitosan composite solutions with different concentrations:

[0092] To explore the effect of different concentrations of chitosan composite solution on the seed setting rate when Lagerstroemia speciosa (light red) is used as the male parent, the following experiment was designed and carried out: Fix the male parent as Lagerstroemia speciosa (light red), and conduct the experiment using a method for improving the seed setting of male sterile Lagerstroemia speciosa 'Yunshang' described in Example 1. The only difference is that in step (5) of cross-pollination, different concentrations of chitosan composite solution are sprayed on the stigma of the female parent before pollination for cross-pollination experiments, and the other steps are the same as those in Example 1. The specific experimental data are shown in Table 1 below:

[0093] Table 1 Results of cross-experiments with different concentrations of chitosan composite solution when Lagerstroemia speciosa (light red) is used as the male parent

[0094]

[0095] Analyzing the data, it can be seen from the above experimental data that in the cross-experiment with Lagerstroemia speciosa (light red) as the male parent, when the concentration of the chitosan composite solution is 0.05% chitosan, 0.01% trehalose, and 0.01% gibberellin solution, all indicators perform the best, the seed setting rate is as high as 38.5%, the number of seeds per single fruit is 162, the thousand-grain weight is 8.3 g, and the germination rate is 13.2%. When the concentration is lower or higher than this standard, indicators such as the seed setting rate and the number of seeds per single fruit all decrease. When the concentration is too low, the functions of the chitosan composite solution in fixing pollen, inhibiting microbial contamination, and promoting pollen tube elongation are weakened; when the concentration is too high, it may have a certain inhibitory effect on the stigma and pollen, affecting the normal germination and fertilization process of pollen, and thus reducing the seed setting rate. Therefore, it is determined that the concentration of 0.05% chitosan + 0.01% trehalose + 0.01% gibberellin solution is the appropriate concentration of the chitosan composite solution.

[0096] The seed setting and germination of the fruits produced in Examples 1-6 and Comparative Examples 1-4 were counted, and the data are shown in Table 2.

[0097] Table 2 Seed setting and germination of cross combinations in Examples 1-6 and Comparative Examples 1-4

[0098]

[0099] Through the data comparison of Examples 1-6, after hybridization with different male parents (Lagerstroemia speciosa (light red), Lagerstroemia speciosa (purple), Red Rocket Lagerstroemia, 'Velvet' Lagerstroemia, Lagerstroemia balansae, Lagerstroemia tomentosa, Lagerstroemia siamica), the indicators are significantly different. In Example 1, when Lagerstroemia speciosa (light red) is used as the male parent, the seed setting rate is the highest, reaching 38.5%, and the number of seeds per single fruit is the largest, reaching 162. This shows that the genetic characteristics of the male parent have a significant impact on the seed setting and seed conditions of the hybrid offspring. Different gene combinations are produced by the hybridization of different male parents and female parents, resulting in different hybridization effects, verifying the importance of selecting a suitable male parent for achieving trait improvement and innovation, and the excellent traits of different male parents can be introduced into the female parent by selection.

[0100] In Example 1, the pollen was activated at low temperature and suspended in a 10% sucrose + 50 mg / L boric acid solution. In Comparative Example 1, these treatments were not carried out. The transverse diameter of the fruit in Comparative Example 1 was 19.81 mm and the longitudinal diameter was 19.50 mm, which were smaller than those in Example 1. The number of seeds per fruit was 152, the fruit setting rate was 25.7%, and the germination rate was 9.5%, which were much lower than those in Example 1. Low-temperature activation can maintain pollen viability, and suspension in a specific solution is beneficial for maintaining pollen activity. Without treatment, the pollen viability decreases significantly, the fertilization success rate is significantly reduced, affecting fruit development and seed formation, resulting in worse indicators such as the fruit setting rate. Therefore, standardized pollen collection and pretreatment operations can effectively maintain pollen viability, significantly improve the fertilization success rate, and thus increase the fruit setting rate.

[0101] In Comparative Example 2, the chitosan composite solution was not sprayed before pollination, and the fruit setting rate of 30.3% was lower than that in Example 1. The chitosan composite solution contains 0.05% chitosan, 0.01% trehalose, and 0.01% gibberellin solution, which is sprayed on the stigma surface before pollination and can form a protective film. This protective film can not only fix the pollen, make the pollen attach to the stigma more stably, increase the contact opportunity between the pollen and the stigma, but also inhibit microbial contamination, reduce the stigma infection rate, and create a good environment for pollen germination. At the same time, the gibberellin in the solution can promote the elongation of heterologous pollen tubes, shorten the fertilization time, improve the effective fertilization rate, make the pollen tubes more likely to reach the ovules to complete fertilization, greatly increasing the chance of successful fertilization, and thus increasing the fruit setting rate. If the chitosan composite solution is not sprayed, the attachment of the pollen will be affected, the risk of stigma infection will increase, and the lack of gibberellin will also limit the elongation of the pollen tubes, reducing the fertilization efficiency. This will interfere with the normal germination of the pollen and the growth of the pollen tubes, ultimately affecting the pollination effect and resulting in a decrease in the fruit setting rate. Therefore, spraying the chitosan composite solution before pollination is crucial for improving the pollination success rate and the fruit setting rate.

[0102] In Comparative Example 3, brassinolide was lacking in the seed soaking, and only the gibberellin solution was used, and the germination rate of 8.9% was lower than that in Example 1. The synergistic effect of brassinolide and gibberellin can break the deep dormancy of seeds and promote seed germination. The lack of brassinolide inhibits seed germination, indicating that their synergy is crucial for increasing the seed germination rate. A high germination rate is an important prerequisite for ensuring the fruit setting rate. Only when the seeds germinate smoothly can the normal growth and fruiting of the plants be further ensured.

[0103] Influence of the lack of comprehensive factors: Comparative Example 4 incorporated issues such as non-low-temperature activation of pollen, suspension in a specific solution, non-spraying of chitosan composite solution before pollination, and lack of brassinolide in seed soaking. The transverse diameter of its fruit was 19.78 mm, the longitudinal diameter was 19.45 mm, the number of seeds per single fruit was 148, the seed setting rate was 22.2%, and the germination rate was 8.7%, all of which were the lowest. The lack or improper treatment of multiple factors seriously affected all aspects of hybridization, from pollen viability, the pollination process to seed germination, resulting in extremely poor hybridization effects.

[0104] In this cross-breeding technical solution, the selection of the male parent is one of the key factors for achieving trait improvement and innovation. Different male parents have a significant impact on the seed setting and seed-related indicators of the hybrid offspring. The operations in the stigma treatment, pollen collection, and pre-treatment links are also crucial for the hybridization results. Stigma treatment, low-temperature activation of pollen, and suspension in a specific solution play key roles in promoting pollen tube elongation and maintaining pollen viability respectively. The lack of these operations will reduce the fertilization success rate and seed quality, affecting the hybridization effect. The synergistic effect of multiple factors is indispensable for the success of cross-breeding. Due to the lack or improper treatment of multiple key factors in Comparative Example 4, the hybridization effect is far inferior to that of Example 1, indicating that all links cooperate with each other to jointly ensure the success rate of cross-breeding and the quality of offspring. The entire technical solution gives full play to the advantages of each link through fine control of each link and the synergistic effect of multiple factors, effectively improving the fertilization success rate and seed quality, thus significantly enhancing the seed setting rate of Lagerstroemia speciosa cross-breeding, and can effectively improve the purity, success rate of Lagerstroemia speciosa cross-breeding, as well as the germination and growth of offspring, with high scientificity and practicality.

[0105] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for improving the fruiting of male sterile Lagerstroemia indica 'Yunshang', characterized in that: The following steps are involved: (1) Selecting the female parent: Select a healthy, disease-free, and insect-free male-sterile Lagerstroemia speciosa ‘Yunshang’ plant as the female parent; (2) Stigma receptivity test: The stigma receptivity of the first flowering stigma of the female plant was tested using the benzidine-hydrogen peroxide method, and the stigma with stronger receptivity was selected as the basis for subsequent pollination; (3) Pollen collection and pretreatment: Collect male fertile crape myrtle pollen with excellent breeding target traits from 8:00 to 12:00 in the morning when the male flowers are open, immediately place the pollen at 4°C for low-temperature activation and store for 24 h, then suspend it in 10% sucrose + 50 mg / L boric acid solution and test its vitality; (4) Selecting the male parent: selecting a male fertile crape myrtle plant that has been tested for vitality in step (3) and whose vitality reaches or exceeds 80% as the male parent; (5) Cross pollination: When the petals of the female parent are fully unfolded and the stigma of the stronger pistil reaches full maturity after the stigma receptivity test in step (2), the pollination operation begins; (6) Strengthening during sowing and seedling stages: After harvesting mature fruits, the peels must be removed to obtain F1 seeds, and their thousand-grain weight must be weighed; then, these seeds are placed in a prepared activator solution for treatment; finally, the treated seeds are sown in peat soil with a moisture content of 80%-90%; during the seedling stage, an alternating treatment of 16 h of light / 8 h of darkness is implemented, with a daily cycle for 30 days; the nitrogen supply fluctuates by ±30% of the standard amount and is adjusted every 10 days to enhance stress resistance.

2. A method for improving the fruit set of male sterile Lagerstroemia indica 'Yunshang' according to claim 1, characterized in that: The specific steps of the step (2) of detecting the receptivity of the stigma are as follows: dropping the prepared 0.5% benzidine-hydrogen peroxide solution on the groove slide, collecting the stigma of the first flowering of the mother plant and immersing it in the solution, and observing it under a microscope after 5 minutes. If the reaction solution turns blue and is accompanied by a large number of bubbles, it means that the stigma has strong receptivity; if only a small number of bubbles are generated, the receptivity of the stigma is weak; if no bubbles are generated, it means that the stigma does not have receptivity.

3. The method for improving the fruit set of male sterile Lagerstroemia indica 'Yunshang' according to claim 1, characterized in that: The specific steps of the activity detection in step (3) are as follows: take a clean glass slide, use a dropper to absorb a small amount of prepared triphenyltetrazolium chloride solution, and drop a drop on the center of the glass slide; use a pollination pen to dip a small amount of processed pollen, sprinkle it on the triphenyltetrazolium chloride solution drop on the glass slide, and cover it with a cover glass; place the prepared glass slide in a culture dish, keep it warm at 35-38°C for 15-30 minutes, and then use a microscope to examine it, and count the pollen that is dyed red or pink, which is viable pollen.

4. The method for improving the fruit set of male sterile Lagerstroemia indica 'Yunshang' according to claim 1, characterized in that: The concentration of the triphenyltetrazolium chloride solution used in the activity detection in step (3) is 0.5-1.0%.

5. The method for improving the fruit set of male sterile Lagerstroemia indica 'Yunshang' according to claim 1, characterized in that: In the step (4), the male parent may be selected from one of Lagerstroemia indica (light red), Lagerstroemia indica (purple), 'Red Rocket' Lagerstroemia, 'Velvet' Lagerstroemia, Lagerstroemia indica, Lagerstroemia indica and Lagerstroemia indica.

6. The method for improving the fruit set of male sterile Lagerstroemia indica 'Yunshang' according to claim 1, characterized in that: The pollination operation of step (5) specifically comprises the following steps: when the petals of the female parent are fully unfolded and the stronger pistil stigma reaches full maturity after the stigma fecundity detection in step (2), the pollination operation is started; precise pollination is carried out for three consecutive days, and before pollination, a chitosan composite solution is sprayed on the female parent stigma to form a bioadhesive film, and the male parent pollen treated with suspension is dipped with a sterilized soft brush, the amount of pollen dipped is such that the tip of the brush is evenly attached to the pollen, and the male parent pollen is evenly applied to the female parent pistil stigma; wherein the chitosan composite solution contains 0.04-0.06% chitosan, 0.008-0.012% trehalose and 0.008-0.012% gibberellin solution.

7. A method for improving the fruit set of male sterile Lagerstroemia indica 'Yunshang' according to claim 6, characterized in that: The specific time of the three-day pollination in step (5) is to carry out the pollination during the first three days after the female parent blooms, from 10:00 to 12:00 every morning when the stigma is most pollinable; the pollination environment is controlled to be 28±2°C, sunny and ≤75% in humidity.

8. The method for improving the fruit set of male sterile Lagerstroemia indica 'Yunshang' according to claim 1, characterized in that: During the entire pollination process of step (5), the plants are regularly checked for the presence of pests and diseases, and yellow sticky insect boards and blue insect traps are set in advance around the plants to monitor and trap pests. Once pests and diseases are found, appropriate biological control or low-toxic chemical control methods are selected according to the specific types of pests and diseases.

9. The method for improving the fruit set of male sterile Lagerstroemia indica 'Yunshang' according to claim 1, characterized in that: After each pollination, the flowers pollinated in step (5) are covered with a waterproof sulfuric acid paper bag with an inner layer coated with a titanium dioxide nano-coating, and the size of the paper bag can completely cover the flowers and part of the pedicels; when the ovary begins to swell, the waterproof sulfuric acid paper bag is removed and replaced with a nylon mesh bag with a light transmittance of ≥80% and a pore size of 0.8 mm.

10. The method for improving the fruit set of male sterile Lagerstroemia indica 'Yunshang' according to claim 1, characterized in that: The specific steps of treating the seeds with the activator solution in step (6) are: adding 1 μM brassinolide to a 48-52 mg / L gibberellin solution to prepare an activator solution, soaking the seeds in the activator solution for 11-13 hours, and then washing with water for 3-5 times.

Images