Distant hybridization cultivation method of hexaploid common wheat disease-resistant and stress-resistant variety

Abstract

The invention discloses a distant hybridization cultivation method of a hexaploid common wheat disease-resistant and stress-resistant variety. The method comprises the following steps: parent screening and determination, cross pollination and fruiting promotion, hybrid immature embryo rescue and test-tube plantlet cultivation, molecular marker-assisted screening, and homozygous stability and comprehensive character identification. The invention relates to the technical field of agricultural crop breeding. The distant hybridization cultivation method of the hexaploid common wheat disease-resistant and stress-resistant variety reaches the parent screening standard, hybridization and embryo rescue parameters, a molecular marker screening system and the like are clear and controllable, and the distant hybridization cultivation method can be popularized to other wheat distant hybridization breeding scenes; a standardized technical normal form is provided for improving wheat characters by using excellent genes of related species such as rye, and the genetic basis and the resource utilization range of wheat breeding are widened.

Description

Technical Field

[0001] This invention relates to the field of agricultural crop breeding technology, specifically to a method for distant hybridization breeding of a hexaploid common wheat variety that is resistant to disease and stress. Background Technology

[0002] Wheat is one of my country's most important food crops, playing a crucial role in ensuring food security, promoting agricultural production, and supporting national economic development. With the upgrading of the flour processing industry and the diversification of market demand, high-quality, strong-gluten wheat, suitable for processing high-end products such as bread and noodles, has become an important direction in wheat breeding. Currently, developed countries focus on breeding high-quality bread wheat, continuously improving variety quality to consolidate their competitive advantage in the international high-quality wheat trade. However, my country's breeding of high-quality, strong-gluten wheat faces many bottlenecks. Existing varieties generally suffer from weak disease and stress resistance, with frequent occurrences of winter frost damage, late spring frosts, and diseases such as powdery mildew, stripe rust, and sheath blight, severely impacting the high and stable yield potential of varieties. Simultaneously, the scarcity of high-quality, strong-gluten wheat resources and the narrow genetic base of existing varieties further restrict breakthroughs in breeding work, making it difficult to simultaneously achieve the three core objectives of "high quality, multiple resistances, and high yield."

[0003] Distant hybridization is an effective breeding approach to broaden the genetic base of wheat and introduce superior genes from closely related species. Rye, as an important crop of the wheat tribe in the Poaceae family, carries a rich set of superior genes for disease resistance, cold resistance, and stress tolerance, making it a valuable resource for improving the stress resistance traits of common wheat. However, traditional distant hybridization breeding techniques between wheat and rye have many limitations, including low hybridization seed setting rate, easy abortion of hybrid embryos, and unstable chromosome doubling success rate, leading to lengthy breeding cycles. At the same time, the lack of precise target gene screening methods makes it difficult to efficiently track superior genes from rye, and trait segregation or loss of superior traits is likely to occur. This makes it extremely difficult to breed new wheat varieties with both high-quality, strong gluten and multiple resistance characteristics through distant hybridization, requiring optimization of the breeding technology system to overcome these bottlenecks. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: a method for breeding hexaploid common wheat disease-resistant and stress-resistant varieties through distant hybridization, comprising the following steps: Step 1: Parental selection and determination: Select a high-quality, strong-gluten common wheat variety with stable agronomic traits and a clear genetic background as the female parent. The female parent should have a test weight ≥780g / L, wet gluten content ≥32%, and stability time ≥10min, making it suitable for winter wheat cultivation in northern regions. Select a diploid rye with chromosome set RR as the male parent. The male parent rye should be highly resistant to powdery mildew, stripe rust, and sheath blight, and have a cold resistance level of 2 or higher, as well as strong resistance to late spring frosts. Step 2, Hybridization and Fruit Set Promotion: After artificial emasculation of the female parent, immediately isolate it by bagging. When the pistils enter the full bloom stage, collect fresh mature pollen from the male parent for uniform artificial pollination. After pollination, spray the female parent wheat ears with a mixed solution of auxin and gibberellin at a mass concentration of 25-35 mg / L, and bag again to retain moisture. The temperature of the pollination and subsequent culture environment should be controlled at 19-23℃, and the relative humidity should be maintained at 65-75%. Step 3, Hybrid Embryo Rescue and Test-tube Seedling Culture: 15-17 days after pollination, dissect the maternal wheat ear to separate the hybrid embryos, select robust spherical to heart-shaped embryos, and inoculate them into modified MS medium. First, culture in the dark at 22-24℃ for 12-15 days, then transfer to a light intensity of 280-320 μmol / m². -2 *s -1 Continue culturing in a constant temperature environment with a light duration of 13-15 h / d for 12-14 days to obtain hybrid F1 test-tube seedlings with uniform growth. The modified MS medium was supplemented with 0.6-1.2 mg / L of 6-benzylaminopurine, 0.52 mg / L of naphthaleneacetic acid, and 2.0 g / L of activated carbon. Step 4: Homozygous stability and comprehensive trait identification: The selected superior single plants are self-pollinated for 4-5 generations to achieve homozygous hybridization. Each generation is self-pollinated with bags on single plants and agronomic traits are recorded. At the same time, field identification of disease resistance by artificial inoculation, identification of cold resistance and stress tolerance, and quality testing are carried out. Trait segregating plants and inferior plants are eliminated to finally obtain a high-quality, strong, disease-resistant, stress-resistant, and high-yielding hexaploid common wheat variety.

[0005] Preferably, the artificial emasculation in step 2 is performed by emasculation. Pollination is completed within 24-36 hours after emasculation. During the pollination process, a sterile brush is used to apply pollen to avoid contamination by foreign pollen.

[0006] Preferably, in step 3, the modified MS medium has a sucrose concentration of 30-35 g / L, an agar concentration of 6.5-7.5 g / L, a pH value adjusted to 5.7-6.0, and a constant temperature of 23±1℃ maintained during both the dark and light culture stages.

[0007] Preferably, the special seedling substrate in step 4 is made by mixing leaf mold, decomposed straw, river sand and perlite in a volume ratio of 4:2:2:1. Before transplanting, add 60-80g of decomposed cake fertilizer to each pot as base fertilizer and maintain the substrate moisture content at 60-65%.

[0008] Preferably, in step 5, the backcrossing process adopts a single-plant-to-single-plant backcrossing mode, and each generation selects a single maternal plant with compact plant type and uniform ear layer as the backcross parent to improve the stability of agronomic traits in the backcross offspring.

[0009] Preferably, in step 5, the SSR molecular markers are selected from the Xgwm series and Xwmc series markers that are closely linked to the rye disease resistance gene, and the SNP molecular markers are selected from functional gene loci related to cold resistance and tolerance to late spring frost. Only single plants that are positive for the marker detection and meet the agronomic traits are allowed to enter the next round of backcrossing.

[0010] Preferably, in step 6, the field disease resistance assessment uses a gradient concentration pathogen inoculation method, with the powdery mildew inoculation spore concentration being 8 × 10⁻⁶. 5 -1.2×10 6 1 spore / mL. For stripe rust, spray inoculation was used, and for sheath blight, sclerotinia soil inoculation was used. The disease incidence was investigated 25 days after inoculation according to national standards, and the severity was determined.

[0011] The national standard mentioned refers to the "National Wheat Variety Approval Standard (2024 Revision)" (implemented on January 1, 2025).

[0012] Preferably, in step 6, the cold resistance and stress tolerance assessment is performed using artificially simulated low-temperature stress. During the overwintering period, the plant is treated with simulated low temperatures of -14 to -16℃ for 24-30 hours, and during the late spring frost, the plant is treated with simulated low temperatures of 0-2℃ for 48 hours. After treatment, the plant survival rate and fruit setting rate are investigated. Individual plants with a survival rate ≥90% and a fruit setting rate ≥85% are included in subsequent screening.

[0013] This invention provides a method for breeding hexaploid common wheat varieties with disease and stress resistance through distant hybridization. It has the following beneficial effects: (I) The distant hybridization breeding method of this hexaploid common wheat disease-resistant and stress-resistant variety involves screening a high-quality strong gluten wheat female parent and a highly resistant diploid rye male parent for distant hybridization. This breaks through the bottleneck of "difficulty in achieving both high quality and disease and stress resistance" in traditional wheat breeding. The resulting wheat variety retains the high-quality strong gluten characteristics of the female parent (≥780g / L test weight, ≥32% wet gluten content, ≥10min stability time) and integrates the male parent's high resistance to powdery mildew, stripe rust, and sheath blight, as well as its cold resistance of level 2 or above and strong resistance to late spring frost, filling the resource gap of high-quality strong gluten and multi-resistant wheat varieties.

[0014] (II) The distant hybridization breeding method of this hexaploid common wheat disease-resistant and stress-resistant variety, through targeted optimization of hybridization pollination and embryo rescue technology, spraying a specific concentration of auxin-gibberellin mixed solution after pollination, combined with modified MS medium (fixed concentration of 0.52 mg / L naphthaleneacetic acid, with added activated carbon) and temperature and light regulation, effectively solves the problems of low seed setting rate and easy embryo abortion in distant hybridization.

[0015] (III) The distant hybridization breeding method of this hexaploid common wheat disease-resistant and stress-resistant variety uses SSR and SNP molecular marker joint detection technology to accurately track the target genes of the paternal parent for disease resistance and stress resistance. Combined with the "single plant to single plant" backcross mode and multiple generations of self-pollination homozygosity, the probability of trait segregation is greatly reduced, ensuring the stable inheritance of the target gene homozygosity. At the same time, through standardized field identification (referring to national approval standards) and agronomic trait screening, the agronomic traits of the variety are made closer to those of the high-quality maternal parent, and the yield and adaptability are improved simultaneously.

[0016] (iv) The distant hybridization breeding method of this hexaploid common wheat disease-resistant and stress-resistant variety, through the standardization of technical details such as artificial emasculation, substrate ratio, and low temperature stress identification in the breeding process, can be directly applied to wheat breeding practices in major producing areas such as the northern winter wheat region and the Huang-Huai wheat region; the bred variety has the characteristics of high quality, strong gluten, multiple resistance, and high yield, which can not only meet the needs of flour processing enterprises for high-quality raw materials, but also resist the threat of winter freezing, late spring cold and various diseases in the producing areas, thereby improving the stability and economic benefits of wheat production.

[0017] (v) The method for breeding disease-resistant and stress-resistant hexaploid common wheat through distant hybridization: The parental screening criteria, hybridization and embryo rescue parameters, molecular marker screening system, etc. of this invention are all clear and controllable, and can be extended to other wheat distant hybridization breeding scenarios. It provides a standardized technical paradigm for improving wheat traits by utilizing the superior genes of closely related species such as rye, and broadens the genetic basis and resource utilization scope of wheat breeding. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] A distant hybridization breeding method for a hexaploid common wheat variety resistant to disease and stress was carried out in experimental fields in the northern winter wheat region (36°-38°N, altitude 50-100m, annual average temperature 12-14℃) for 8 years. The entire process followed standardized breeding procedures, and all reagents used were commercially available analytical grade. The instruments and equipment were conventional breeding and molecular biology detection equipment (including constant temperature incubator, near-infrared grain analyzer, agarose gel electrophoresis system, gene sequencer, etc.).

[0020] The method includes the following steps: Step 1: Parental selection and identification; Maternal parent screening: From high-quality strong gluten wheat resources in the northern winter wheat region, a hexaploid common wheat line with stable agronomic traits and clear genetic background was screened. After testing, the test weight of this line was 792 g / L, the wet gluten content was 34.5%, the plant type was compact, the ear layer was uniform, and the maturity period was consistent. It was suitable for cultivation in the northern winter wheat region and was determined to be the hybrid maternal parent.

[0021] Male parent selection: Diploid rye lines with chromosome set RR were selected. After field disease resistance identification and stress resistance evaluation, the lines were found to be highly resistant to powdery mildew, stripe rust, and sheath blight (all disease severity level 1, as determined by the "National Wheat Variety Approval Standard (2024 Revision)"). They also showed cold resistance level 2, and the seed setting rate remained above 88% under late spring frost stress. They were determined to be the male parent for hybridization.

[0022] Step 2: Hybrid pollination and fruit set promotion; Artificial emasculation: After the female wheat head emerges and before flowering (before the anthers dehisce), emasculation is performed using the glume-cutting method. Use sterilized scissors to cut off the top 1 / 3 of the glumes of the wheat ear, and then use sterile tweezers to gently remove all the anthers, ensuring that there are no anther residues and stigma damage. After emasculation, immediately cover the ear with a breathable sulfuric acid paper bag for isolation, and label it with the emasculation date and the female parent number.

[0023] Pollen collection and pollination: 30 hours after emasculation (when the pistil is in full bloom and the stigma secretes a transparent mucus), collect fresh, mature ears of wheat from the male parent (the anthers have just dehisced and the pollen viability is over 90%), place them in a sterile petri dish, gently rub them, and sieve to collect pure pollen free of impurities; use a sterile brush to pick up the pollen and evenly apply it to the surface of the stigma of the female parent, ensuring that the stigma is fully covered with pollen, thus completing the pollination operation.

[0024] Fruit setting promotion: After pollination, immediately spray the female wheat ears with a mixed solution of auxin and gibberellin with a mass concentration of 30 mg / L (volume ratio 1:1). The amount of spray should be enough to moisten the wheat ears without dripping water. Then cover the bags again to keep them moist. The temperature of the culture environment during pollination and the following 7 days should be controlled at 21℃ and the relative humidity should be maintained at 70%. Check the humidity inside the bags regularly every day and replenish water in time to avoid pollen mold or stigma dehydration.

[0025] Step 3: Hybrid embryo rescue and in vitro seedling cultivation; Embryo removal: 16 days after pollination, select healthy and disease-free female wheat ears, dissect and remove hybrid embryos on a sterile operating table, select healthy spherical to heart-shaped embryos (0.5-0.8 mm in size, with normal endosperm development), rinse 3 times with sterile water (30 seconds each time), and dry the surface moisture with sterile filter paper for later use.

[0026] Culture medium preparation: Prepare modified MS medium with the following components: sucrose 32 g / L, agar 7.0 g / L, 6-benzylaminopurine 0.9 mg / L, naphthaleneacetic acid 0.52 mg / L, activated carbon 2.0 g / L. Adjust the pH to 5.8 with 0.1 mol / L NaOH or HCl. Dispense into culture flasks (50 mL per flask), autoclave at 121°C for 20 min, cool to about 45°C, pour into plates, and allow to solidify before use.

[0027] In vitro seedling culture: Treated immature embryos were inoculated into modified MS medium, with 8-10 immature embryos inoculated per plate (embryo tip facing upward, avoid inversion), and cultured in the dark at 23°C for 13 days, followed by transfer to a light intensity of 300 μmol / m². -2 *s -1 The plants were cultured for another 13 days in a constant temperature incubator with a light duration of 14 hours / day and a temperature of 23℃ to obtain hybrid F1 test-tube seedlings with uniform growth, a plant height of 3-5cm, and well-developed root systems. The seedling survival rate reached 82%.

[0028] Step 4: Homozygous stability and comprehensive trait identification; Self-pollination homozygosity: The selected superior single plants were subjected to four generations of self-pollination homozygosity. Each generation was self-pollinated by bagging single plants (bagging after heading to avoid cross-pollination). At the same time, agronomic traits such as plant height, ear length, thousand-grain weight, maturity period, and number of tillers were systematically recorded. Plants with segregating traits, inferior plants, and susceptible plants were eliminated to ensure the consistency and stability of agronomic traits.

[0029] Field disease resistance assessment: The gradient concentration pathogen inoculation method was used for assessment. The spore concentration for powdery mildew inoculation was 1.0 × 10⁻⁶. 6 Spraying inoculate leaves with 8 × 10⁶ spores / mL; for stripe rust inoculation, the spore concentration is 8 × 10⁶ spores / mL. 5 Spray inoculation with 1 spore / mL; for sheath blight, use sclerotinia soil inoculation method, inoculating each plant with 5-8 sclerotia (sclerotinia viability ≥90%); 25 days after inoculation, investigate the disease incidence according to the "National Wheat Variety Approval Standard (2024 Revision)" (implemented on January 1, 2025), determine the disease severity, and screen for single plants that are highly resistant to powdery mildew and stripe rust (disease severity level 1) or moderately resistant to sheath blight (disease severity level 3 or below).

[0030] Cold resistance and stress tolerance assessment: The artificial low temperature stress method was used. During the overwintering period, the plants were placed in an artificial climate chamber and treated with simulated low temperature of -15℃ for 27 hours; during the late spring cold period, the plants were treated with simulated low temperature of 0-2℃ for 48 hours; after the low temperature treatment, the plants were restored to normal growth conditions (temperature 20-22℃, light 12h / d). The plant survival rate was investigated after 15 days, and the fruit setting rate was investigated after maturity. Individual plants with a survival rate ≥90% and a fruit setting rate ≥85% were selected.

[0031] Quality testing: Near-infrared grain analyzer and stretching tester are used for quality testing. The test indicators and standards are as follows: protein content ≥15%, sedimentation value ≥40mL, stretching area ≥120cm². Only those three indicators that meet the standards can be included in the stable variety range.

[0032] Cultivation results; Through the above-mentioned breeding process, a high-quality, strong-gluten, disease-resistant, and stress-resistant hexaploid common wheat variety with good yield was finally obtained. This variety has a chromosome set of AABBDD and carries disease-resistant and stress-resistant genes derived from rye. Field identification results show that it is highly resistant to powdery mildew, slow stripe rust, and leaf rust, moderately resistant to Fusarium head blight and sheath blight, has a cold resistance of level 2, and strong resistance to late spring frost. Quality test results show that the protein content is 15.6%, wet gluten content is 33.2%, sedimentation value is 42.3 mL, stability time is 17.0 min, stretching area is 128 cm², and test weight is 788 g / L. The agronomic traits are stable, and the yield can reach 620 kg per mu. It is suitable for cultivation in the northern winter wheat area and the Huang-Huai wheat area.

[0033] Comparative analysis of traditional distant hybridization breeding methods Using conventional distant hybridization breeding methods (without spraying a mixture of auxin and gibberellin hormones, using basal MS medium, and without molecular marker-assisted screening), hybridization was carried out with the same maternal and paternal parents as in the example. The results showed that the hybridization seed setting rate was only 6.2%, the embryo rescue survival rate was 45%, and continuous backcrossing improvement was required, with a breeding cycle of more than 20 years. The final bred variety had unstable disease resistance, and some individual plants showed signs of powdery mildew susceptibility (disease level 4). The overall traits were significantly inferior to the bred variety of this invention.

[0034] In summary, this invention significantly improves the efficiency of distant hybridization breeding and the comprehensive traits of varieties by optimizing key technologies such as cross-pollination to promote seed setting and improving the embryo rescue system. The wheat varieties bred have the characteristics of high quality, strong gluten, multiple resistances, and high yield. It solves the problems of poor disease resistance and stress resistance and narrow genetic base in traditional high-quality wheat breeding, and has significant application value.

[0035] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0036] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for breeding a hexaploid common wheat variety with disease resistance and stress tolerance through distant hybridization, characterized in that, Includes the following steps: Step 1, Parental selection and determination: Select hexaploid common wheat as the female parent, with a test weight ≥780g / L, wet gluten content ≥32%, and stability time ≥10min; select diploid rye with chromosome set RR as the male parent, with cold resistance level 2 or above. Step 2, Hybridization and Fruit Set Promotion: After artificial emasculation of the female parent, immediately isolate it by bagging. When the pistils enter the full bloom stage, collect fresh mature pollen from the male parent for repeated artificial pollination. After pollination, spray the female parent wheat ears with a mixed solution of auxin and gibberellin at a mass concentration of 25-35 mg / L, and bag again to retain moisture. The temperature of the pollination and subsequent culture environment should be controlled at 19-23℃, and the relative humidity should be maintained at 65-75%. Step 3, Hybrid Embryo Rescue and In Vitro Seedling Culture: 15-17 days after pollination, dissect the maternal wheat ear to separate the hybrid embryos, select spherical to heart-shaped embryos, and inoculate them into modified MS medium. First, culture in the dark at 22-24℃ for 12-15 days, then transfer to a light intensity of 280-320 μmol / m². -2 *s -1 Continue culturing in a constant temperature environment with a light duration of 13-15 h / d for 12-14 days to obtain hybrid F1 test-tube seedlings; The modified MS medium was supplemented with 0.6-1.2 mg / L of 6-benzylaminopurine, 0.52 mg / L of naphthaleneacetic acid, and 2.0 g / L of activated carbon. Step 4: Homozygous stability and comprehensive trait identification: The selected superior single plants are self-pollinated for 4-5 generations to achieve homozygous hybridization. Each generation is self-pollinated with bags on single plants and agronomic traits are recorded. At the same time, field identification of disease resistance by artificial inoculation, identification of cold resistance and stress tolerance, and quality testing are carried out. Trait segregating plants and inferior plants are eliminated to finally obtain a high-quality, strong, disease-resistant, stress-resistant, and high-yielding hexaploid common wheat variety.

2. The method for breeding a hexaploid common wheat disease-resistant and stress-resistant variety through distant hybridization according to claim 1, characterized in that: In step 2, the artificial emasculation is performed by clipping the eustachian tubes. Pollination is completed within 24-36 hours after emasculation. During the pollination process, a sterile brush is used to apply pollen to avoid contamination by foreign pollen.

3. The method for breeding a hexaploid common wheat disease-resistant and stress-resistant variety through distant hybridization according to claim 1, characterized in that: In step 3, the modified MS medium has a sucrose concentration of 30-35 g / L, an agar concentration of 6.5-7.5 g / L, and a pH value adjusted to 5.7-6.

0. The temperature is kept constant at 23±1℃ during both the dark and light culture stages.

4. The method for breeding a hexaploid common wheat disease-resistant and stress-resistant variety through distant hybridization according to claim 1, characterized in that: In step 4, the field disease resistance assessment uses a gradient concentration pathogen inoculation method, with the powdery mildew inoculation spore concentration being 8 × 10⁻⁶. 5 -1.2×10 6 1 spore / mL. For stripe rust, spray inoculation was used, and for sheath blight, sclerotium soil inoculation was used. Disease incidence was investigated 25 days after inoculation.

5. The method for distant hybridization breeding of a hexaploid common wheat variety resistant to disease and stress according to claim 4, characterized in that: In step 4, the cold resistance and stress tolerance assessment was conducted using artificially simulated low-temperature stress. During the overwintering period, the plants were treated with simulated low temperatures of -14 to -16℃ for 24-30 hours, and during the late spring frost, they were treated with simulated low temperatures of 0-2℃ for 48 hours. After treatment, the plant survival rate and fruit setting rate were investigated. Individual plants with a survival rate ≥90% and a fruit setting rate ≥85% were included in the subsequent screening.