Breeding method of rice germplasm with high resistance to imidazolinone herbicides

Abstract

The invention discloses a breeding method of rice germplasm with high resistance to imidazolinone herbicides. The breeding method comprises the following steps: hybridizing an excellent receptor variety and herbicide-resistant rice germplasm to obtain an F1 generation; carrying out backcross and selfing on the F1 generation by using the recurrent parent, carrying out molecular marker and herbicide treatment detection, selecting strains with consistent backgrounds, carrying out comprehensive evaluation, and selecting an advanced generation backcross improved line; performing a field experiment, and observing the performance; comprehensively evaluating agriculture, quality, rice blast resistance and herbicide resistance, and selecting final germplasm. According to the invention, the ALS627N gene in Jinjing 818 is introduced into six rice varieties owned by the reclamation seed industry through a molecular marker-assisted selection method to obtain a series of advanced backcross stable improved lines which have the background basically consistent with that of a receptor parent and carry the ALS627N gene, and then herbicide resistance identification is carried out. By combining field basic agronomic trait investigation, quality trait analysis and rice blast resistance level detection, effective polymerization of multiple target breeding traits such as resistance, high yield and stress resistance is realized.

Description

Technical Field

[0001] The invention relates to the technical field of rice breeding, and in particular to a method for breeding rice germplasm with high resistance to imidazolinone herbicides. Background Art

[0002] Rice is the main food crop in my country and even in the world, and is the main source of calories for nearly half of the world's population. In rice fields in all rice-growing areas in my country, there have always been a large number of weeds. According to statistics, there are more than 200 species of weeds in rice fields across the country, of which about 40 species are common, serious and most common (10 to 20 species are distributed in each rice-growing area). In my country, the main methods for controlling weeds and weedy rice in rice fields are manual weeding, weeding with agricultural tools and chemical weeding. Manual weeding and weeding with agricultural tools are labor-intensive and have low efficiency. They cannot be controlled in a large area in a timely manner. The current chemical means cannot remove weedy rice in the field, and improper spraying will affect the growth of rice. Therefore, planting herbicide-resistant rice and using chemical means to control field weeds and weedy rice is the most effective way.

[0003] The resistance of rice germplasm to imidazolinone herbicides is due to the base mutation of the ALS gene. Acetolactate synthase (ALS) is the primary key enzyme in the branched-chain amino acid synthesis pathway. Herbicides that target it are divided into: sulfonylureas, imidazolinones, pyrimidine salicylates and sulfonamides. Imidazolinone herbicides have excellent herbicidal activity against sedge weeds, annual or perennial monocotyledonous weeds, broad-leaved weeds and weedy rice. This type of herbicide has the characteristics of high activity, broad spectrum of weed control and strong selectivity, and is currently an ideal herbicide. In recent years, scientists have obtained a number of non-transgenic imidazolinone herbicide-resistant rice mutants through various channels. Some of the germplasm materials with better resistance have been successfully used in rice breeding.

[0004] In the current breeding process with existing technology, multiple backcrossing and self-pollination are required. Each selection is based on personnel experience and the performance of each generation is screened. Multiple selections are time-consuming and labor-intensive, and are not thorough enough.

[0005] Therefore, it is necessary to provide a method for breeding rice germplasm with high resistance to imidazolinone herbicides to solve the above technical problems. Summary of the invention

[0006] The invention overcomes the shortcomings of the prior art and provides a method for breeding rice germplasm with high resistance to imidazolinone herbicides.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a method for breeding rice germplasm with high resistance to imidazolinone herbicides, comprising the following steps:

[0008] S1. In the greenhouse, select the recipient variety with excellent agronomic traits as the female parent and cross it with Jinjing 818 to obtain F 1 generation;

[0009] S2. In the greenhouse, the recipient varieties were used to treat F 1 Continuous backcrossing and selfing are performed for each generation. During each backcrossing or selfing, the target improved line is selected based on herbicide treatment, MAS prospect selection and germplasm evaluation methods, and finally a high-generation backcross improved line is bred;

[0010] S3. Conduct field trials on the selected high-generation backcross improved lines to observe and record their performance in the actual production environment;

[0011] S4. Comprehensively evaluate agronomic traits, quality traits, rice blast resistance and herbicide resistance to select the final rice germplasm with high resistance to imidazolinone herbicides.

[0012] In a preferred embodiment of the present invention, in said S1, the recipient varieties include: Shanghai Soft Jade No. 1, Zhejiang Fengnuo 188, Yingxiang No. 1, Shangnongjing 927, Shangnongjing 219 and Guangmingnuo No. 6.

[0013] In a preferred embodiment of the present invention, in S1 and S2, the growth process of hybrid offspring is carried out in an artificial intelligence greenhouse, and breeding is accelerated by controlling the temperature, humidity and light conditions in the artificial intelligence greenhouse.

[0014] In a preferred embodiment of the present invention, in S1 and S2, the rice is emasculated by a method in which the anthers swell and lose activity after the anthers are cut and exposed to water.

[0015] In a preferred embodiment of the present invention, in S2, the original recipient variety, that is, the parent of the F1 generation, is used to perform continuous backcrossing and selfing with the F1 generation, and the specific process includes:

[0016] 1. The recipient variety × Jinjing 818, hybridization to obtain F 1 generation;

[0017] 2. 5 consecutive backcrosses:

[0018] 1. Receptor species × F 1 Generation, herbicide treatment, BC 1 F 1 generation;

[0019] 2. Receptor species × BC 1 F 1 generation, herbicide treatment and MAS prospect selection to obtain BC 2 F 1 generation;

[0020] 3. Receptor species × BC2 F 1 Generation, herbicide treatment, MAS prospect selection and first seed quality evaluation selection to obtain BC 3 F 1 generation;

[0021] 4. Receptor species × BC 3 F 1 Generation, herbicide treatment, MAS prospect selection and first seed quality evaluation selection to obtain BC 4 F 1 generation;

[0022] 5. Receptor species × BC 4 F 1 Generation, herbicide treatment, MAS prospect selection and first seed quality evaluation selection to obtain BC 5 F 1 generation;

[0023] 3. Continuous 3 self-pollinations:

[0024] 1. BC 5 F 1 Generation × BC 5 F 1 Generation, herbicide treatment, MAS prospect selection and second germplasm evaluation selection to obtain BC 5 F 2 generation;

[0025] 2. BC 5 F 2 Generation × BC 5 F 2 The second germplasm evaluation and selection resulted in BC 5 F 3 generation;

[0026] 3. BC 5 F 3 Generation × BC 5 F 3 The second germplasm evaluation selects high-generation backcross improved lines.

[0027] In a preferred embodiment of the present invention, the herbicide treatment is specifically as follows: the same variety of rice is divided into a control area and a treatment area, the control area is planted normally, and the treatment area is treated by diluting 1000 times the imidazolinone herbicide 5% imidazolinone aqueous solution when the rice has 3 leaves and 1 heart, and spraying the shallow water layer in the field is 3 to 5 cm, and maintained for 5 to 7 days. Each plot has an area of ​​4m 2 , 3 times repeated;

[0028] Ten days after application, the occurrence of rice leaf damage was observed and recorded according to the five-level damage method, and the rice plant height and tiller number were measured on the 45th day;

[0029] Based on the data collected during the herbicide treatment, the herbicide sensitivity of rice was calculated:

[0030]

[0031] In a preferred embodiment of the present invention, the first quality evaluation is to conduct the first quality evaluation on a number of rice plants selected through MAS prospects, and the first quality evaluation includes: similarity of agronomic trait expression and herbicide sensitivity.

[0032] In a preferred embodiment of the present invention, the second germplasm evaluation includes: similarity of agronomic trait expression and stability of herbicide resistance gene expression; wherein, the stability of herbicide resistance gene expression is specifically: the average of the herbicide sensitivity of the current rice and the herbicide sensitivity of all previous generations of the same family of rice.

[0033] In a preferred embodiment of the present invention, the agronomic trait expression similarity is specifically the similarity value between the agronomic trait expression of the hybrid offspring rice and the agronomic trait expression of the recipient variety (female parent), and the calculation process of the similarity value S includes:

[0034] Assume that the agronomic trait vector of the recipient variety is X = (x 1 , x 2 , x 3 ,...,x n ), the agronomic trait characteristic vector of hybrid rice is Y = (y 1 ,y 2 ,y 3 ,...,y n );

[0035] The Euclidean distance between X and Y is Among them, x i and i are the i-th components of vector X and vector Y, i.e., the i-th agronomic trait performance value, respectively, and n is the number of components in the vector (the number of observed agronomic traits);

[0036] d represents the distance between X and Y. The smaller the distance, the greater the similarity between X and Y. The larger the distance, the smaller the similarity between X and Y.

[0037] In a preferred embodiment of the present invention, the similarity of agronomic trait expression and herbicide sensitivity are used as the horizontal axis and the vertical axis respectively to construct a first quality evaluation matrix, and a scatter plot is drawn in the first quality evaluation matrix based on the similarity of agronomic trait expression and herbicide sensitivity data, and each point represents a rice plant;

[0038] Based on the thresholds of similarity in expression of agronomic traits and herbicide sensitivity, the map was divided into different regions to visually identify which rice plants performed better or worse in both aspects;

[0039] The rice in the area with good performance is selected for the next backcrossing.

[0040] The present invention solves the defects existing in the background technology and has the following beneficial effects:

[0041] (1) The present invention provides a method for breeding rice germplasm with high resistance to imidazolinone herbicides, by 627N The gene was introduced into six rice varieties owned by Zhongken Seed, and gene selection and the first germplasm evaluation were carried out to obtain a series of high-generation backcross stable improved lines with a background basically consistent with the recipient parent and carrying the ALS627N gene. This part was subjected to field trials, and agronomic traits, quality traits, rice blast resistance and herbicide resistance were comprehensively evaluated to select the final high-resistance to imidazolinone herbicide rice germplasm, achieving the effective aggregation of multiple target breeding traits such as resistance, high yield, high quality, green color and stress resistance.

[0042] (2) The present invention provides a method for breeding rice germplasm with high resistance to imidazolinone herbicides. The method successfully breeds rice germplasm resistant to imidazolinone herbicides through advanced genetic engineering technology. On this basis, the present invention uses a method of emasculating rice by cutting the glumes and touching water to cause the anthers to swell and lose their activity during rice hybridization. This method can ensure that all hybrid seeds are derived from the hybrid offspring of the selected female parent, thereby avoiding the complicated manual emasculation step.

[0043] (3) In the breeding process, the present invention uses continuous backcrossing and selfing methods, combined with efficient molecular marker-assisted selection, herbicide sensitivity and herbicide resistance gene expression stability screening technology, to achieve rapid aggregation of multiple excellent traits. This not only simplifies the hybridization and seed selection process, but also significantly accelerates the breeding process of rice germplasm with high resistance to imidazolinone herbicides. The implementation of the present invention will help improve the herbicide resistance of rice and provide more high-quality and efficient germplasm resources for agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work.

[0045] Figure 1The present invention is a flow chart of a method for breeding rice germplasm with high resistance to imidazolinone herbicides according to a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0048] like Figure 1 As shown, the present invention provides a method for breeding rice germplasm with high resistance to imidazolinone herbicides, comprising the following steps:

[0049] S1. In the greenhouse, select recipient varieties with excellent agronomic traits as female parents and cross them with Jinjing 818 to obtain the F1 generation;

[0050] S2. In the greenhouse, the recipient varieties are used to perform continuous backcrossing and selfing on the F1 generation. During each backcrossing or selfing, the target improved lines are selected based on herbicide treatment, MAS prospect selection and germplasm evaluation methods, and finally the high-generation backcross improved lines are selected;

[0051] S3. Conduct field trials on the selected high-generation backcross improved lines to observe and record their performance in the actual production environment;

[0052] S4. Comprehensively evaluate agronomic traits, quality traits, rice blast resistance and herbicide resistance to select the final rice germplasm with high resistance to imidazolinone herbicides.

[0053] The present invention provides a method for breeding rice germplasm with high resistance to imidazolinone herbicides, wherein the ALS in Jinjing 818 is grown in an artificial intelligence greenhouse. 627N The gene was introduced into 6 rice varieties owned by Zhongken Seeds, and gene selection and first quality evaluation were carried out to obtain a series of rice varieties with a background basically consistent with the recipient parent and carrying ALS. 627N The high-generation backcross stable improved line of the gene will be subjected to field trials, and agronomic traits, quality traits, rice blast resistance and herbicide resistance will be comprehensively evaluated to select the final high-resistance to imidazolinone herbicide rice germplasm, realizing the effective aggregation of multiple target breeding traits such as resistance to insecticides, high yield, high quality, green color and stress resistance.

[0054] Each step is described in detail below.

[0055] In step S1, in a greenhouse, a recipient variety with excellent agronomic traits is selected as the female parent and hybridized with Jinjing 818 to obtain the F1 generation.

[0056] That is to say, in the artificial intelligence greenhouse, the recipient variety with excellent agronomic traits is first selected as the female parent, and the rice variety Jinjing 818 is used as the male parent for hybridization, and rapid generation technology is used in the artificial intelligence greenhouse to shorten the growth cycle of the hybrid offspring.

[0057] The purpose of selecting Jinjing 818 as the male parent for hybridization in the present invention is that the herbicide resistance of Jinjing 818 is one of its greatest advantages. 627N Gene, that is, herbicide resistance gene, Jinjing 818 can be matched with imidazolinone herbicides to effectively control weeds.

[0058] The recipient varieties for hybridization with Jinjing 818 include: Huruanyu No. 1, Zhefengnuo No. 188, Yingxiang No. 1, Shangnongjing No. 927, Shangnongjing No. 219 and Guangmingnuo No. 6, all of which are rice varieties owned by Zhongken Seed Industry, and have been verified by the market and are good rice varieties. The hybridization process includes: artificial pollination of the emasculated female rice panicles on the same day or the next day, and the best time for pollination is from 11 am to 1 pm. The male panicles in full bloom are shaken on the cut female panicles to make the pollen fall on the stigma of the female panicle flowers to achieve the purpose of pollination.

[0059] It is worth noting that one day before or in the morning of the flowering of the female parent rice ear, about 1 / 3 is cut obliquely from the upper part of the lemma of the floret with scissors to expose the stamens and pistils inside. Be careful not to damage the stigma when cutting the lemma to avoid affecting the subsequent pollination process. After cutting the lemma, the female ear is immediately subjected to water immersion treatment. The female ear is placed in clean water and soaked for a period of time, or the female ear is sprayed with water using a water pipe. Water immersion or water spraying can stimulate the female ear to develop further, and cause the anther to absorb water and swell and lose activity. The present invention uses this method to emasculate, which can ensure that all hybrid seeds are derived from the hybrid offspring of the selected female parent, thereby avoiding complicated manual emasculation steps.

[0060] In step S2, in a greenhouse, the F1 generation is continuously backcrossed and selfed using the recipient variety. During each backcross or selfing, the target improved line is selected based on herbicide treatment, MAS prospect selection and germplasm evaluation methods, and finally a high-generation backcross improved line is bred.

[0061] That is to say, in the AI ​​greenhouse, the original recipient variety, that is, the parent of the F1 generation, is used to perform continuous backcrossing and self-pollination with the F1 generation. The specific process includes:

[0062] 1. The recipient variety × Jinjing 818, hybridization to obtain F1 generation;

[0063] 2. 5 consecutive backcrosses:

[0064] 1. Receptor species × F 1 Generation, herbicide treatment, BC 1 F 1 generation;

[0065] 2. Receptor species × BC 1 F 1 generation, herbicide treatment and MAS prospect selection to obtain BC 2 F 1 generation;

[0066] 3. Receptor species × BC 2 F 1 Generation, herbicide treatment, MAS prospect selection and first seed quality evaluation selection to obtain BC 3 F 1 generation;

[0067] 4. Receptor species × BC 3 F 1 Generation, herbicide treatment, MAS prospect selection and first seed quality evaluation selection to obtain BC 4 F 1 generation;

[0068] 5. Receptor species × BC 4 F 1 Generation, herbicide treatment, MAS prospect selection and first seed quality evaluation selection to obtain BC 5 F 1 generation;

[0069] 3. Continuous 3 self-pollinations:

[0070] 1. BC 5 F 1 Generation × BC 5 F 1 Generation, herbicide treatment, MAS prospect selection and second germplasm evaluation selection to obtain BC 5 F 2 generation;

[0071] 2. BC 5 F 2 Generation × BC 5 F 2 The second germplasm evaluation and selection resulted in BC 5 F 3 generation;

[0072] 3. BC 5 F 3 Generation × BC 5 F 3The second germplasm evaluation selects high-generation backcross improved lines.

[0073] Specifically, the herbicide treatment is as follows: the same variety of rice is divided into a control area and a treatment area. The control area is planted normally, while the treatment area is treated with a 1000-fold dilution of 5% imidazolinone herbicide imidazolinone when the rice has 3 leaves and 1 heart. The shallow water layer in the field is 3 to 5 cm and maintained for 5 to 7 days. Each plot is 4m2. 2 , repeated 3 times. Ten days after application, the occurrence of rice leaf damage was observed and recorded according to the five-level damage method, and the height and tiller number of rice were measured on the 45th day. Specifically, the degree of rice leaf damage was visually observed using the five-level damage method 10 days after application, namely: Level 0: green leaves, normal growth; Level 1: yellowing of leaf tips; Level 2: yellowing area of ​​leaves accounts for 1 / 5 of the total leaf area; Level 3: yellowing area of ​​leaves accounts for 2 / 5 of the total leaf area; Level 4: yellowing area of ​​leaves accounts for 3 / 5 of the total leaf area; Level 5: the entire leaf is completely yellowed. 45 days after application, a rectangular wire frame with a side length of 0.7m*0.7m was taken, and the types and quantities of weeds in the rice field were investigated using the 5-point sampling method, and the rice yield was measured at the rice maturity stage.

[0074] Furthermore, based on the data collected during the herbicide treatment, the herbicide sensitivity of rice was calculated:

[0075] The MAS prospect selection is specifically for molecular marker-assisted selection, marking the target gene in rice (ALS 627N MAS prospect selection can be used to select rice individuals with the target gene directly at the DNA level without relying on phenotype. 627N Genetic plants.

[0076] In the present invention, the first quality evaluation is to conduct the first quality evaluation on a number of rice plants selected through MAS prospects, and the first quality evaluation includes: similarity of agronomic trait expression and herbicide sensitivity.

[0077] The similarity of agronomic trait expression is specifically the similarity value between the agronomic trait expression of the hybrid offspring rice and the agronomic trait expression of the recipient variety (female parent). The calculation process of the similarity value S includes:

[0078] Assume that the agronomic trait vector of the recipient variety is X = (x 1 , x 2 , x 3 ,...,x n ), the agronomic trait characteristic vector of hybrid rice is Y = (y 1 ,y 2 ,y 3 ,...,yn );

[0079] The Euclidean distance between X and Y is Among them, x i and i are the i-th components of vector X and vector Y, that is, the i-th agronomic trait performance value, and n is the number of components in the vector (the number of observed agronomic traits).

[0080] d represents the distance between X and Y. The smaller the distance, the greater the similarity between X and Y; the larger the distance, the smaller the similarity between X and Y.

[0081] Furthermore, the similarity of agronomic trait expression and herbicide sensitivity were used as the horizontal and vertical axes, respectively, to construct the first quality evaluation matrix. Based on the agronomic trait expression similarity and herbicide sensitivity data, a scatter plot was drawn in the first quality evaluation matrix, and each point represented a rice plant. Furthermore, according to the thresholds of agronomic trait expression similarity and herbicide sensitivity, the graph was divided into different areas to intuitively identify which rice plants performed better or worse in both aspects. The rice in the area with good performance was selected for the next backcrossing. While ensuring the excellent agronomic traits of the original recipient variety, it also had better herbicide resistance. After three backcrossings, the target improved line could be bred accurately and efficiently.

[0082] The second quality evaluation includes: agronomic trait expression similarity and herbicide resistance gene expression stability; among them, the herbicide resistance gene expression stability is specifically: the average of the herbicide sensitivity of the current rice and the herbicide sensitivity of all previous generations of the same family of rice. The herbicide sensitivity of the current rice and the herbicide sensitivity of the current rice father are aggregated to BC 1 F 1 The stability of herbicide resistance gene expression in multiple generations of rice in the same lineage is expressed in the following ways: The herbicide sensitivity of the current rice is compared with the average of the herbicide sensitivity of the previous generation of the same family of rice. If the difference between the two is not large, it means that the expression of the herbicide resistance gene of the current rice is stable; if the difference is large, it may indicate that the expression of the herbicide resistance gene of the current rice is unstable or mutated. By screening the stability of the expression of the herbicide resistance gene, the continuity of the expression of the herbicide resistance gene can be screened out, which can avoid the mutation or failure of the herbicide resistance gene.

[0083] Furthermore, the similarity of agronomic trait expression and the stability of herbicide resistance gene expression were used as the horizontal and vertical axes, respectively, to construct a second germplasm evaluation matrix. Based on the data of agronomic trait expression similarity and herbicide resistance gene expression stability, a scatter plot was drawn in the second germplasm evaluation matrix, and each point represented a rice plant. Furthermore, according to the thresholds of agronomic trait expression similarity and herbicide resistance gene expression stability, the graph was divided into different areas to intuitively identify which rice plants performed better or worse in both aspects. The rice in the area with good performance was selected for the next self-pollination. While ensuring the excellent agronomic traits of the original recipient variety, the expression ability of its herbicide resistance gene was also relatively stable. After three rounds of self-pollination, a high-generation backcross improvement line could be accurately and efficiently bred.

[0084] Furthermore, the growth process of hybrid offspring was carried out in an artificial intelligence greenhouse, and breeding was accelerated by controlling the temperature, humidity, and light conditions in the artificial intelligence greenhouse. Specifically, using light flux density of about 550umol m-1, a photoperiod of 22 hours of light + 2 hours of darkness, and a culture temperature of 22°C (light) + 17°C (darkness), combined with the method of early seed harvesting, the growth of multiple generations of rice in one year was achieved, accelerating the breeding rate of rice germplasm.

[0085] In one embodiment, backcrossing and selfing are performed directly in a practical environment to obtain lines whose genetic background is consistent with that of the recurrent parent.

[0086] In the breeding process, the present invention adopts continuous backcrossing and selfing means, combined with efficient molecular marker-assisted selection, herbicide sensitivity and herbicide resistance gene expression stability screening technology, to achieve rapid aggregation of multiple excellent traits. This not only simplifies the hybridization and seed selection process, but also significantly accelerates the breeding process of rice germplasm with high resistance to imidazolinone herbicides. The implementation of the present invention will help improve the herbicide resistance of rice and provide more high-quality and efficient germplasm resources for agricultural production.

[0087] In step S3, the selected high-generation backcross improved lines are subjected to field trials to observe and record their performance in the actual production environment.

[0088] The field experimental conditions included: a completely randomized block design with three replications and 450 plants planted in each plot;

[0089] Field inspection of basic agronomic traits, including plant height, full growth period, effective ears, number of grains per ear, fruit setting rate, 1000-grain weight and actual yield;

[0090] Quality traits are tested indoors, including: whole polished rice rate, chalky grain rate, chalkiness and taste value.

[0091] The method for identifying rice blast resistance is to inject 1 ml of bacterial mixture into each ear of rice during the heading stage, inoculate 5 young ears of each variety, and investigate the results according to the national unified standards after maturity. The specific steps include:

[0092] Rice blast is a major disease of rice, caused by the fungus Piricularia oryzae, which seriously affects the yield and quality of rice. Identification of rice blast resistance is an important part of breeding and disease management. The identification method you mentioned is a commonly used inoculation test method. The specific steps are as follows:

[0093] Preparation of strains: First, the strains of rice blast fungus need to be prepared. This usually involves selecting from a laboratory strain bank or isolating from diseased plants in the field.

[0094] Strain culture: Culture the strain on a suitable culture medium and allow it to grow to an appropriate concentration and activity.

[0095] Mixed solution preparation: Mix the cultured bacteria with an appropriate amount of culture solution to make a mixed solution. Usually, the concentration of the mixed solution needs to be adjusted to ensure the inoculation amount and bacterial activity.

[0096] Inoculation: During the rice booting stage, select healthy young ears for inoculation. Inject 1 ml of the bacterial mixture into each ear. This usually needs to be done under sterile conditions to avoid contamination by other microorganisms.

[0097] Cultivation and observation: After inoculation, the rice plants are cultivated under suitable conditions and the development of the disease is observed.

[0098] Disease survey: After maturity, disease survey is conducted according to national unified standards, which usually includes the determination of disease incidence, disease index, disease grade and other indicators.

[0099] Data analysis: Based on the survey results, the disease resistance of different varieties was compared and analyzed.

[0100] Report writing: Finally, write a detailed identification report based on the experimental results. The report should include experimental design, operation steps, result analysis and conclusions, etc.

[0101] The resistance of rice blast in this example is graded as follows:

[0102] Resistance level: Based on the number and size of lesions on rice leaves and the loss rate from panicle blast, the disease resistance is divided into levels 0 to 9, where level 0 means disease-free and level 9 means the most serious losses.

[0103] Disease Index (DI): The disease index is an indicator to measure the severity of the disease. According to the size of the disease index, the disease resistance is divided into different levels. For example, a disease index of less than or equal to 11.1% is highly resistant, and a value greater than 77.9% is highly susceptible.

[0104] Population disease incidence resistance classification: According to the percentage of diseased ears in the rice population, disease resistance is divided into levels such as immune (I), resistant (R), moderately resistant (MR), moderately susceptible (MS), susceptible (S) and highly susceptible (HS).

[0105] Resistance index: Resistance index is another indicator to evaluate disease resistance. According to the size of the resistance index, disease resistance is divided into levels such as immune, resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible.

[0106] These grading standards help researchers and breeders evaluate the disease resistance of rice varieties and screen and cultivate better disease-resistant rice varieties.

[0107] Specifically for the classification of rice blast resistance, the disease resistance classification is as follows:

[0108] Rice panicle blast population incidence and disease resistance classification:

[0109] -Level 0: Diseased ear rate 0%, immune (I)

[0110] -1 level: diseased ear rate 0.1% to 5.0%, resistant (R)

[0111] -Level 3: Diseased ear rate 5.1% to 10.0%, medium resistance (MR)

[0112] -5: Diseased ear rate 10.1% to 25.0%, moderately susceptible (MS)

[0113] -7: Diseased ear rate 25.1% to 50.0%, susceptible (S)

[0114] -9: Diseased ear rate ≥ 50.1%, high susceptibility (HS)

[0115] Rice blast resistance index classification:

[0116] -Level 0: Disease resistance index 0, immunity (I)

[0117] -1 level: disease resistance index 0.1~2.0, resistance (R)

[0118] -Level 3: Disease resistance index 1.1-4.0, medium resistance (MR)

[0119] -Level 5: Disease resistance index 4.1-6.0, moderately susceptible (MS)

[0120] -7: Disease resistance index 6.1~7.5, susceptible (S)

[0121] -Level 9: Disease resistance index>6.5, high sensitivity (HS)

[0122] These ratings provide a standardized method for evaluating disease resistance in rice varieties, helping to guide breeding and disease management decisions.

[0123] The herbicide resistance identification method in this embodiment is to direct-seed plant the high-generation backcross improved lines and the control varieties, and when they have 3 leaves and 1 heart, they are sprayed with a 1000-fold dilution of 5% imidazolinone herbicide imidacloprid aqueous solution, and observations are recorded.

[0124] It is worth noting that the identification of herbicide resistance includes the following steps:

[0125] Experimental design: Select a suitable experimental site and time to ensure that environmental conditions are suitable for rice cultivation and herbicide treatment; design an experimental plan, including an experimental group (high-generation backcross improved lines) and a control group (control varieties with known resistance levels).

[0126] Sowing and seedling raising: Sow high-generation backcross improved lines and control varieties according to the experimental design requirements; carry out appropriate field management to ensure the healthy growth of seedlings.

[0127] Seedling management: observe the growth of seedlings and record growth data, such as emergence rate, growth rate, etc.; maintain water and nutrient supply in the field to promote balanced growth of seedlings.

[0128] Herbicide treatment: When the seedlings reach the 3-leaf 1-heart stage (i.e., have 3 fully expanded leaves and 1 heart leaf), prepare for herbicide treatment; dilute 5% imidacloprid aqueous solution at a ratio of 1000 times and prepare for spray treatment.

[0129] Application: Use a sprayer to evenly spray the diluted herbicide on the rice in the experimental and control groups; ensure that the application is uniform and avoid repeated spraying or omissions.

[0130] Observation and recording: After applying the pesticide, regularly observe the growth of rice and record changes in leaves, stems and other treated parts; record the growth status of rice and any abnormal reactions within a certain period of time (such as 24 hours, 48 ​​hours, 72 hours, etc.) after herbicide treatment.

[0131] Resistance evaluation: Based on the observation results, the resistance of rice to imidazolinone herbicides was evaluated; the resistance performance of the experimental group and the control group was compared to determine the resistance level of the improved line.

[0132] Data analysis: Conduct statistical analysis on the collected data, including growth rate, degree of damage, recovery ability, etc.; use statistical methods to evaluate the differences in herbicide resistance.

[0133] The present invention provides a method for breeding rice germplasm with high resistance to imidazolinone herbicides. During each seed selection, the comprehensive evaluation method proposed in the present invention is used to express the agronomic traits, quality traits, rice blast resistance and herbicide resistance of the rice germplasm as specific comprehensive scores, which can more intuitively evaluate the comprehensive performance of each combination hybrid offspring, and provide accurate data basis for breeding rice germplasm with high resistance to imidazolinone herbicides.

[0134] The above is based on the ideal embodiment of the present invention. Through the above description, relevant personnel can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of the present invention is not limited to the content in the specification, and the technical scope must be determined according to the scope of the claims.

Claims

1. A method for breeding rice germplasm with high resistance to imidazolinone herbicides, It is characterized in that The following steps are involved: S1. In the greenhouse, select the recipient variety with excellent agronomic traits as the female parent and cross it with Jinjing 818 to obtain F 1 generation; S2. In the greenhouse, the recipient varieties were used to treat F 1 Continuous backcrossing and selfing are performed for each generation. During each backcrossing or selfing, the target improved line is selected based on herbicide treatment, MAS prospect selection and germplasm evaluation methods, and finally a high-generation backcross improved line is bred; S3. Conduct field trials on the selected high-generation backcross improved lines to observe and record their performance in the actual production environment; S4. Comprehensively evaluate agronomic traits, quality traits, rice blast resistance and herbicide resistance to select the final rice germplasm with high resistance to imidazolinone herbicides.

2. The method for breeding rice germplasm with high resistance to imidazolinone herbicides according to claim 1, Features: In the S1, the recipient varieties include: Shanghai Soft Jade No. 1, Zhejiang Fengnuo 188, Yingxiang No. 1, Shangnongjing 927, Shangnongjing 219 and Guangmingnuo No.

6.

3. The method for breeding rice germplasm with high resistance to imidazolinone herbicides according to claim 1, Features: In S1 and S2, the growth process of hybrid offspring is carried out in an artificial intelligence greenhouse, and breeding is accelerated by controlling the temperature, humidity and light conditions in the artificial intelligence greenhouse.

4. The method for breeding rice germplasm with high resistance to imidazolinone herbicides according to claim 1, Features: In S1 and S2, rice is emasculated by cutting the glumes and touching water to cause the anthers to swell and lose their activity.

5. The method for breeding rice germplasm with high resistance to imidazolinone herbicides according to claim 1, Features: In the S2, the original recipient variety, that is, the parent of the F1 generation, is used to perform continuous backcrossing and selfing with the F1 generation, and the specific process includes:

1. The recipient variety × Jinjing 818, hybridization to obtain F 1 generation; 2. 5 consecutive backcrosses:

1. Receptor species × F 1 Generation, herbicide treatment, BC 1 F 1 generation; 2. Receptor species × BC 1 F 1 generation, herbicide treatment and MAS prospect selection to obtain BC 2 F 1 generation; 3. Receptor species × BC 2 F 1 Generation, herbicide treatment, MAS prospect selection and first seed quality evaluation selection to obtain BC 3 F 1 generation; 4. Receptor species × BC 3 F 1 Generation, herbicide treatment, MAS prospect selection and first seed quality evaluation selection to obtain BC 4 F 1 generation; 5. Receptor species × BC 4 F 1 Generation, herbicide treatment, MAS prospect selection and first seed quality evaluation selection to obtain BC 5 F 1 generation; 3. Continuous 3 self-pollinations:

1. BC 5 F 1 Generation × BC 5 F 1 Generation, herbicide treatment, MAS prospect selection and second germplasm evaluation selection to obtain BC 5 F 2 generation; 2. BC 5 F 2 Generation × BC 5 F 2 The second germplasm evaluation and selection resulted in BC 5 F 3 generation; 3. BC 5 F 3 Generation × BC 5 F 3 The second germplasm evaluation was used to select high-generation backcross improved lines.

6. The method for breeding rice germplasm with high resistance to imidazolinone herbicides according to claim 5, Features: The herbicide treatment is specifically as follows: the same variety of rice is divided into a control area and a treatment area. The control area is planted normally, while the treatment area is treated with a 1000-fold dilution of 5% imidazolinone herbicide imidazolinone when the rice has 3 leaves and 1 heart. The shallow water layer in the field is 3 to 5 cm and maintained for 5 to 7 days. Each plot has an area of ​​4m 2 , 3 times repeated; Ten days after application, the occurrence of rice leaf damage was observed and recorded according to the five-level damage method, and the rice plant height and tiller number were measured on the 45th day; Based on the data collected during the herbicide treatment, the herbicide sensitivity of rice was calculated:

7. The method for breeding rice germplasm with high resistance to imidazolinone herbicides according to claim 5, Features: The first quality evaluation is for several rice plants selected through MAS prospects, and then the first quality evaluation is carried out. The first quality evaluation includes: similarity of agronomic trait expression and herbicide sensitivity.

8. The method for breeding rice germplasm with high resistance to imidazolinone herbicides according to claim 5, Features: The second germplasm evaluation includes: the similarity of agronomic trait expression and the stability of herbicide resistance gene expression; among them, the stability of herbicide resistance gene expression is specifically: the average of the herbicide sensitivity of the current rice and the herbicide sensitivity of all previous generations of the same family of rice.

9. The method for breeding rice germplasm with high resistance to imidazolinone herbicides according to claim 7, Features: The agronomic trait expression similarity is specifically the similarity value between the agronomic trait expression of the hybrid offspring rice and the agronomic trait expression of the recipient variety (female parent). The calculation process of the similarity value S includes: Assume that the agronomic trait vector of the recipient variety is X = (x 1 , x 2 , x 3 ,…,x n ), the agronomic trait characteristic vector of hybrid rice is Y = (y 1 ,y 2 ,y 3 ,…,y n ); The Euclidean distance between X and Y is Among them, x i and i are the i-th components of vector X and vector Y, i.e., the i-th agronomic trait performance value, respectively, and n is the number of components in the vector (the number of observed agronomic traits); d represents the distance between X and Y. The smaller the distance, the greater the similarity between X and Y. The larger the distance, the smaller the similarity between X and Y.

10. The method for breeding rice germplasm with high resistance to imidazolinone herbicides according to claim 7, Features: The agronomic trait expression similarity and herbicide sensitivity were used as the horizontal axis and the vertical axis respectively to construct the first quality evaluation matrix. Based on the agronomic trait expression similarity and herbicide sensitivity data, a scatter plot was drawn in the first quality evaluation matrix, and each point represented a rice plant. Based on the thresholds of similarity in expression of agronomic traits and herbicide sensitivity, the map was divided into different regions to visually identify which rice plants performed better or worse in both aspects; The rice in the area with good performance is selected for the next backcrossing.

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