Production method of hybrid rice seeds

Abstract

The invention discloses a production method of hybrid rice seeds, and belongs to the field of agricultural breeding. According to the method, high-purity hybrid seeds are produced through the technical means of excellent female parent reproductive line cultivation, reproductive line reproduction, preliminary fluorescent sorting, further fine seed screening, artificial seed shell adding, hybrid seed production and the like. According to the method, the technical problem that the sorting effect and the seed purity of colorless seeds and colored fluorescent seeds are influenced due to the fact that the precision of a color sorting technology cannot reach 100% due to difference or abnormal development of maturity of reproductive line seeds of a common nuclear male sterile line can be solved, and sterile line seeds meeting the market standard are obtained.

Description

Technical Field

[0001] The invention belongs to the field of agricultural breeding, and in particular relates to a method for producing rice hybrid seeds. Background Art

[0002] Rice is currently the largest food crop in my country's cereal crop production, with the widest distribution, largest planting area, and highest yield per unit area. The application of common nuclear sterile line breeding technology can make the use of rice hybrid advantages more popular, which plays a major strategic role in ensuring my country's food security. Hybrid rice with common nuclear sterile lines as genetic tools, that is, common recessive nuclear male sterile lines that can be reproduced on a large scale obtained through genetic transformation, are stable and sterile in any region and at any time. DuPont Pioneer Seed Company in the United States has established a molecular design technology for intelligent sterility in corn, which uses the recessive nuclear sterility phenomenon that is common in plants to solve technical problems such as unstable fertility and maintenance of sterile lines in hybrid breeding.

[0003] Common nuclear male sterility is simple in inheritance, free in pairing, and stable in fertility. Its fertility is generally controlled by only a pair of recessive genes. Any strain can be transformed into a sterile line without being restricted by genetic background; any strain can be used as a restorer of the common nuclear sterile line; as long as the selected gene has a complete abortive effect, the pollen of its offspring common nuclear sterile line will be completely aborted, the sterility will be stable, and it will not be affected by the environment. The use of common nuclear sterility not only has the advantages of stable fertility of three-line sterile lines and free in pairing of two-line sterile lines, but also overcomes the disadvantages of limited in pairing of three-line sterile lines and risks in breeding and seed production of two-line sterile lines. It is an ideal way to utilize hybrid advantages of rice. Common recessive nuclear male sterile rice lines that can be commercially bred on a large scale are referred to as common nuclear sterile lines.

[0004] 1. Basic standards for excellent common nuclear male sterile rice lines

[0005] Due to the particularity of common nuclear male sterile lines in genetic basis and trait expression, breeders have strict selection standards for excellent common nuclear male sterile lines of rice. The basic standards for excellent common nuclear male sterile lines of rice mainly include the following four aspects:

[0006] First, the excellent common nuclear male sterile lines of rice must maintain stable male fertility expression characteristics. Based on the characteristics of the SPT technology of linked expression of pollen fertility genes, fluorescent protein genes and pollen lethal genes, Chinese scholar Li Xinqi clearly proposed the basic idea of ​​in-depth research on the use of common nuclear sterility in hybrid rice at the beginning of this century. Li Xinqi used the rice pollen fertility gene EAT1 and the corresponding rice nuclear male sterile mutant as research materials, and referred to the SPT technology operation procedures to construct a ternary vector for linked expression of fertility genes, red fluorescent protein genes and pollen lethal genes, and then successfully transformed the male sterile mutant eat1. After years of hard exploration, a transgenic plant with normal fertility was screened out from tens of thousands of positive rice plants. In 2015, the common nuclear male sterile line Gt1s and the breeding line Gt1S that met the standards of rice male sterile lines were successfully created, and the seed combination for the use of common nuclear sterility was formulated.

[0007] The common recessive nuclear male sterile lines of rice that can be commercially propagated on a large scale obtained by genetic engineering methods have certain fertility expression characteristics, that is, the male sterile mutants can be propagated by self-pollination to produce transgenic plants with normal fertility (i.e., common nuclear male sterile breeding lines), so as to achieve the purpose of maintaining their special genetic basis. Through self-pollination, half of the seeds produced on each rice ear of the common nuclear sterile breeding line are colored seeds and the other half are colorless seeds. The colorless seeds have 100% sterility and 100% sterile plant rate, and maintain the male sterility characteristics, and can be used for hybrid rice seed production (because they are non-transgenic seeds, and the hybrid rice seeds obtained after hybrid seed production with them as the mother parent are also non-transgenic seeds); the colored seeds have fertile characteristics, and half of the colored seeds and half of the colorless seeds will be separated in the next generation population, and the two can be separated using a color sorter. It can be seen that in the technical system of utilizing common nuclear male sterility, the use of this type of common nuclear male sterile line will make hybrid seed production and common nuclear male sterile line reproduction very simple and easy. Therefore, maintaining stable fertility expression characteristics during hybrid seed production and common nuclear male sterile line reproduction under normal ecological conditions is an essential characteristic of excellent rice common nuclear male sterile lines.

[0008] Secondly, the excellent common nuclear male sterile lines of rice must have good comprehensive agronomic traits. Whether the common nuclear male sterile lines can show strong heterosis effects is closely related to whether their parents have excellent comprehensive agronomic traits. Good comprehensive agronomic traits mainly include relatively ideal plant and leaf morphology, appropriate growth period, relatively high yield potential, reasonable panicle-grain combination, relatively strong stress resistance and adaptability, etc. In hybrid rice breeding, whether the parents have ideal plant type is directly related to whether the first generation of hybrid populations can efficiently utilize light energy and obtain as much dry matter as possible per unit area and per unit time. This is the material basis for the high yield of hybrid rice, that is, to maximize the organic relationship between the "source", "flow" and "sink" of hybrid rice in its growth and development process, so as to achieve the highest economic yield to the greatest extent. Therefore, having good comprehensive agronomic traits is a must-have characteristic of excellent common nuclear male sterile lines of rice.

[0009] Third, the excellent common nuclear male sterile lines of rice must have good combining ability. The combining ability of male sterile lines is a very important technical indicator in the process of hybrid rice parent selection. The combining ability of rice varieties (lines) refers to the yield performance or potential of the first generation of hybrids after a variety (line) is hybridized with other varieties (lines). The one with relatively high yield or relatively large yield potential is called high combining ability, otherwise it is low combining ability. The combining ability of rice varieties (lines) can be divided into two types (i.e. general combining ability and special combining ability). In the process of hybrid rice selection, general combining ability refers to the average performance of the yield potential (or other quantitative trait phenotypes) of the first generation of hybrids of a tested parent variety (line) in a series of hybrid combinations produced by combining with other varieties (lines). General combining ability is mainly caused by the additive effect of genes in genetics, and the characteristic of phenotype is that the effect of genes is a cumulative effect (can be stably inherited), which is usually said to rise with the tide.

[0010] Special combining ability refers to the yield potential of the first generation of hybrids composed of two specific hybrid parents. When measuring the combining ability of rice varieties, the varieties, inbred lines, sterile lines, restorer lines, hybrids, etc. used to cross with the tested lines (varieties) are collectively referred to as test varieties. This type of hybridization is called test cross (test cross), and the resulting hybrids are called test cross varieties. Special combining ability mainly comes from the non-additive effects of genes, mainly including genetic effects such as dominant effects, overdominant effects and epistatic effects. The recessive adverse gene effects of the parents are masked by the dominant favorable gene effects (dominant effect), or derived from the heterogeneous combination of the parental genotypes to cause interactions between alleles (overdominant effect), thereby causing the hybrid F1 to show an over-parent advantage effect.

[0011] The main methods for determining combining ability are the common test variety method, double row hybridization or reciprocal cross method and multi-line test cross method. When using the common test variety method to determine combining ability, 1 to 3 materials are usually selected as common test varieties, which are hybridized with the tested lines (variety) respectively. When using the double row hybridization or reciprocal cross method, each tested line is a test variety for each other, and two of them are hybridized with each other. When using the multi-line test cross method, several excellent lines or backbone lines with known combining ability are selected as test varieties, and they are tested and crossed with a series of tested lines to form several single cross combinations. In the second year, they are arranged in order, and a yield comparison test is carried out to obtain the average yield of each hybrid combination, from which the general combining ability and special combining ability can be calculated.

[0012] When breeding excellent common nuclear male sterile lines of rice, its combining ability must be carefully identified. Only by selecting common nuclear male sterile lines with high combining ability as hybrid female parents can it be possible to configure hybrid combinations with obvious heterosis effects and high yield potential.

[0013] Fourth, the excellent common nuclear male sterile lines of rice must have good hybrid seed production and parental reproduction characteristics. Hybrid seed production and parental reproduction are important links in its application technology system, and their yield and seed quality are directly related to the application prospects of hybrid rice. In the application technology system of the first generation of hybrid rice and the second generation of hybrid rice, hybrid seed production areas and male sterile line reproduction areas must be set up every year. When using common nuclear male sterile lines to configure hybrid first-generation seeds and carry out parental reproduction, two isolation areas, namely, common nuclear male sterile line hybrid seed production and reproduction areas and parental reproduction areas, need to be set up every year. Therefore, whether in the hybrid seed production area or in the parental reproduction area, the excellent common nuclear male sterile lines of rice must have good seed production and reproduction characteristics (i.e., stable male sterile characteristics, excellent flowering habits and outcrossing characteristics, high-yield population structure and high yield, relatively strong stress resistance and adaptability, etc.), so as to obtain the highest possible hybrid seed production yield and parental reproduction yield, and reduce seed production costs.

[0014] 2. Seed propagation method of rice common nuclear male sterile line

[0015] The breeding line of the common nuclear male sterile line is obtained through biological genetic engineering methods, and contains transgenic components in its genetic composition (it is particularly worth noting that due to the role of the lethal gene in its genetic composition, the male gametes or male gametophytes carrying transgenes can be eliminated during the breeding process of the common nuclear male sterile line, and the hybrid rice produced through hybrid seed production is the first generation of non-transgenic hybrids). Therefore, in accordance with the relevant provisions of the Regulations on the Safety Management of Transgenics of the Ministry of Agriculture and Rural Affairs of my country, its production safety must be strengthened to prevent it from mixing into the conventional male sterile rice line population.

[0016] In the process of seed propagation of rice genetically engineered nuclear male sterile lines, in order to solve the technical problem of low red fluorescent protein expression in individual seeds due to environmental influences and other reasons, there is a low probability of mixing transgenic breeding line seeds when color sorting rice ordinary nuclear male sterile line seeds. There are two breeding methods for producing rice genetically engineered nuclear male sterile line seeds:

[0017] One is the self-fertilization method. According to the conventional rice cultivation method, the rice common nuclear male sterile breeding line is planted, and after self-fertilization, a progeny group with mixed characteristics is produced, that is, the rice common nuclear male sterile line seeds and the corresponding breeding line seeds each account for 50%. Subsequently, the mixed progeny group seeds are separated through the color sorting link using fluorescent color sorting technology and a specific color sorter, thereby obtaining the rice common nuclear male sterile line seeds and the corresponding breeding line seeds. In production practice, the rice common nuclear male sterile breeding line plants are planted in the breeding field according to the planting method of conventional rice varieties, and the individual plants are planted (the spacing between the plants and the rows is 30cm × 20cm), and the usual field management measures are adopted. Half of the rice ears produced by the rice common nuclear male sterile breeding line plants through self-fertilization are the common nuclear male sterile line seeds, and the other half are the corresponding breeding line seeds. This production method is simple in the early planting and cultivation, and only needs to harvest all the seeds on the breeding line plants in the later stage. The subsequent technical focus is to color sort the harvested mixed seeds to screen out the non-fluorescent rice common nuclear male sterile seeds and the red fluorescent breeding seeds. Due to the limitations of the accuracy of existing color sorting technology and devices, it is still possible that a small amount of breeding seeds containing genetically modified components will be mixed into the common nuclear male sterile seeds during the color sorting process. Therefore, it is necessary to conduct in-depth research on the color sorting link in this production method in order to improve the accuracy (100% non-genetically modified) and throughput of color sorting, which will lay a good foundation for the industrial production of common nuclear sterile rice.

[0018] The second is the hybrid breeding method. This breeding method is similar to the breeding method of the nuclear-cytoplasmic interaction male sterile line in the "three-line" hybrid rice technology system. The ordinary nuclear male sterile line is used as the hybrid female parent, and the ordinary nuclear male sterile breeding line is used as the hybrid male parent to breed the ordinary nuclear male sterile line through hybridization. In the specific breeding field, the ordinary nuclear male sterile line and the ordinary nuclear sterile breeding line are planted alternately in rows and planted individually. Artificial assisted pollination is carried out during heading and flowering. After pollination, the ordinary nuclear male sterile breeding line plants are removed, and only the seeds borne on the ordinary nuclear male sterile line plants are retained and harvested. In theory, the seeds borne on the ordinary nuclear male sterile line plants do not contain any genetically modified components. Compared with the production method of the first breeding method, the production method of this breeding method omits the technical link of color sorting of harvested seeds. However, the early stages of cultivation and assisted pollination have certain technical difficulties. It is necessary to study the optimal distance and proportion of alternating planting of common nuclear male sterile lines and corresponding breeding lines, as well as the feasibility and specific operating standards of artificial pollination or drone-assisted pollination.

[0019] In 2015, Li Xinqi and others successfully created the common nuclear male sterile line Gt1s, the breeding line Gt1S and the hybrid rice they formulated. At present, a series of common nuclear male sterile lines and their hybrid combinations have been cultivated, including excellent indica rice sterile lines, japonica rice sterile lines, salt-tolerant sterile lines and high-quality sterile lines with the eat1 sterile gene. "Sanyou No. 1" is the first strong and superior hybrid rice combination with common nuclear male sterile lines, with an acreage yield of 1046.3kg; in 2020, the average acreage yield was 911.7kg when planted as a double-season late rice. The common nuclear male sterile line G3-9s has free combination and has some indica and japonica genetic backgrounds. The hybrid combination shows strong heterosis effects and yield potential. The hybrids prepared have excellent plant type, strong tillering ability, large number of ears, large ears and many grains, strong stems, fertilizer resistance and lodging resistance, green leaves and yellow seeds in the later stage, no premature aging, vigorous functions of the last three leaves, and full and plump grains. Some of the medium-season rice hybrid combinations prepared have shown a yield of over 1100kg / 667m 2 The yield potential of some late rice hybrid combinations is 1000kg / 667m 2 The yield potential of this variety is great and the rice quality is excellent.

[0020] The first generation of hybrid rice was a three-line method using nuclear-cytoplasmic interaction male sterile lines as genetic tools, while the second generation of hybrid rice was a two-line method using photoperiod-temperature-sensitive male sterile lines as genetic tools. Limited by the restoration relationship, the available excellent parents are limited and the pairing is not free. The frequency of wild abortive maintenance lines is less than 0.1%, and the frequency of restorer lines is less than 5%. The genetic differences between sterile lines are small and the types are similar, which makes it difficult to break through the advantages of hybrid F1. The parent that hybridizes with the sterile line to produce completely sterile F1 is not necessarily the maintenance line. It is difficult to eliminate the minor effect restoration gene, which makes it difficult to identify and select the maintenance line. Some sterile lines are easily affected by high temperature and produce self-pollination. The restoration gene shows incomplete dominance, which makes it difficult for the hybrid seed setting rate to reach the level of conventional varieties. The flowering and pollination are affected by bad weather, and the fruit setting rate is not stable enough. The fertility of the second generation of photoperiod-temperature-sensitive nuclear male sterile lines is sensitive to the environment and the temperature drift of the sterility threshold, which easily causes the photoperiod-temperature-sensitive sterile lines to self-pollinate and reduce the purity of hybrid seed production. Weather changes are large-scale, and so are the risks. The fertility sensitive period of photothermosensitive sterile seed production is arranged in the high temperature season. The problem brought about by this is that the cross-breeding habits deteriorate under the high temperature weather during the sensitive period, and the seed production yield is low. The more complete the sterility, the worse the flowering habit. The common recessive nuclear male sterile system achieves the reproduction of common nuclear sterility by closely linking the fertility gene with the screening marker so that the fertile trait and the screening trait are co-separated, and then the fertile seeds and the sterile seeds are separated by the screening marker (see the SPT technology of Pioneer Seed Company in the United States). Half of the seeds on the rice ears of the common nuclear sterile breeding system are "colorless" seeds, and the other half are "fluorescent" seeds. "Colorless" seeds are non-GMO and male sterile, and can be used as female parents for hybrid seed production. The hybrid seeds produced are also non-GMO; "fluorescent" seeds are GMO and fertile, and can be used to breed genetically engineered male sterile lines. The rice ears of their self-pollinated offspring are half "colorless" seeds and half "fluorescent" seeds. The two can be separated by a color sorter, and the sterile lines and maintenance lines are produced in this way. Hybrid rice has vigorous physiological metabolism. Under adverse conditions, hybrid rice can better show its superior potential, has stronger stress resistance, and its yield is relatively less affected by adversity. Ordinary nuclear sterile lines are matched with salt-tolerant varieties to select salt-tolerant and high-yield combinations. Salt-tolerant varieties are transformed into ordinary nuclear sterile lines, matched with high-yield and excellent rice varieties, and salt-tolerant and high-yield hybrid rice salt-tolerant ordinary nuclear sterile lines are selected and hybridized with salt-tolerant varieties to improve salt tolerance and promote them in saline-alkali land with rice planting potential. This is a new way to ensure my country's food security. The three-excellent series of hybrid seawater rice cultivated under 0.6% seawater irrigation conditions have good growth performance and have the potential for large-scale application. At the same time, high-yield varieties that can still grow normally after seawater backflow have been cultivated. Hybrid seawater rice still grows well 5 days after seawater backflow.

[0021] 3. Hybrid seed sorting technology

[0022] Fluorescent color sorters use the optical property differences between materials and photoelectric technology to automatically sort heterochromatic particles in granular materials, effectively removing particles containing black spots or other colors. The device is equipped with an ultra-high-speed sensor electronic eye that can accurately identify tiny heterochromatic areas as small as 0.08 square millimeters, and has the advantages of being easy to use and simple to operate. It is widely used in the field of rice and other grain processing, and can completely remove chalky grains in rice, with a processing speed of 3 to 10 tons per hour. In the seed production process of the common nuclear male sterile line of rice, the sorting effect of the color sorter is very important. The sorting of breeding line seeds requires very high color sorter accuracy. However, the current color sorter technology cannot achieve 100% separation of general breeding line seeds after sorting, and fluorescent color sorting technology needs to be further improved.

[0023] In order to further improve the color sorting accuracy and ensure that the breeding line seeds can be completely separated, the method uses a fluorescence detector to detect and remove non-fluorescent non-breeding line plants at night in the breeding field, uses a fluorescence detector to detect and remove fluorescent breeding line plants at night in the seed production field, and humidifies the fluorescent and non-fluorescent seeds to distinguish fluorescent seeds and non-fluorescent seeds with high accuracy; at the same time, the pre-screened seeds are dried and shelled, and the residual fluorescent seeds, seeds with poor development and insect damage and impurities are further removed by a color sorter, and finally the visible fluorescent seeds are removed by naked eyes, so that 100% of the fluorescent seeds and non-fluorescent seeds can be separated. In addition, the shelled seeds are wrapped with artificial seed shells containing nutrients, bactericidal ingredients and protective functions, which significantly improves the seed emergence rate. Through the combination of the above methods, a large number of sterile female parents without colored fluorescent seeds can be effectively obtained for hybrid rice seed production and production, and the purity of hybrid rice seeds can be effectively improved. Summary of the invention

[0024] The purpose of the present invention is to provide a method for producing rice hybrid seeds, which can significantly improve the production efficiency and seed quality of rice hybrid seeds, obtain high-purity hybrid seeds, and reduce production costs.

[0025] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0026] A method for producing rice hybrid seeds comprises the following steps:

[0027] (1) Cultivating superior maternal reproductive lines: Cultivating a common nuclear sterile maternal reproductive line with a fluorescent gene linked to a fertile gene, and pairing the common nuclear sterile maternal reproductive line with superior parents to obtain superior hybrid combinations for production applications;

[0028] (2) Propagation by breeding lines: Select a breeding base based on ecological conditions to ensure that there are no major diseases and insect pests during the breeding stage, and that there is sufficient sunlight and suitable temperature, and avoid heavy rainfall during the maturity period; adopt single-plant planting and strict weed control for the obtained excellent hybrid combinations, and use a fluorescence detector to search and remove non-fluorescent plants at night during the maturity period to ensure the purity of the retained plants, and harvest self-pollinated seeds after they are fully mature;

[0029] (3) Fluorescence preliminary sorting: For the harvested self-pollinated seeds, use a fluorescent color sorter to perform preliminary sorting of the reproductive line seeds and the sterile line seeds to separate the fluorescent seeds from the colorless seeds;

[0030] (4) Seeds are further finely screened: the colorless seeds after preliminary sorting are dried and shelled, and then re-selected using a fluorescent color sorter to remove residual fluorescent seeds, seeds that are stunted or damaged by pests and diseases, and other impurities. Fluorescent seeds visible to the naked eye are manually removed; then after drying and high-temperature sterilization, the purity and quality of the seeds are sampled and tested according to standards, and stored in a low-temperature environment;

[0031] (5) Adding artificial seed shell: coating the surface of the seed with a layer of artificial seed shell, wherein the components of the artificial seed shell include matrix material, functional ingredients, adhesive, dye and filler;

[0032] (6) Hybrid seed production: Use colorless seeds with artificial seed shells for high-density sowing, seedling raising and transplanting, and strictly manage the weed removal process throughout the entire growth stage; use fluorescent detectors to search at night during the maturity stage of the seed production field, remove fluorescent plants, and produce high-purity hybrid seeds.

[0033] Furthermore, in step (1), a common nuclear sterile female breeding line having a fluorescent gene linked to a fertile gene is cultivated by hybridization, backcrossing, recrossing or molecular biological means.

[0034] Furthermore, in step (1), the female reproductive line of the common nuclear sterile line is the reproductive line Gt1S of the common nuclear male sterile line Gt1s.

[0035] Furthermore, the sufficient sunlight in step (2) refers to a light intensity of 30,000-50,000 lux, and the suitable temperature refers to an ambient temperature of 28-35°C.

[0036] Furthermore, in step (2), transplanting is performed when the seedlings have 4 to 6 leaves, with the best time being a sunny afternoon or a cloudy day; the transplanting density is controlled to be 10,000 to 20,000 holes per mu according to the variety and soil conditions; weeding is performed throughout the growth period, and strict weeding is performed before harvesting to remove abnormal plants.

[0037] Furthermore, in step (2), the harvested seeds are promptly dried and selected to remove impurities, shriveled seeds and moldy seeds, and then the selected seeds are stored in a ventilated and dry warehouse to reduce moisture content, prevent mold, and avoid moisture and insect pests.

[0038] Furthermore, in step (3), before sorting, the selfed seeds are sprayed with water or soaked in water to keep their surface moist, thereby enhancing the fluorescent color development effect.

[0039] Furthermore, in step (3), the fluorescent color sorter is specifically a color CCD fluorescent color sorter.

[0040] Furthermore, the colorless seeds in step (3) refer to seeds with natural color and no fluorescence.

[0041] Furthermore, in step (3), the seeds are tested for non-transgenic molecules, and if the purity reaches 100%, steps (4) and (5) are omitted.

[0042] Furthermore, in step (4), the temperature of high temperature sterilization is 50-55°C, and the temperature of low temperature storage is below 8°C.

[0043] Furthermore, in step (4), the fluorescent color sorter is used for reselection until sterile seeds with a purity of more than 99% are obtained.

[0044] Furthermore, in step (4), a DNA test is performed on the seeds to determine that the colorless seeds do not contain colored fluorescent seeds.

[0045] Furthermore, the artificial seed shell in step (5) specifically includes the following components:

[0046] Matrix materials, such as sodium alginate, starch, cellulose, polyvinyl alcohol (PVA), polylactic acid (PLA), gelatin, etc., are used to form the basic structure of the artificial seed coat;

[0047] Functional ingredients, such as pesticides, fungicides, growth regulators, fertilizers, trace elements, etc., are used to provide additional protection and promote growth;

[0048] Adhesives, such as gum arabic, polyvinyl alcohol, carboxymethyl cellulose (CMC), etc., are used to enhance the adhesion and stability of the seed coat;

[0049] dyes, used to mark and differentiate different types of seeds;

[0050] Fillers, such as talcum powder and diatomaceous earth, are used to adjust the physical properties of the seed coat.

[0051] The present invention has achieved the following technical effects:

[0052] The present invention significantly improves the seed purity and production efficiency of hybrid rice seed production and production through a series of innovative measures. Specifically, the method designs a linkage system of fluorescent genes and fertility genes to ensure high-precision sorting of breeding line seeds and precise management of the female parent during seed production. To further improve the color sorting accuracy, the following technical means are used:

[0053] 1. In the breeding field, the present invention uses a fluorescence detector at night to detect and remove non-fluorescent non-breeding plants; in the seed production field, the fluorescence detector is used at night to detect and remove fluorescent breeding plants, and the fluorescence characteristics are used to efficiently distinguish between impure plants and fluorescent seeds, thereby realizing rapid weed removal in large fields. The fluorescence detector has high resolution and high detection efficiency at night, which can avoid the low efficiency and high cost problems caused by post-harvest screening, and ensure seed purity and production efficiency.

[0054] 2. In the process of screening fluorescent and non-fluorescent seeds, the present invention improves the fluorescence effect by humidification treatment, and achieves high-precision distinction between fluorescent seeds and non-fluorescent seeds. After the initial screening, the seeds are further dried and shelled, and a color sorter is used to remove residual fluorescent seeds, stunted seeds, seeds infested by pests and diseases, and impurities. At the same time, the visible fluorescent seeds are removed by the naked eye, and finally the complete separation of fluorescent seeds and non-fluorescent seeds is achieved, and the purity can reach 100%.

[0055] 3. The present invention coats the shelled seeds with artificial seed shells containing nutrients, bactericidal ingredients and protective functions, effectively improving the germination rate, emergence rate and seedling rate of the seeds, while preventing pests and diseases from invading during sowing and seedling raising, further improving the reliability of seed production and use. The mechanism of action of the artificial seed shell is: sodium alginate and calcium ions (Ca 2 +) will quickly form a gel, which is due to the cross-linking reaction between the carboxyl group (-COO-) in the sodium alginate molecule and the calcium ion. When the sodium alginate solution comes into contact with the calcium ion solution, the calcium ions will enter the gaps between the sodium alginate molecules. The calcium ions combine with the carboxyl group (-COO-) in the sodium alginate molecule to form a bridging structure, thereby connecting multiple sodium alginate molecules together. When enough calcium ions combine with the carboxyl group in the sodium alginate molecule, a three-dimensional network structure will be formed, so that the solution will quickly solidify into a gel. Use natural polymers such as sodium alginate, cellulose derivatives, starch, collagen, and synthetic polymers such as polyvinyl alcohol and polylactic acid; and add diatomaceous earth, nano-silica, nano-clay and other materials to improve the barrier properties and mechanical strength. Add fertilizers, pesticides, growth regulators, seed coating agents, etc. to form a protective layer outside the breeding system brown rice, and improve the growth effect of ordinary sterile lines.

[0056] 4. The present invention can not only obtain a large number of high-purity sterile female plants without fluorescent seeds for hybrid rice seed production, but also effectively solve the technical problem that the ordinary nuclear male sterile breeding seeds are affected by the difference in maturity or abnormal development and the screening effect is affected, thereby ensuring that there are no breeding plants in the seed production field and promoting the healthy and rapid application and development of the ordinary nuclear sterile line. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a diagram showing the early stage of rice panicle maturity of the rice breeding line G3-8S;

[0058] Figure 2 This is a graph showing that the reproductive line Gt1s sterile line blooms earlier than the male parent;

[0059] Figure 3 The performance of wheat breeding lines under natural light (a) and under a fluorescence detector at night (b);

[0060] Figure 4 This is a graph showing the performance of wheat breeding line seeds under a fluorescence detector at night;

[0061] Figure 5 This is the performance diagram of rice fluorescent seeds and ordinary seeds under the fluorescence detector;

[0062] Figure 6 This is a color difference chart of the sterile and reproductive lines of hulled rice. DETAILED DESCRIPTION

[0063] The present invention is further described in detail below in conjunction with examples, but these examples should not be used to limit the scope of protection of the present invention.

[0064] The present invention specifically proposes a method for producing rice hybrid seeds, which mainly comprises the following steps:

[0065] (1) Cultivate a female reproductive line of a common nuclear sterile line having a fluorescent gene as a selection marker linked to a fertile gene by hybridization, backcrossing, recrossing, cell biology or molecular biology (see Figure 1 ). It is paired with an excellent parent (preferably the breeding line Gt1s of the common nuclear male sterile line Gt1s with early flowering habit as the parent material) to obtain an excellent hybrid combination that can be promoted and applied, the so-called third-generation hybrid rice. Gt1s is usually selected as the sterility gene donor because it carries the early flowering gene and has an early flowering habit. Transferring the early flowering habit of Gt1s to the sterile line with different genetic backgrounds can coordinate the flowering time of the parents to meet, increase seed production, and solve the problem of delayed flowering and asynchronous flowering with the father due to the impact on the developmental physiology of the sterile line (see Figure 2 ).

[0066] (2) Select an excellent breeding base for breeding lines based on the characteristics of ecological conditions and perennial meteorological data. Ensure that there are no major diseases and insect pests when the breeding lines enter the reproductive development stage, that there is sufficient sunlight and suitable temperature conditions (28-35℃), and that there is no heavy or moderate rain during the maturity period, so as to promote the full maturity and health of the self-pollinated seeds on the rice ears. Transplant the seedlings when they are 4-6 leaves, the best time is a sunny afternoon or cloudy day, and plant them alone. Control the appropriate transplanting density according to the variety and soil conditions, generally 10,000-20,000 holes per mu. Adopt moist management, medium to high fertility levels, and strict disease and pest control. Ensure that the seeds in the breeding rice ears are fully mature, so that the boundary between colorless seeds and colored fluorescent seeds is clear. During the entire growth period, remove the weeds in time, and strictly remove weeds and remove the deformed plants before harvesting.

[0067] During the maturity period, a fluorescence detector is used to remove the non-fluorescent plants at night. The applicant found that the fluorescent protein is not obvious under strong natural light and has low resolution; at night, the fluorescent protein seeds have an ideal display effect and can easily distinguish fluorescent seeds from ordinary seeds (see Figure 3 and Figure 4 ). During the reproduction process of the breeding line, the fluorescence detector can remove the non-fluorescent plants, thereby completely removing the non-breeding line hybrids, ensuring the purity of the retained fluorescent plants, and thus ensuring that the sterile line seeds for field use are not mixed with fertile seeds. After harvest, the breeding line seeds are routinely selected to ensure that they are quickly harvested after full maturity, and are dried in time to remove impurities, shriveled grains and moldy seeds. The selected seeds are stored in a ventilated and dry warehouse to reduce the moisture content and prevent mold and insect pests.

[0068] (3) Use fluorescent color sorting machines to mechanically sort breeding line seeds and sterile line seeds. The sorting machine uses color as the sorting mark trait (see Figure 6 ), add a fluorescent excitation light source to the ordinary color sorter (refer to Anke 6SX2-126LED color CCD fluorescent color sorter), and achieve separation by exciting the fluorescent seeds to produce fluorescence.

[0069] Before fluorescent screening, the self-pollinated seeds are sprayed with water or soaked in water to maintain a certain humidity to enhance the fluorescent color development effect; after absorbing water, the fluorescent protein can show fluorescent color through the husk, which is closer to the color of the husk-free colored fluorescent seeds, thus helping to distinguish the colored fluorescent seeds from the colorless seeds. Through the initial selection of the fluorescent color sorter, most of the colorless sterile seeds are separated from the fluorescent breeding seeds; then the color sorter is used for reselection until the sterile seeds with a purity of more than 99% are obtained. After the seed purity is tested to meet the use standard, the obtained colorless seeds can be directly used for seed production. If the purity reaches 100%, steps (4) and (5) can be omitted.

[0070] (4) The colorless seeds obtained by sorting are dried and then shelled, and the remaining fluorescent seeds, underdeveloped seeds or seeds damaged by pests and diseases and impurities are removed again by a color sorter, and the visible fluorescent seeds are manually removed (see Figure 5 ). According to national standards, samples are taken for DNA testing to ensure that colorless seeds do not contain colored fluorescent seeds. The seeds are then dried, sterilized at high temperatures, and sampled for seed purity and quality, and finally stored in a low-temperature environment.

[0071] (5) Adding artificial seed husks to seeds to form a protective layer on the outside of the seeds. By adding specific biochemical agents (such as insect repellents or fungicides), seeds are helped to resist the influence of adverse environmental conditions such as pests and diseases. Artificial seed husks contain substances that promote seed germination (such as plant growth regulators) and nutrients such as nitrogen, phosphorus, and potassium, which provide seeds with the nutrients needed for initial growth, thereby promoting the healthy growth of seedlings. Sterile artificial seed husks imitate the shell function of natural seeds, provide protection and support for seeds, and promote their successful germination under suitable conditions. If brown rice is sown directly, the germination rate is usually not affected and may even be improved. However, due to the lack of shell protection, seeds are susceptible to pests and diseases during the seedling process and die. The artificial husk designed by the applicant is different from the coating agent of ordinary seeds and the seed coat of artificial seeds. It has the characteristics of both in terms of composition and function. It mainly acts as a husk to inhibit pests and diseases and resist adverse environments.

[0072] (6) When used for hybrid seed production, high-density sowing, seedling raising and transplanting are required, and strict weed removal is required throughout the growth stage. During the maturity period of the seed production field, a fluorescence detector is used at night to remove fluorescent single plants. There may be fluorescent breeding seeds in the seed production field, so they need to be 100% removed. The applicant found that the fluorescent protein is not obvious and has low resolution under strong natural light, but the detection effect is ideal at night, and it is easy to distinguish fluorescent seeds from ordinary seeds. During the maturity period of seeds in the seed production field, using a fluorescence detector to quickly detect and remove fluorescent plants in the field is an efficient weed removal method that can ensure that hybrid seeds in the field are not mixed with breeding seeds. The fluorescence detector has high detection efficiency, and one technician can detect more than 100 acres of seed production fields. If detection and sorting are done after harvest, not only will the effect be poor and the efficiency be low, but the cost will also be high.

[0073] Specific embodiments are listed below. The crop varieties (lines) used in each embodiment are all varieties (lines) conventionally used in the breeding field, have passed national approval or technical appraisal, and can be obtained in the variety resource library or purchased on the market.

[0074] Embodiment 1:

[0075] 1. Construction of sterile and reproductive lines

[0076] Taking the pollen fertility gene of common nuclear sterile rice and the corresponding sterile mutant as the object, a ternary vector was constructed for the linked expression of the fluorescent protein DsRed gene driven by the endosperm-specific promoter LDP2, the common nuclear sterile fertility gene EAT1 and the pollen lethal gene ZMAA1 driven by Pg47, and transferred into the rice common nuclear sterile mutant eat1 through Agrobacterium-mediated transformation. One plant with normal fertility was screened out from tens of thousands of positive rice plants. The common nuclear male sterile line Gt1s and its breeding line Gt1S that meet the standards of rice male sterile lines were created.

[0077] 2. Planting, harvesting and handling of propagation lines

[0078] Gt1S was planted in winter in Yacheng, Sanya, Hainan. The local climate and soil conditions are very suitable for rice growth. Sowing was done on December 6 to cultivate strong seedlings. Transplanting was done when the seedlings were 4-6 leaves, in the afternoon on sunny days and on cloudy days. Single planting. 15,000 holes per mu. Moisture management, medium-high fertility level, strict pest and disease control, weeding was done during the entire growth period, and before harvesting, weeding was done strictly to remove abnormal plants. At around 8 p.m. during the maturity period of Gt1S, an LED fluorescence excitation device (LUYOR-3415GHand-Held Lamp) was used to detect and remove single plants without fluorescence in the field. Harvest quickly after full maturity. After self-pollination of the common nuclear male sterile breeding line Gt1S, each rice ear produced half of the sterile line seeds and half of the breeding line seeds. The seeds that were solid on the plants of the common nuclear breeding line were harvested. After harvesting, they were dried in time to reduce the moisture content and prevent mildew, and were carefully selected to remove impurities, shrunken grains and mildewed seeds. Store the selected rice in a ventilated, dry warehouse to avoid moisture and insect infestation.

[0079] 3. Sorting of reproductive line seeds and sterile line seeds

[0080] (1) Use 6SX2-126LED color CCD fluorescent color sorter to sort most of the colorless sterile seeds from the fluorescent breeding seeds; then use the color sorter to sort again until more than 98% of the seeds are separated. The first sorting obtains colorless seeds with a purity of 98.9%, and the second sorting obtains colorless seeds with a purity of 99.7%.

[0081] (2) The colorless seeds are hulled with a brown rice machine, and then the colorless sterile seeds and the remaining fluorescent breeding seeds are separated by a color sorter until the colorless seed ratio no longer increases. Then, the colorless seeds are checked by naked eyes to confirm that the colored fluorescent seeds are not contained in the colorless seeds.

[0082] 4. Drying and sterilization

[0083] The colorless seeds are dried in a drying oven and sterilized at a high temperature of 53 degrees Celsius.

[0084] 5. Add artificial seed shell.

[0085] Prepare materials: sodium alginate powder, carbendazim 0.3g / kg seeds, calcium chloride (CaCl2), deionized water or distilled water, G3-8S colorless brown rice seeds, stirrer or magnetic stirrer, weighing apparatus, container, syringe or dropper, filter or funnel.

[0086] Production steps:

[0087] (1) Preparation of sodium alginate solution

[0088] Weigh an appropriate amount of sodium alginate powder according to the required concentration. The concentration is 1.5%.

[0089] Dissolve sodium alginate: Slowly add sodium alginate powder into deionized water, stirring with a stirrer to prevent lumps. Continue stirring until it is completely dissolved to form a uniform transparent solution.

[0090] (2) Preparation of seed mixture

[0091] Select G3-8S colorless brown rice seeds and make sure the seeds are dry and free of impurities. Add the seeds to the prepared sodium alginate solution and stir gently to evenly distribute the seeds in the solution. Make sure each seed is covered by the solution.

[0092] (3) Preparation of calcium ion solution

[0093] Weigh an appropriate amount of calcium chloride powder according to the required concentration. The concentration is 0.8%. Add the calcium chloride powder into deionized water and stir until it is completely dissolved.

[0094] (4) Forming an artificial seed shell

[0095] Add the mixture drop by drop: Use a syringe or dropper to drop the sodium alginate solution containing the seeds into the calcium ion solution drop by drop. The drop will quickly form a gelled artificial seed shell when it comes into contact with the calcium ion solution. Let the formed artificial seed shell soak in the calcium ion solution for 1-2 minutes to ensure complete solidification. Use a strainer or funnel to remove the gelled artificial seed shell from the calcium ion solution and rinse it with clean water several times to remove excess calcium ions. Make sure that each artificial seed shell is completely solidified and has a smooth surface. Place the prepared seeds in a clean container and store in a cool and dry place.

[0096] 6. Seed production

[0097] Hybrid seed production using the rice sterile line Gt1s and the restorer line 19z-1:

[0098] (1) Sowing at the right time to ensure a safe flowering period. In the technical system of the "three-line method" hybrid rice, the female parent reproduction of the nuclear-cytoplasmic interaction male sterile line is the same as hybrid seed production. The most suitable rice ear temperature for the female parent to bloom is 28-32°C, and the relative humidity in the field is 75%-85%. It requires clear weather without more than three consecutive days of rain. When arranging the heading and flowering period, it is necessary to avoid the "plum rain" season in mid-June and the high temperature season of "fire south wind" in late July each year. Therefore, the heading and flowering of seed production should be arranged from late June to early July.

[0099] (2) Cultivate strong seedlings of appropriate age. When breeding from parents, the seedling age of both parents is short (about 20d to 25d), so it is more important to cultivate strong seedlings of appropriate age with multiple tillers. First, select a nursery field with enough seedlings, with the ratio of nursery field to field being 1:3. Secondly, apply enough base fertilizer to the nursery field. Apply 60kg of compound fertilizer to the nursery field. Apply 10kg of compound fertilizer to promote tillering per mu. Apply 5kg of urea per mu 5d to 7d before transplanting. Sow sparsely and evenly, and strictly control the sowing amount of both parents per mu of nursery field to less than 15kg. After sowing, collapse the valley into mud and cover with residue fertilizer, herbal ash or fine cow dung. It is best to use two-stage seedling raising for the male parent to promote early tillering and more tillering. The female parent seedling is required to be about 20d old and have more than 2 tillers. The first-stage male parent seedlings are about 23 days old, with more than 2.5 tillers; the second-stage male parent seedlings are 20 days old, with more than 2.0 tillers.

[0100] (3) Establish a reasonable high-yield population and increase the number of effective panicles. When producing seeds, the row ratio of the parent plant is 2:10; the row spacing of the female plant is 16.6cm×13.3cm, and two rice seedlings are planted in each hole, so that the effective panicles of the female plant per hectare can reach 3 million; the row spacing of the male plant is 13.3cm×26.7cm, and the distance between the male plant and the female plant on both sides is 20cm. 2-3 rice seedlings are planted in each hole of the male plant, so that the effective panicles of the male plant per mu can reach about 90,000.

[0101] (4) Rational fertilization to coordinate the balanced growth of the parents. The fertilizer requirement of the common nuclear male sterile line is relatively large, mainly basal fertilizer, with a reasonable combination of nitrogen fertilizer, phosphorus fertilizer and potassium fertilizer. In terms of fertilization methods, basal fertilizer accounts for 70% and topdressing accounts for 30%. 5 to 7 days after the male parent is transplanted, 5 kg of urea plus 6 kg of superphosphate and 4 kg of potassium chloride are applied per mu. After the field is sun-dried and re-watered, it depends on the growth of the seedlings. For the female parent with heavy yellowing and weak growth, 3 kg to 5 kg of urea and 5 kg to 6 kg of potassium chloride are applied per mu to ensure stable growth without excessive growth, green leaves and strong stalks without premature aging. In terms of water management, shallow water is frequently irrigated in the early stage to promote rapid tillering; in the middle stage, the seedlings are sun-dried; from the 4th stage of young spike differentiation to the heading and flowering stage, a 4 cm water layer is maintained in the field; in the later stage, intermittent irrigation is carried out to maintain the vitality of the root system and prevent premature aging of the root system.

[0102] (5) Apply "920" scientifically, 8 grams per mu for the male plant and 6 grams per mu for the female plant.

[0103] (6) Strictly prevent weeds and maintain purity to ensure seed quality. Establish a strictly isolated breeding area, and completely remove weeds during the growth and development process to ensure the quality of the breeding seeds. Select a space with good natural conditions for isolation, which should be more than 100m. Within this range, no other rice varieties can be planted except for the common nuclear male sterile breeding line. During the entire growth stage of the common nuclear male sterile line, the breeding line, recovery line and foreign plants mixed in it must be repeatedly and thoroughly removed. At the same time, the breeding line must also be cleaned and purified. Fine weed removal is an important technical measure to ensure the purity of hybrid rice seeds. Weeds and mutants in the field must be removed before the first panicle sheds pollen.

[0104] Other cultivation and management measures for Gt1s seed production are the same as those for general hybrid rice seed production. Before harvest, strict weeding is carried out to remove abnormal plants. At around 8 p.m. during the maturity period, fluorescent excitation devices are used to detect and remove fluorescent plants in the field. Through the above measures, the purity of Gt1s seed production has reached 99.5%, with no colored fluorescent seeds, and a yield of 165.2 kg per mu.

[0105] Embodiment 2:

[0106] 1. Gt1S was hybridized with rice variety Pei'ai 64, and excellent breeding line plants with early flowering traits of Gt1s were selected in the offspring. Through hybrid breeding, ordinary nuclear sterile breeding line G3-8S and G3-8S were bred. They were sown in the Chunhua transgenic experimental field in Changsha, Hunan Province on May 12, and transplanted when the seedlings were 4-6 leaves, in the afternoon of sunny days and cloudy days. Single planting. According to the variety and soil conditions, 15,000 holes were planted per mu. Moisture management, medium-high fertility level, and other cultivation measures were the same as general field production. Heading on August 16, with suitable high temperature and high light conditions. Strict pest and disease control, weeds were removed during the entire growth period, and before harvest, weeds were strictly removed to remove abnormal plants. The maturity period was at night after dark, and a fluorescence excitation device (LUYOR-3415G Hand-Held Lamp) was used to detect and remove single plants without fluorescence in the field. Harvest quickly after full maturity, and dry in time after harvest to reduce moisture content and prevent mildew. Harvest seeds from ordinary nuclear propagation plants and select them with ordinary rice selectors. Store the selected rice in a ventilated and dry warehouse to avoid moisture and insect pests.

[0107] 2. Sorting of reproductive line seeds and sterile line seeds

[0108] G3-8S self-pollination seeds were soaked in sterile water for one hour to make the seeds more fully colored, and then drained. The seeds were preliminarily sorted under the 6SX2-126LED color CCD fluorescent color sorter to separate most of the colorless sterile seeds from the fluorescent breeding seeds; the color sorter was then used for re-selection until 98.9% of the colorless seed samples were obtained. After sorting, the sorted colorless seeds were quickly dried.

[0109] 3. Use a rice husker to shell the colorless seed samples, and use the 6SX2-126LED color CCD color sorter to sort the colorless sterile seeds and the remaining fluorescent breeding seeds, and reselect again until there are no fluorescent seeds in the colorless seeds. Through visual inspection, no fluorescent seeds are mixed in the colorless seeds.

[0110] 4. Drying and sterilization

[0111] Sterilize and dry at 53 degrees Celsius in a drying oven.

[0112] 5. Add artificial seed shell

[0113] (1) Preparation of matrix solution

[0114] 50 g of starch and 50 g of polyvinyl alcohol (PVA) were dissolved in 500 ml of water, heated to 80° C. and stirred until completely dissolved to form a uniform matrix solution.

[0115] (2) Functional ingredient blending

[0116] Mix 5 g of nano-TiO2, 2.5 g of thiamethoxam, 0.5 g of carbendazim, and 0.1 mg of 6-benzylaminopurine evenly and add them into the matrix solution.

[0117] (3) Addition of adhesive

[0118] Add 5 g of gum arabic to the base solution and stir well to ensure that the adhesive is fully dissolved.

[0119] Add the filler talc and stir well to ensure all ingredients are evenly mixed.

[0120] (4) Coating operation

[0121] Remove impurities and dust from the hull-less rice seeds. Dry them with drying equipment. Put the dried hull-less rice seeds into the seed coating machine. Start the coating machine and spray the prepared coating liquid evenly on the surface of the seeds. The stirring device in the coating machine will constantly turn the seeds to ensure that the coating liquid is evenly distributed. The coating time is 10-15 minutes to ensure that each seed is fully exposed to the coating liquid.

[0122] (5) Drying and curing

[0123] After coating is completed, continue to run the coating machine and use hot air or other drying methods to preliminarily dry the coating liquid on the surface of the seeds. The preliminary drying time is 10-15 minutes. Transfer the preliminarily dried seeds to the drying equipment for thorough drying. The drying temperature is 30-40℃ and the drying time is 2-4 hours to ensure that the coating is completely cured.

[0124] (6) Check quality

[0125] Visually inspect the surface of the hull-less rice seeds to see if they are evenly coated with a layer of coating to ensure that there are no missing bags or uneven coating. Take a small amount of coated hull-less rice seeds and sow them in the nursery field for germination rate test to ensure that the coating process does not affect the germination ability of the seeds. Evaluate the coating effect.

[0126] (7) Packaging and storage

[0127] Package the coated rice hull-less seeds according to the specifications, and ensure that the packaging materials are well sealed to prevent external contamination. Label the seed variety, coating ingredients, production date and other information. Store the packaged rice hull-less seeds in a cool, dry, ventilated place, avoiding high temperature and humidity. The storage temperature is 5-10℃ and the relative humidity is 50-60%.

[0128] Other cultivation and management measures for G3-8s seed production are the same as those for general hybrid rice seed production. Before harvest, strict weeding is carried out to remove abnormal plants. At around 8 p.m. during the maturity period, fluorescent plants are detected and removed using a fluorescence excitation device in the field. Through the above measures, the purity of G3-8s seed production reached 99.8%, with no colored fluorescent seeds, and a yield of 167.3 kg per mu.

[0129] Embodiment three:

[0130] 1. Cultivation and management of G3-9S breeding line

[0131] The G3-9S breeding line was bred by hybridizing Gt1S with the rice variety Minghui 63. It has the early flowering trait of Gt1s. G3-9s usually blooms around 11 a.m., 30-60 minutes earlier than the male parent. The breeding line was sown on May 16 in the Chunhua transgenic experimental field in Changsha, Hunan. To ensure the best growth state, transplanting was carried out when the seedlings grew to the 4-6 leaf stage, and it was carried out on a sunny afternoon or cloudy day to reduce damage to the seedlings. The single plant planting method was adopted, and 15,000 plants were planted per mu of land according to different variety characteristics and soil conditions. The soil was kept moist, medium to high fertilizer levels were provided, and conventional field production measures were followed. On August 21, G3-9S began to head, which was a season of high temperature and sufficient light, providing an ideal growth environment for G3-9S. In order to ensure the healthy growth of G3-9S, strict disease and pest control measures were implemented, and weeds were removed immediately when they were found. Before harvesting, a thorough weeding was carried out, with special attention paid to removing plants with abnormal appearance. At night, a fluorescence excitation device (LUYOR-3415G hand lamp) was used to detect and remove plants that did not fluoresce. G3-9S was harvested quickly when it was fully mature and dried immediately to reduce moisture and avoid mold. The collected seeds were carefully screened and finally stored in a well-ventilated and dry warehouse to prevent moisture and insects.

[0132] 2. Separation of reproductive line seeds and sterile line seeds

[0133] In order to effectively distinguish the reproductive and sterile seeds from the seeds produced by G3-9S self-pollination, the seeds were first soaked in sterile water for about an hour to better show their fluorescent color characteristics. Subsequently, a 6SX2-126LED color CCD fluorescence color sorter was used for preliminary screening, the main purpose of which was to separate the mostly colorless sterile seeds from the fluorescent reproductive seeds. Next, a secondary screening was performed using the color sorter to obtain colorless seeds with a purity of 98.9%. After the screening was completed, the colorless seeds were immediately dried.

[0134] 3. Further processing of colorless seeds

[0135] The colorless seeds obtained after the initial screening are husked by a rice husker, and then further screened by a 6SX2-126LED color CCD color sorter to completely eliminate any residual fluorescent breeding seeds. This process needs to be repeated many times until it is confirmed that the colorless seeds no longer contain fluorescent seeds. Finally, a visual inspection is performed to ensure that there are no luminous seeds mixed in with the colorless seeds.

[0136] 4. Drying and sterilization

[0137] Sterilize and dry at 53 degrees Celsius in a drying oven.

[0138] 5. Add artificial cellulose derivatives and mix with sodium alginate to make artificial seed shells

[0139] Mixing sodium alginate with cellulose derivatives not only ensures the rapid formation of gel, but also improves the mechanical strength and durability of the seed coat. Mixing sodium alginate with cellulose derivatives can significantly improve the performance of artificial seed coat, especially in terms of mechanical strength, flexibility and biocompatibility. Sodium alginate is a natural polysaccharide extracted from brown algae. It can react with calcium ions (Ca 2 +) and other divalent cations to form gels, which are also widely used in the food industry, pharmaceutical field and other fields. The following are the basic steps for using sodium alginate solution and calcium ion solution to form gels:

[0140] (1) Prepare materials

[0141] Sodium alginate powder, sodium benzoate, Thiram;

[0142] General dosage: 0.5g-2g / kg seeds;

[0143] Calcium chloride (CaCl2) solution, deionized or distilled water, stirrer or magnetic stirrer, weighing equipment, container, syringe or dropper. During the preparation process, make sure all the equipment used is clean to avoid contamination.

[0144] (2) Preparation of sodium alginate solution

[0145] Weigh a certain amount of sodium alginate powder according to the required concentration.

[0146] Sodium alginate powder was slowly added into deionized water while stirring well with a stirrer to avoid agglomeration.

[0147] Continue stirring until the sodium alginate is completely dissolved to form a uniform solution. This process takes some time because sodium alginate dissolves slowly.

[0148] (3) Preparation of calcium ion solution

[0149] Select a suitable calcium salt, such as calcium chloride (CaCl2), according to the needs, and weigh the corresponding weight according to the required concentration. Dissolve the weighed calcium salt in deionized water and stir until it is completely dissolved.

[0150] (4) Gel formation

[0151] Control the temperature of the reaction environment. Too high a temperature may cause sodium alginate to degrade. Drop the prepared sodium alginate solution into the calcium ion solution through a dropper or syringe. When the sodium alginate droplets come into contact with the calcium ion solution, the surface will quickly undergo a cross-linking reaction with the calcium ions to form gel beads. Adjust the speed and method of dripping as needed to control the size and shape of the gel beads. Allow the formed gel beads to stay in the calcium ion solution for a period of time to complete the cross-linking process. Use a filtration method to separate the gel beads from the calcium ion solution and wash them with deionized water to remove excess calcium ions. Adjust the concentrations of the sodium alginate solution and the calcium ion solution to change the properties of the final gel, such as hardness and elasticity.

[0152] In the production and seed production of G3-9s, in addition to taking the same cultivation and management measures as conventional hybrid rice seed production, special emphasis is placed on strict weed removal before harvest. Specifically, a comprehensive weed removal operation is carried out before harvest, with an emphasis on removing deformed plants in the field. When the crop is close to maturity, at around 8 p.m., a fluorescent excitation device is used to conduct detailed inspections in the field to identify and remove plants that exhibit fluorescent characteristics. By implementing these precise management measures, G3-9s used in seed production not only achieves a high purity of 99.5%, ensuring that the seeds do not contain any individuals with fluorescent markers, but also achieves a good yield of 126.8 kilograms per mu.

[0153] Although the present invention has been disclosed as above by way of embodiments, it is not intended to limit the present invention. Appropriate modifications or equivalent substitutions of the technical solutions of the present invention made by ordinary technicians in the field should all be included in the protection scope of the present invention. The protection scope of the present invention shall be based on what is defined in the claims.

Claims

1. A method for producing rice hybrid seeds, characterized in that: The following steps are involved: (1) Cultivating superior maternal reproductive lines: Cultivating a common nuclear sterile maternal reproductive line with a fluorescent gene linked to a fertile gene, and pairing the common nuclear sterile maternal reproductive line with superior parents to obtain superior hybrid combinations for production applications; (2) Propagation by breeding lines: Select a breeding base based on ecological conditions to ensure that there are no major diseases and insect pests during the breeding stage, and that there is sufficient sunlight and suitable temperature, and avoid heavy rainfall during the maturity period; adopt single-plant planting and strict weed control for the obtained excellent hybrid combinations, and use a fluorescence detector to search and remove non-fluorescent plants at night during the maturity period to ensure the purity of the retained plants, and harvest self-pollinated seeds after they are fully mature; (3) Fluorescence preliminary sorting: For the harvested self-pollinated seeds, use a fluorescent color sorter to perform preliminary sorting of the reproductive line seeds and the sterile line seeds to separate the fluorescent seeds from the colorless seeds; (4) Seeds are further finely screened: the colorless seeds after preliminary sorting are dried and shelled, and then re-selected using a fluorescent color sorter to remove residual fluorescent seeds, seeds that are stunted or damaged by pests and diseases, and other impurities. Fluorescent seeds visible to the naked eye are manually removed; then after drying and high-temperature sterilization, the purity and quality of the seeds are sampled and tested according to standards, and stored in a low-temperature environment; (5) Adding artificial seed shell: coating the surface of the seed with a layer of artificial seed shell, wherein the components of the artificial seed shell include matrix material, functional ingredients, adhesive, dye and filler; (6) Hybrid seed production: Use colorless seeds with artificial seed shells for high-density sowing, seedling raising and transplanting, and strictly manage the process of removing impurities throughout the growth stage; During the maturity stage of seed production fields, fluorescent detectors are used to search at night, remove fluorescent plants, and produce high-purity hybrid seeds.

2. The method according to claim 1, characterized in that In step (1), a common nuclear sterile female breeding line having a fluorescent gene linked to a fertile gene is cultivated by hybridization, backcrossing, recrossing or molecular biological means.

3. The method according to claim 1, characterized in that In step (1), the common nuclear sterile female reproductive line is the reproductive line Gt1S of the common nuclear male sterile line Gt1s.

4. The method according to claim 1, characterized in that The sufficient sunlight in step (2) refers to a light intensity of 30,000-50,000 lux, and the suitable temperature refers to an ambient temperature of 28-35°C; the high-temperature sterilization temperature in step (4) is 50-55°C, and the low-temperature storage temperature is below 8°C.

5. The method according to claim 1, characterized in that In step (2), transplanting is performed when the seedlings have 4 to 6 leaves, and the best time is in the afternoon on a sunny day or on a cloudy day; the transplanting density is controlled to be 10,000 to 20,000 holes per mu according to the variety and soil conditions; weeding is performed throughout the growth period, and strict weeding is performed before harvesting to remove abnormal plants; The harvested seeds are dried in time and selected to remove impurities, shriveled grains and moldy seeds. The selected seeds are then stored in a ventilated and dry warehouse to reduce the moisture content, prevent mold, and avoid moisture and insect pests.

6. The method according to claim 1, characterized in that In step (3), before sorting, the selfed seeds are sprayed with water or soaked in water to keep their surface moist, thereby enhancing the fluorescent color development effect.

7. The method according to claim 1, characterized in that In step (3), the fluorescent color sorter is specifically a color CCD fluorescent color sorter; the colorless seeds refer to seeds with natural color and no fluorescence.

8. The method according to claim 1, characterized in that In step (3), the seeds are tested for non-transgenic molecules. If the purity reaches 100%, steps (4) and (5) are omitted.

9. The method according to claim 1, characterized in that In step (4), the fluorescent color sorter is used for reselection until sterile seeds with a purity of more than 99% are obtained; and a DNA test is performed on the seeds to determine that the colorless seeds do not contain colored fluorescent seeds.

10. The method according to claim 1, characterized in that In step (5), the matrix material of the artificial seed shell is selected from one or more of sodium alginate, starch, cellulose, polyvinyl alcohol, polylactic acid, and gelatin; the functional ingredients are selected from one or more of pesticides, fungicides, growth regulators, fertilizers, and trace elements; the adhesive is selected from one or more of gum arabic, polyvinyl alcohol, and carboxymethyl cellulose; and the filler is selected from one or more of talc and diatomaceous earth.

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