Method for purifying generation-adding breeding hybrid rice three-line sterile line by utilizing regeneration method

Abstract

The invention relates to the technical field of rice heterosis utilization, in particular to a method for purification and generation-adding breeding of a hybrid rice three-line sterile line by using a regeneration method. According to the method disclosed by the invention, the sterile line is rapidly purified and propagated by adopting a'regeneration method 'under the condition that the purity is reduced after the hybrid rice three-line sterile line is subjected to multi-generation propagation and the purity is lower than 99.5% and does not reach the standard. The method is easy to operate, short in period and good in purification effect, the purity of the sterile line can be remarkably improved, sufficient seed sources can be obtained, and the hybrid seed production requirement is met.

Description

Technical Field

[0001] This invention relates to the field of rice heterosis utilization technology, specifically to a method for purifying and propagating hybrid rice three-line sterile lines using a regeneration method. Background Technology

[0002] With the rapid development of hybrid rice and the increasing number of newly approved varieties each year, rice growers have increasingly higher requirements for rice seed quality, and production units are also continuously raising their requirements for the purity and quantity of three-line male-sterile lines in rice. The general production process for three-line male-sterile lines in hybrid rice is as follows: selection of original seeds for three-line male-sterile lines → propagation of original male-sterile lines → generational propagation of male-sterile lines → purification and rejuvenation through a "multi-field system" → propagation of male-sterile lines. In actual production, production units need to propagate the provided original seeds to meet production needs. To meet the required number of male-sterile seeds for first-generation hybrid rice production, production units continuously propagate through multiple generations. However, during each generation, several factors lead to a decrease in the purity of the male-sterile seeds, such as mechanical mixing during harvesting and drying, mixing of fallen grains from the previous season's rice, mixing of maintainer lines, and biological cross-pollination of hybrid plants. As the number of generations increases, the purity of the male-sterile lines continuously declines, eventually failing to meet the standards (seed purity not reaching 99.5%). However, there is still a need for this three-line male-sterile rice in production. In the past, the male-sterile line was purified using the "multi-field system". However, the "multi-field system" is technically demanding, has a long cycle, and produces a limited number of seeds, which can easily lead to a mismatch between demand and supply.

[0003] Therefore, there is an urgent need to develop a method that can rapidly improve the purity of the three-line male-sterile line in hybrid rice and increase the number of male-sterile line seeds. Summary of the Invention

[0004] To address the above problems, this invention provides a method for purifying and propagating hybrid rice three-line sterile lines using a regeneration method.

[0005] This invention is achieved through the following technical solution: A method for purifying and propagating hybrid rice three-line male-sterile lines using a regeneration method includes the following steps: S1. Seedling raising in the seedbed: Sow sterile line A and maintainer line B in the seedbed to cultivate strong seedlings.

[0006] S2. Transplanting: When the seedlings have 5.1 to 5.8 leaves, they are lifted from the seedbed and transplanted to the main field.

[0007] S3. First removal of hybrid plants: After transplanting to the main field until the 5th to 6th stage of young spikelet differentiation, remove non-variety plants in the rows of sterile line A and maintainer line B according to the differences in plant morphology and leaf color.

[0008] S4. Second weeding and first "green cutting" treatment: During the heading stage to full heading stage of male-sterile line A and maintainer line B, do not spray gibberellin. Use the necking phenomenon of male-sterile line A to identify and remove hybrid plants in the row of male-sterile line A that do not have a neck and whose agronomic traits are different from those of male-sterile line A. At the same time, remove hybrid plants in the row of maintainer line B according to the differences in agronomic traits. After weeding, cut off all male-sterile lines A and maintainer line B.

[0009] S5. Promote regeneration: After harvesting, ensure that there is a water layer of 4 to 5 centimeters in the field to promote the regeneration of rice stubble.

[0010] S6. Management of regenerated seedlings and third weeding: When the flag leaves of the regenerated sterile line A and the maintainer line B have grown to 50%~60%, apply fertilizer and remove any stray plants according to the differences in agronomic traits.

[0011] S7. Flowering period regulation and gibberellin spraying: First application: When 20%–30% of the regenerating sterile line A shows white markings, spray only line A with gibberellin at a rate of 100 mg / 666.7 m³. 2 The dosage is 10g~12g.

[0012] Second application: When 40%–50% of the regenerated male-sterile line A has headed, simultaneously spray gibberellin onto both regenerated male-sterile line A and regenerated maintainer line B, at a rate of 100 mg / m². 2 The dosage is 10g~12g.

[0013] Third time: One day after the second spray, gibberellin was sprayed only on the regeneration maintainer line B, at a rate of 100 mg / m². 2 The dosage is 4g.

[0014] By spraying gibberellin three times, it can be ensured that the regenerated maintainer line B is more than 10 cm taller than the regenerated sterile line A.

[0015] S8. Artificial pollination and removal of male parent: During the flowering period of the regenerated maintainer line B, the pollen of the regenerated maintainer line B is artificially assisted to drift to the regenerated sterile line A for pollination; after the pollination period, all regenerated maintainer lines B are cut off and removed from the field.

[0016] S9. Harvesting and Purification: After the hybrid seeds on the regenerated sterile line A mature, they are harvested and dried separately.

[0017] Preferably, maintainer line B in S1 is sown in two phases. The first phase is designated as maintainer line B1, and the second phase is designated as maintainer line B2. Maintainer line B1 is sown 4 to 5 days later than sterile line A, and maintainer line B2 is sown 6 to 7 days later than sterile line A. Due to the influence of local climate and temperature, the development progress of the vegetative growth period and reproductive growth period of the maintainer line and sterile line is not consistent. In order to better match the flowering period of the maintainer line and sterile line, the maintainer line is sown in two phases.

[0018] Preferably, the seeding rate of sterile line A in S1 is 1 kg / 666.7 m². 2 The seeding rate for line B was maintained at 0.5 kg / 666.7 m². 2 .

[0019] Preferably, the specifications for transplanting in S2 are: Sterile line A: Single-planted, one plant per hole, with a plant spacing of 13.3cm × 13.3cm.

[0020] Maintainer B: 1 to 2 plants can be planted per hole, with a plant spacing of 13.3cm × 16.7cm.

[0021] Row ratio: The ratio of the number of rows of maintainer line B to the number of rows of sterile line A is 2:7~8.

[0022] Row spacing: The row spacing between line B and line A is 16.7 cm.

[0023] Preferably, Wufeng B2 and Wufeng A have a walkway at the back, with a walkway width of 23.3cm, to facilitate field operations.

[0024] Preferably, the method for identifying hybrid plants using the necking phenomenon of sterile line A described in S4 is as follows: under conditions without gibberellin spraying, sterile line A exhibits necking, while maintainer lines or other hybrid plants mixed in the row of sterile line A exhibit non-necking. Compared with necked plants, non-necked plants grow more vigorously, are taller, and their male flowers shed pollen. At the same time, hybrid plants in the row of maintainer line B are identified and removed based on differences in plant agronomic traits.

[0025] Preferably, the agronomic traits include plant and leaf morphology and leaf color.

[0026] Preferably, when removing sterile line A and maintainer line B in S4, the cutting height is 2-3 cm below the main panicle neck; the cutting order is as follows: first remove sterile line A, then remove the row of maintainer line B closest to sterile line A after 1-2 days, and then remove the row of maintainer line B closest to the walkway after another 2-3 days.

[0027] Preferably, in S8, artificial pollination is carried out by shaking the holding line B with a bamboo pole to allow pollen to drift to the sterile line A, and this is done 2 to 3 times a day during the flowering period.

[0028] Preferably, the sterile line A includes Wufeng A or Yexiang A; the maintainer line B includes Wufeng B or Yexiang B.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for rapidly purifying and propagating hybrid rice three-line male-sterile lines, comprising the following steps: First crop planting: sowing male-sterile line A and maintainer line B, and conducting field management, including removing impurities from the young panicle differentiation stage to the heading stage; First crop heading treatment: not spraying gibberellin from the first heading stage to the full heading stage, using the "neck-wrapping" phenomenon of male-sterile line A to distinguish impurities, and thoroughly removing impurities; Green stubble regeneration: after removing impurities, cutting off the above-ground parts of male-sterile line A and maintainer line B, retaining the stubble, and maintaining the field water layer to promote regeneration; Regeneration season management: during the regeneration season, conducting field management on the regenerated male-sterile line A and maintainer line B, including spraying foliar fertilizer, spraying gibberellin, and artificial pollination to produce high-purity male-sterile line seeds.

[0030] Through two years of trials, the "regeneration method" for rapidly purifying and propagating hybrid rice three-line male-sterile lines has proven to be more efficient than the "multi-field system" method, yielding a greater quantity of male-sterile seeds more quickly. The regeneration method is simple to operate, rapidly expands the variety population, accelerates the propagation coefficient, and significantly shortens the purification cycle. Agricultural technicians utilize the period from the initial heading to full heading and flowering of the male-sterile lines, until the removal of the male-sterile and maintainer lines, to remove fallen grains, maintainer line contamination, and all other plants with cross-pollination, thus accelerating the improvement of the purity of the three-line male-sterile rice. During harvesting, it can also remove male-sterile lines contaminated by cross-pollination from the first crop due to delayed removal of contaminants. Furthermore, the strong regenerative capacity of the male-sterile lines ensures a sufficient quantity of seeds for propagation. If the purity of hybrid rice three-line male-sterile seeds produced by a production unit declines after years of propagation, falling below the standard (seed purity less than 99.5%), the "regeneration method" provided by this invention offers a simple and feasible way to quickly obtain high-purity male-sterile seeds. Detailed Implementation

[0031] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0033] The inventive concept of this invention is as follows: During the propagation of three-line male-sterile hybrid rice, mechanical mixing and previous crop spoilage due to continuous generation of the original seed can easily lead to biological contamination. During the fifth to sixth stage of panicle differentiation, hybrid plants with different plant and leaf morphologies begin to appear, requiring field weeding. This process continues until the male-sterile line finishes flowering, with various types of hybrid plants remaining. This demands high technical skills and involves a heavy workload for agricultural technicians. The time from the beginning of panicle emergence to full heading and flowering is tight; if weeds are not removed promptly, biological contamination is likely to occur. After multiple generations of propagation, the purity of the male-sterile line becomes difficult to guarantee.

[0034] In the current technology for producing three-line male-sterile hybrid rice, the general production process is as follows: selection of original seeds of the three-line male-sterile hybrid rice → propagation of original seeds of the male-sterile line → generational propagation of the male-sterile line → purification and rejuvenation through a "multi-field system" → propagation of the male-sterile line. However, the "multi-field system" purification and rejuvenation method is cumbersome, time-consuming, labor-intensive, costly, and technically demanding in actual production, and cannot provide the required quantity of male-sterile line seeds to production units in a timely manner.

[0035] Based on this, the present invention provides a method for rapidly purifying and propagating hybrid rice three-line male-sterile lines using a "regeneration method." In this process, if gibberellin is not sprayed after the first heading of the male-sterile line during the first planting, a "neck-wrapping" phenomenon will occur, followed by panicle aging and a halt in growth. However, hybrid plants will not exhibit this phenomenon, making it easy to distinguish between male-sterile and hybrid plants. Sufficient time is available for removing impurities from the male-sterile line during this period. Using this method, agricultural technicians can fully utilize the time from the beginning of heading to the flowering stage of the male-sterile line until the removal of the male-sterile and maintainer lines to remove all hybrid plants from the male-sterile and maintainer lines. During the operation, hybrid plants must be removed along with their stubble to prevent regeneration and maintainer line purity. Furthermore, harvesting the male-sterile line during this period also removes male-sterile lines contaminated by cross-pollination from hybrid plants that were not removed in time during the first planting. This method also demonstrates strong regeneration ability and ensures a sufficient quantity of seeds for male-sterile line propagation.

[0036] In contrast, the "multi-field system" purification process requires two to three generations of purification, resulting in a significantly smaller seed quantity compared to the rapid purification and propagation via the "regeneration method." The "regeneration method" is simple and feasible, requiring only a small amount of sterile lines with insufficient purity (below 99.5%) for a single generation of rapid purification and propagation. This achieves a sterile line purity of over 99.8%, producing a substantial quantity of seeds. This provides sufficient seed resources for the next stage of sterile line propagation, thus meeting the large-scale sterile line seed requirements of hybrid rice production units. Furthermore, the "regeneration method" provides the necessary sterile line seeds to production units one to two generations earlier than the "multi-field system."

[0037] The Wufeng A and Wufeng B varieties of this invention were bred by the Rice Research Institute of Guangdong Academy of Agricultural Sciences, while Yexiang A and Yexiang B varieties were bred by Guangxi Zhuang Autonomous Region Green Sea Seed Industry Co., Ltd.

[0038] The base fertilizer is a nitrogen-phosphorus-potassium ternary compound fertilizer with a total nutrient content (N-P2O5-K2O) ≥45% and a ratio of 15-15-15. It was purchased from Anhui Sierte Fertilizer Co., Ltd.

[0039] The tillering fertilizer used is a nitrogen-phosphorus-potassium ternary compound fertilizer with a total nutrient content (N-P2O5-K2O) ≥45% and a ratio of 15-15-15. It was purchased from Anhui Sierte Fertilizer Co., Ltd.

[0040] The gibberellin used was Guoguang Dingyue Gibberellin 920, with an active ingredient gibberellic acid content of 3%. It was purchased from Sichuan Runer Technology Co., Ltd. The gibberellin was diluted with water before spraying.

[0041] The foliar fertilizer is potassium dihydrogen phosphate, with a main content (KH2PO4 on a dry basis) ≥98.0%, of which P2O5 ≥51% and K2O ≥34%. It is diluted 500 times with water for foliar spraying and was purchased from Sichuan Guoguang Agrochemical Co., Ltd.

[0042] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0043] Example 1 A method for purifying and propagating hybrid rice three-line male-sterile lines using a "regeneration method" is disclosed. The method uses the male-sterile line Wufeng A as the female parent to be purified and the maintainer line Wufeng B as the male parent. The purification and propagation are carried out using the "regeneration method" to obtain a high-purity, abundant hybrid rice three-line male-sterile line Wufeng A. The specific steps include: Phase 1: First crop planting.

[0044] S1. Seedling raising in the seedbed: Using a moist seedling raising method, sow Wufeng A and Wufeng B seeds in the seedbed to cultivate strong seedlings. Wufeng B is sown in two stages: the first stage is designated Wufeng B1, and the second stage is designated Wufeng B2. Wufeng B1 is sown 4 days later than Wufeng A, and Wufeng B2 is sown 6 days later than Wufeng A. The sowing rate for Wufeng A is 1 kg / 666.7 m². 2 The total seeding rate for Wufeng B is 0.5 kg / 666.7 m². 2 .

[0045] Honda pretreatment: In the fields where rice seedlings are planned to be transplanted, drain the water 20 days before the transplanting period to encourage the germination of residual weed seeds and scattered "fallen rice" from the previous season. After thoroughly removing these, perform the first tilling. One day before transplanting, apply 35 kg of compound fertilizer as base fertilizer during the second harrowing of the fields.

[0046] S2. Transplanting: When the seedlings reach 5.1 leaves, lift them from the nursery and transplant them to the pre-treated paddy field. Transplanting specifications are as follows:

[0047] Wufeng A: Strictly single-stem planting, one plant per hole, with a plant spacing of 13.3cm × 13.3cm.

[0048] Wufeng B: 1 to 2 plants can be planted per hole, with a plant spacing of 13.3cm × 16.7cm.

[0049] Row ratio configuration: The row ratio of Wufeng B to Wufeng A is 2:8.

[0050] Row spacing and walkways: The row spacing between Wufeng B row and Wufeng A row is 16.7cm. A walkway with a width of 23.3cm is left behind Wufeng B2 and Wufeng A rows to facilitate field operations.

[0051] S3. Early field management and first weeding: After the seedlings turn green, apply 15 kg of tillering fertilizer in time and maintain a shallow water layer of 2 cm in the field. From the time of transplanting to the fifth stage of panicle differentiation, repeatedly identify and remove non-variety plants in rows A and B of Wufeng based on plant and leaf morphology and leaf color (this weeding method is a common weeding method in this field).

[0052] S4. Second weeding and first "green cutting" treatment: Before the beginning of heading, drain and dry the field for 3 days, then re-irrigate, per 666.7m². 2 Apply 7.5 kg of 45% NPK compound fertilizer (15-15-15). Do not spray gibberellin during the heading stage to full heading stage of Wufeng A and Wufeng B. Utilizing the "neck-wrapping" phenomenon when the male-sterile line is not sprayed with gibberellin, identify and remove non-necked plants with different agronomical traits in the Wufeng A row; simultaneously, continue to remove non-necked plants in the Wufeng B row based on agronomical differences (this weeding method is common in this field). After weeding, first cut all Wufeng A plants. One day after cutting Wufeng A plants, cut the Wufeng B1 plant closest to the Wufeng A row; three days later, cut the Wufeng B2 plant closest to the aisle. During the green rice harvesting process, remove all cut rice seedlings from the field. All plants should be cut to a height of 2 cm below the neck of the main panicle.

[0053] Phase Two: Regeneration Crop Planting.

[0054] S5. Promote regeneration: After harvesting, ensure that there is a 5 cm water layer in the field to promote the regeneration of rice stubble.

[0055] S6. Management of regenerated seedlings and third weed removal: When the sword leaves of regenerated Wufeng A and Wufeng B have grown to 60%, spray 50g of foliar fertilizer. Two days later, spray 50g of foliar fertilizer again to strengthen the seedlings. During this process, remove any stray weeds according to the plant and leaf morphology and leaf color (this weed removal method is a common weed removal method in this field).

[0056] S7. Flowering period regulation and gibberellin spraying: First application: When the white residue of regenerated Wufeng A reaches 25%, spray only regenerated Wufeng A with gibberellin at a rate of 1:10 per 666.7 m³. 2 The dosage is 10g.

[0057] Second application: When 40% of the ears of Regenerated Wufeng A have emerged, spray both Regenerated Wufeng A and Regenerated Wufeng B with gibberellin simultaneously, at a rate of 100 mg / m². 2 The dosage is 10g.

[0058] Third time: One day after the second spray, apply gibberellin only to the regenerated Wufeng B, at a rate of 666.7m³. 2 The dosage is 4g.

[0059] The above spraying method resulted in Regenerated Wufeng B seedlings being more than 10cm taller than Regenerated Wufeng A seedlings.

[0060] S8. Artificial pollination and removal of the male parent: During the peak flowering period of Regenerated Wufeng B, manually shake B twice a day with a bamboo pole to allow its pollen to drift towards Regenerated Wufeng A for pollination. After the pollination period, promptly cut down all Regenerated Wufeng B plants and remove them from the field.

[0061] S9. Harvesting and Purification: After the hybrid seeds from Regenerated Wufeng A mature, harvest them separately and dry them in the sun.

[0062] Throughout the entire operation and drying process, farm tools and sites must be thoroughly cleaned to prevent mixing with other rice varieties and ensure that the obtained seeds are high-purity sterile line Wufeng A.

[0063] Example 2 A method for purifying and propagating hybrid rice three-line male-sterile lines using a "regeneration method" is disclosed. The method uses the male-sterile line Yexiang A as the female parent to be purified and the maintainer line Yexiang B as the male parent. The method involves purification and propagation using a "regeneration method" to obtain a high-purity, abundant hybrid rice three-line male-sterile line, Yexiang A. The specific steps include: Phase 1: First crop planting.

[0064] S1. Seedling raising in the seedbed: Wild Fragrance A and Wild Fragrance B are sown in the seedbed using a moist seedling raising method to cultivate strong seedlings. Wild Fragrance B is sown in two stages: the first stage is designated Wild Fragrance B1, and the second stage is designated Wild Fragrance B2. Wild Fragrance B1 is sown 4 days later than Wild Fragrance A, and Wild Fragrance B2 is sown 6 days later than Wild Fragrance A. The sowing rate for Wild Fragrance A is 1 kg / 666.7 m². 2 The total seeding rate for Wild Fragrance B was 0.5 kg / 666.7 m². 2 .

[0065] Honda pretreatment: In the fields where rice seedlings are planned to be transplanted, drain the water 20 days before the transplanting period to encourage the germination of residual weed seeds and scattered "fallen rice" from the previous season. After thoroughly removing these, perform the first tilling. One day before transplanting, apply 35 kg of compound fertilizer as base fertilizer during the second harrowing of the fields.

[0066] S2. Transplanting: When the seedlings reach 5.1 leaves, lift them from the nursery and transplant them to the pre-treated paddy field. Transplanting specifications are as follows:

[0067] Wild Fragrance A: Strictly planted as a single stem, one plant per hole, with a spacing of 13.3cm × 13.3cm; Wild Fragrance B: 1 to 2 plants can be planted per hole, with a spacing of 13.3cm × 16.7cm; Row ratio configuration: The row ratio of Wild Fragrance B to Wild Fragrance A is 2:8; Row spacing and walkways: The row spacing between Wild Fragrance B row and Wild Fragrance A row is 16.7cm. A walkway with a width of 23.3cm is left behind Wild Fragrance B2 and Wild Fragrance A to facilitate field operations.

[0068] S3. Early field management and first weeding: After the seedlings turn green, apply 15 kg of tillering fertilizer in time and maintain a shallow water layer of 2 cm in the field. From the time of transplanting to the fifth stage of young spikelet differentiation, based on the differences in agronomic traits, repeatedly identify and remove non-varietal hybrid plants in the rows of Wild Fragrance A and Wild Fragrance B (this weeding method is a common weeding method in this field).

[0069] S4. Second weeding and first "green cutting" treatment: Before the beginning of heading, drain and dry the field for 3 days, then re-irrigate, per 666.7m². 2 Apply 7.5 kg of 45% NPK compound fertilizer (15-15-15). Do not spray gibberellin during the heading stage to full heading stage of Wild Fragrance A and Wild Fragrance B. Utilizing the "neck-wrapping" phenomenon when the sterile line is not sprayed with gibberellin, identify and remove non-necked plants with different agronomical traits in the Wild Fragrance A row; simultaneously, continue removing non-necked plants in the Wild Fragrance B row based on agronomical differences (this weeding method is common in this field). After weeding, first cut down all Wild Fragrance A plants. One day after cutting down Wild Fragrance A, cut down Wild Fragrance B1 plants closest to the Wild Fragrance A row; three days later, cut down Wild Fragrance B2 plants closest to the aisle. During the green rice harvesting process, remove all cut rice seedlings from the field. All plants should be cut 2 cm below the neck of the main panicle.

[0070] Phase Two: Regeneration Crop Planting.

[0071] S5. Promote regeneration: After harvesting, ensure that there is a 5 cm water layer in the field to promote the regeneration of rice stubble.

[0072] S6. Management of regenerated seedlings and third weeding: When the sword leaves of regenerated wild sage A and wild sage B have grown to 60%, spray 50g of foliar fertilizer. Two days later, spray 50g of foliar fertilizer again to strengthen the seedlings. During this process, remove any stray weeds according to the differences in agronomic traits (this weeding method is a common weeding method in this field).

[0073] S7. Flowering period regulation and gibberellin spraying: First application: When the regenerated wild herb A shows 30% whitening, spray only wild herb A with gibberellin at a rate of 100 mg / 666.7 m³. 2 The dosage is 10g.

[0074] Second application: When 40% of the regenerated wild clover A has emerged, simultaneously spray gibberellin on both regenerated wild clover A and regenerated wild clover B, at a rate of 100 mg / m². 2 The dosage is 10g.

[0075] Third time: One day after the second spray, apply gibberellin only to the regenerated wild sage B, at a rate of 666.7m². 2 The dosage is 4g.

[0076] The above spraying method resulted in the regenerated wild sage B seedlings being more than 10cm taller than the regenerated wild sage A seedlings.

[0077] S8. Artificial pollination and removal of the male parent: During the peak flowering period of the regenerated wild clover B, manually shake B twice a day with a bamboo pole to allow its pollen to drift towards the regenerated wild clover A for pollination. After the pollination period, promptly cut down all regenerated wild clover B and remove it from the field.

[0078] S9. Harvesting and Purification: After the hybrid seeds on the regenerated wild sage A mature, harvest them separately and dry them.

[0079] Throughout the entire operation and drying process, farm tools and sites must be thoroughly cleaned to prevent mixing with other rice varieties and ensure that the obtained seeds are high-purity sterile line Wild Fragrance A.

[0080] It should be noted that the present invention can be achieved under the following conditions: In S1, maintainer line B is sown in two stages, with maintainer line B1 sown 4-5 days later than male-sterile line A, and maintainer line B2 sown 6-7 days later than male-sterile line A; In S2, the seedlings are 5.1-5.8 leaves old at transplanting; In S3, the seedlings are transplanted to the 5th-6th stage of young panicle differentiation; In S4, the cutting height is 2-3 cm below the main panicle neck, and the cutting order is as follows: first, male-sterile line A is cut off, then 1-2 days later, the row of maintainer line B closest to male-sterile line A is cut off, and then 2-3 days later, the row of maintainer line B closest to the walkway is cut off; In S5, the water layer thickness is 4-5 cm; In S6, fertilization and weeding are carried out when the flag leaves of the regenerated seedlings have grown to 50%-60%; In S7, the gibberellin spraying scheme is as follows: the first application is when the regenerated male-sterile line A has grown 20%-30% of its white leaves, per 666.7 m² 2Dosage: 10g~12g; The second application should be made when 40%~50% of the male-sterile line A has headed, simultaneously spraying both male-sterile line A and maintainer line B, at a rate of 10g per 666.7m². 2 Dosage: 10g~12g; The third application should be made one day after the second application, and should only be applied to maintenance system B, at a rate of 10g per 666.7m². 2 Dosage: 4g; artificial pollination in S8 is performed 2-3 times daily. The technical effects of this invention can be achieved by selecting any specific parameters within the above range.

[0081] Experimental Example 1 In 2021, the experimental site was the Yichun Academy of Sciences, Yuanzhou District, Yichun City, Jiangxi Province (latitude: 27.785366, longitude: 114.416350). The previous crop in the experimental field was rapeseed. The field was flat, with uniform fertility, and the soil type was clay soil. The experimental field area was 0.4 hm². 2 The combination is Wufeng A, and the experimental field area is 0.2 hm². 2 Wild Fragrance A, the experimental field area is 0.2 hm² 2 Seed quantity: 1 kg / 666.7 m² for sterile lines. 2 The maintenance coefficient is 0.5 kg / 666.7 m. 2 The plot was prepared using the "regeneration method" described in Examples 1 and 2 to purify and propagate hybrid rice three-line sterile lines. A total of 214.5 kg of seeds were harvested, equivalent to a yield of 1072.50 kg / hm². 2 A total of 211.20 kg of wild jasmine A was harvested, which translates to a yield of 1056 kg / hm². 2 This demonstrates the technology's excellent reproductive and production capabilities. To scientifically evaluate the purification effect, in 2022, the purified seeds from the experimental group were planted simultaneously with the untreated original seed source under the same conditions, and field purity was assessed. The results are shown in Table 1 below. The varietal purity of Wufeng A and Yexiang A, treated with the "regeneration method," reached 99.80%. In contrast, the purity of the original Wufeng A seed source (as a control) was 99.20% (3 different varieties, 2 maintainer lines, and 3 variants were found among 997 plants surveyed), and the purity of the original Yexiang A seed source was also 99.20% (4 different varieties, 3 maintainer lines, and 1 variant were found among 997 plants surveyed). This result indicates that a single round of purification using the "regeneration method" can significantly increase the purity of sterile line seeds by 0.60%, effectively removing unwanted plants in the field.

[0082] Table 1. Results of Field Purity Identification in 2021 Note: Variety purity (%) = (Total number of plants tested - Number of hybrid plants) / Total number of plants tested × 100; “Source A” is the original unpurified control.

[0083] Experimental Example 2 In 2022, the experimental site was the Yichun Academy of Sciences, Yuanzhou District, Yichun City, Jiangxi Province (latitude: 27.785366, longitude: 114.416350). The previous crop in the experimental field was late rice from the previous year. The field was flat, with uniform fertility, and the soil type was clay soil. The experimental field area was 0.4 hm². 2 The combination is Wufeng A, and the experimental field area is 0.2 hm². 2 Wild Fragrance A, the experimental field area is 0.2 hm² 2 Seed quantity: 1 kg / 666.7 m² for sterile lines. 2 The maintenance coefficient is 0.5 kg / 666.7 m. 2 The plot was prepared using the "regeneration method" described in Examples 1 and 2 to purify and propagate the three-line sterile hybrid rice line. A total of 220.5 kg of Wufeng A seeds were harvested, equivalent to a yield of 1102.50 kg / hm². 2 A total of 210.60 kg of wild jasmine A was harvested, which translates to a yield of 1053 kg / hm². 2 This demonstrates that the technology possesses excellent reproductive and production capabilities. To scientifically evaluate the purification effect, in 2022, the purified seeds produced in the experimental group were planted simultaneously with the unpurified original seed source under the same conditions, and field purity assessments were conducted. The results are shown in Table 2 below. The purity of the purified Wufeng A and Yexiang A seeds stabilized again at 99.80%, while the purity of the original seed source used that year remained at 99.20% (the Wufeng A original seed source hybrids included 4 different varieties, 2 maintainer lines, and 2 variants; the Yexiang A original seed source hybrids included 5 different varieties, 3 maintainer lines, and 1 variant), with the purity increase also being 0.60%.

[0084] Table 2. Field purity assessment results in 2022 Variety purity (%) = (Total number of plants tested - Number of hybrid plants) / Total number of plants tested × 100; “Source A” is the original unpurified control.

[0085] In summary, two consecutive years of repeated trials have fully demonstrated that the "regeneration method" is a stable and reproducible purification and generation technology. It not only achieves rapid seed propagation and generation through the agronomical measure of "harvesting and regeneration," but also systematically increases the field purity of sterile lines from 99.20% to 99.80% through multiple rounds of rigorous impurity removal throughout the process, while also increasing the number of sterile line seeds. This has reliable application value for ensuring the quality of hybrid rice parent seeds and improving the safety of hybrid seed production.

[0086] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0087] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A method for purifying and propagating hybrid rice three-line sterile lines using a regeneration method, characterized in that, Includes the following steps: S1. Seedling raising in the seedbed: Sow sterile line A and maintainer line B in the seedbed to cultivate strong seedlings; S2. Transplanting: When the seedlings have 5.1 to 5.8 leaves, they are lifted from the seedbed and transplanted to the main field. S3. First removal of hybrid plants: After transplanting to the main field until the 5th to 6th stage of young spikelet differentiation, remove non-variety plants in the rows of sterile line A and maintainer line B according to the differences in plant and leaf morphology and leaf color. S4. Second weeding and first "green cutting" treatment: During the heading stage to full heading stage of male-sterile line A and maintainer line B, do not spray gibberellin. Identify and remove hybrid plants in the row of male-sterile line A that do not have a neck and whose agronomic traits are different from those of male-sterile line A by using the necking phenomenon of male-sterile line A. At the same time, remove hybrid plants in the row of maintainer line B according to the differences in agronomic traits. After weeding, cut off all male-sterile lines A and maintainer line B. S5. Promote regeneration: After harvesting, ensure that there is a 4-5 cm water layer in the field to promote the regeneration of rice stubble; S6. Management of regenerated seedlings and third weeding: When the flag leaves of the regenerated sterile line A and the maintainer line B have grown to 50%~60%, apply fertilizer and remove any stray plants according to the differences in plant and leaf morphology and leaf color. S7. Flowering period regulation and gibberellin spraying: First application: When 20%–30% of the regenerating sterile line A shows white markings, spray only line A with gibberellin at a rate of 100 mg / 666.7 m³. 2 The dosage is 10g~12g; Second application: When 40%–50% of the regenerated male-sterile line A has headed, simultaneously spray gibberellin onto both regenerated male-sterile line A and regenerated maintainer line B, at a rate of 100 mg / m². 2 The dosage is 10g~12g; Third time: One day after the second spray, gibberellin was sprayed only on the regeneration maintainer line B, at a rate of 1 per 666.7 m³. 2 The dosage is 4g; S8. Artificial pollination and removal of male parent: During the flowering period of the regenerated maintainer line B, the pollen of the regenerated maintainer line B is artificially assisted to drift to the regenerated sterile line A for pollination; after the pollination period, all regenerated maintainer lines B are cut off and removed from the field. S9. Harvesting and Purification: After the hybrid seeds on the regenerated sterile line A mature, they are harvested and dried separately.

2. The method according to claim 1, characterized in that, In S1, maintainer line B is sown in two phases. The first phase is designated as maintainer line B1, and the second phase is designated as maintainer line B2. Maintainer line B1 is sown 4 to 5 days later than sterile line A, and maintainer line B2 is sown 6 to 7 days later than sterile line A.

3. The method according to claim 1, characterized in that, The seeding rate for sterile line A in S1 is 1 kg / 666.7 m². 2 The seeding rate for line B was maintained at 0.5 kg / 666.7 m². 2 .

4. The method according to claim 1, characterized in that, The specifications for transplanting in S2 are as follows: Sterile line A: Single-stem planting, one plant per hole, with a spacing of 13.3cm × 13.3cm; Maintainer strain B: 1 to 2 plants can be planted per hole, with a plant spacing of 13.3cm × 16.7cm; Row ratio configuration: The row ratio of line B to sterile line A is 2:7~8; Row spacing: The row spacing between line B and line A is 16.7 cm.

5. The method according to claim 1, characterized in that, The method for identifying hybrid plants using the necking phenomenon of sterile line A as described in S4 is as follows: Under conditions without gibberellin spraying, sterile line A exhibits necking, while maintainer lines or other hybrid plants mixed in the row of sterile line A exhibit non-necking. Compared with necked plants, non-necked plants grow more vigorously, are taller, and their male flowers shed pollen. At the same time, hybrid plants in the row of maintainer line B are identified and removed based on differences in agronomic traits.

6. The method according to claim 1, characterized in that, When removing sterile line A and maintainer line B in S4, the cutting height should be 2-3 cm below the main panicle neck.

7. The method according to claim 1, characterized in that, In S8, artificial pollination is achieved by shaking a bamboo pole to keep line B in place, allowing pollen to drift towards sterile line A. This is done 2 to 3 times a day during the flowering period.

8. The method according to claim 1, characterized in that, The sterile line A includes Wufeng A or Yexiang A; the maintainer line B includes Wufeng B or Yexiang B.

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