A breeding method for plant gene editing

Abstract

The application discloses a plant gene editing breeding method and belongs to the technical field of plant genetic breeding. The method comprises six steps of breeding parent screening, gene editing vector construction, gene editing transformation, positive plant screening, trait stable cultivation and breeding identification screening. Through the screening of high-quality parents, the construction of specific gene editing vectors, the realization of target gene directional editing by adopting the agrobacterium-mediated method, the combination of gradient environment domestication and stable cultivation, and finally the screening of breeding plants with excellent traits and genetic stability, the method has the advantages of strong targeting and high breeding efficiency, can effectively solve the problems of long traditional breeding cycle and limited trait improvement effect, is suitable for various plants, can quickly cultivate new varieties with excellent target traits, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant genetics and breeding technology, specifically involving a breeding method for plant gene editing. It is applicable to various crops and economic crops such as rice, wheat, corn, soybean, tomato, and cotton. It can rapidly and directionally improve target traits of plants such as stress resistance, yield, quality, and disease resistance, cultivate superior new plant varieties, and promote high-quality development of agricultural production. Background Technology

[0002] Plant breeding is a core technology for ensuring agricultural production and improving crop yield and quality. Traditional plant breeding methods mainly include hybridization breeding and mutation breeding. However, hybridization breeding has problems such as long cycle, low breeding efficiency, and difficulty in accurately aggregating superior traits. Mutation breeding has defects such as strong randomness of mutations, low rate of superior mutations, and easy generation of unfavorable mutations, which make it difficult to meet the needs of modern agriculture for the rapid cultivation of superior varieties.

[0003] With the development of gene editing technology, CRISPR-Cas9 and other gene editing technologies have been widely used in the field of plant genetic improvement due to their advantages of strong targeting, simple operation, high efficiency, and low cost. However, existing gene editing breeding methods still have many shortcomings. For example, the lack of precise screening of high-quality parents results in limited improvement in traits of edited plants; the lack of systematic trait stabilization cultivation after gene editing leads to unstable expression of target traits and poor heritability; and unclear identification and screening criteria make it difficult to quickly screen out breeding plants that meet the requirements. Therefore, there is an urgent need for a standardized, precise, efficient, and trait-stable plant gene editing breeding method to address the shortcomings of existing technologies. Summary of the Invention

[0004] The purpose of this invention

[0005] To address the problems of low breeding efficiency, unstable target traits, poor heritability, and unclear screening criteria in existing plant gene editing breeding methods, this invention provides a plant gene editing breeding method. Through the synergistic effect of high-quality parent selection, precise gene editing, systematic stable cultivation, and rigorous identification and screening, it achieves targeted improvement of plant target traits, rapidly cultivates new plant varieties with excellent traits and stable heritability, and is simple to operate, highly reproducible, and adaptable to a variety of plants.

[0006] Technical solution

[0007] The specific steps of the plant gene editing breeding method of the present invention are as follows:

[0008] (1) Selection of breeding parents: Select robust plants with good growth, no pests or diseases and excellent agronomic traits as candidate parents. Use PCR technology to detect the target trait-related genes of the candidate parents and screen out high-quality parents with complete gene sequences, no natural mutations and normal expression of target genes. At the same time, investigate the agronomic traits of the candidate parents such as plant height, number of tillers, and fruit setting rate to ensure that the parents have good basic traits.

[0009] (2) Gene editing vector construction: Based on the target breeding traits (stress resistance, high yield, high quality, disease resistance, etc.), the target editing gene is determined, and a specific sgRNA is designed to ensure that the sgRNA and the target gene sequence match ≥98% to avoid off-target effects. The designed sgRNA is ligated to the SpCas9 or SaCas9 protein-coding gene and inserted into the pCas9-sgRNA vector to construct a recombinant gene editing vector. The correctness of the vector construction is verified by sequencing.

[0010] (3) Gene editing transformation: The recombinant gene editing vector was introduced into Agrobacterium, and Agrobacterium culture was obtained. The OD600 of the culture was adjusted to 0.5-0.7. Callus tissue or young organs (leaves, young spikes, etc.) of high-quality parent plants were selected and infected using the Agrobacterium-mediated method. The infection time was 10-20 min. After infection, the plants were co-cultured in the dark at 22-25℃ for 2-3 days to promote the integration of the gene editing vector into the plant genome and obtain gene-edited transformed plants.

[0011] (4) Screening of positive plants: The transformed plants after co-culture were transplanted into the screening medium for resistance screening. Then, the target editing fragment was amplified by PCR technology and verified by sequencing to confirm that the target gene had been edited as expected, and gene editing positive plants were obtained. At the same time, the positive plants were preliminarily screened for the target traits, and plants that were deformed, had poor growth, or did not show the target traits were removed.

[0012] (5) Trait stabilization cultivation: The selected positive plants were transplanted into the cultivation substrate for gradient environment acclimatization and field cultivation. Gradual acclimatization is divided into an adaptation period, an enhancement period, and a stabilization period. During the adaptation period, conventional cultivation conditions were used to help the plants recover their growth. During the enhancement period, field adverse environments (such as mild salt stress and drought stress) were simulated to enhance the plants' resistance to stress. During the stabilization period, conventional cultivation conditions were restored to promote the stable expression of the target trait. During the cultivation period, plant growth regulators were sprayed every 3-4 days to control suitable cultivation environment parameters until the plants matured.

[0013] (6) Breeding identification and screening: Comprehensive identification and screening of mature plants is carried out, including target trait identification, agronomic trait detection and genetic stability analysis. Target trait identification is carried out by physiological index detection and phenotypic observation to confirm that the target trait is significantly superior to the parent. Agronomic trait detection includes indicators such as plant height, yield and quality. Genetic stability analysis is carried out by planting for 2-3 consecutive generations to detect the expression level of target genes and the performance of target traits, and to screen out target breeding plants with excellent traits and genetic stability for subsequent propagation and promotion.

[0014] Beneficial effects

[0015] 1. This invention first screens high-quality parents to ensure that the parents have good basic traits and complete target genes, laying the foundation for subsequent gene editing and trait improvement, effectively improving the breeding success rate and trait improvement effect. Compared with traditional gene editing breeding methods, the breeding efficiency is increased by more than 40%.

[0016] 2. This invention uses specific sgRNA to construct gene editing vectors, which have strong targeting and can achieve precise editing of target genes, effectively reducing off-target effects, ensuring targeted improvement of target traits, and avoiding the generation of unfavorable mutations.

[0017] 3. This invention adds a trait stabilization cultivation step, which promotes stable expression of target genes and improves the genetic stability of target traits through gradient environmental acclimatization and regulation by plant growth regulators, thus solving the problems of unstable target traits and poor heritability in existing methods.

[0018] 4. This method has a standardized process, is easy to operate, and is highly repeatable. It is suitable for a variety of crops and cash crops, and can quickly cultivate new varieties with excellent traits such as stress resistance, high yield, and high quality. It is of great significance for promoting the upgrading of the agricultural industry and ensuring food security. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments. Experimental methods not specified in the embodiments are carried out in accordance with conventional methods and existing technologies; all reagents used are conventional reagents that can be purchased through commercial channels.

[0020] Example 1: Gene Editing Breeding Method for High-Yielding Traits in Rice

[0021] This embodiment uses Yuan Longping's super rice as the parent, and directionally edits the OsSPL14 gene to cultivate a new high-yield rice variety. The specific steps are as follows:

[0022] (1) Selection of breeding parents: Yuan Longping super rice plants with strong growth, no diseases and pests, many tillers and high seed setting rate were selected as candidate parents. The OsSPL14 gene (high yield related gene) was detected by PCR technology. High-quality parents with complete gene sequence and no mutation were selected. The height of the parent plants was 95-105cm, the number of tillers was 18-22 / plant, and the seed setting rate was ≥85%.

[0023] (2) Gene editing vector construction: The OsSPL14 gene was used as the target gene for editing. A specific sgRNA with a matching degree of 99% was designed. The sgRNA was linked to the SpCas9 protein-coding gene and inserted into the pCas9-sgRNA vector to construct a recombinant gene editing vector. Sequencing was used to verify that the vector was constructed correctly.

[0024] (3) Gene editing transformation: The recombinant vector was introduced into Agrobacterium EHA105, and Agrobacterium culture was obtained. The OD600 was adjusted to 0.6. High-quality parental rice callus tissue was selected and infected with Agrobacterium-mediated transformation for 15 min. The cells were co-cultured in the dark at 23℃ for 2 days to obtain gene-edited transformed rice plants.

[0025] (4) Screening of positive plants: Transformed plants were transplanted into a screening medium containing 50 mg / L hygromycin. After two weeks of resistance screening, the OsSPL14 editing fragment was amplified by PCR technology and sequenced to confirm that the target gene had been edited as expected, and gene-edited positive rice plants were obtained. Plants with abnormality and tillering were initially screened out.

[0026] (5) Trait stabilization cultivation: Positive plants were transplanted into a cultivation substrate for gradient environmental acclimatization. Adaptation period: Conventional cultivation conditions (temperature 25℃, light intensity 4000 lux, light duration 13h / d, air humidity 65%), treatment for 4 days; Enhancement period: Mild drought stress (soil moisture content 15%), treatment for 6 days; Stabilization period: Return to conventional cultivation conditions, treatment for 7 days. During the cultivation period, plant growth regulators (gibberellin 75mg / L, salicylic acid 25mg / L) were sprayed every 3 days, with each spraying amount being 12mL / plant, until the rice reached the tillering stage and matured.

[0027] (6) Breeding identification and screening: Mature plants were identified and screened. The target trait identification showed that the number of tillers of the edited rice reached 28-32 / plant, which was more than 30% higher than that of the parents. The seed setting rate was ≥90%, the thousand-grain weight was increased by 12%, and the high-yield trait was significantly better than that of the parents. Agronomic trait detection showed that the plant height was 100-108cm, the growth was robust, and there were no diseases or pests. Genetic stability analysis showed that after three consecutive generations of planting, the expression of the OsSPL14 gene was stable, the high-yield trait showed no obvious segregation, and the genetic stability was 98%. High-yield target breeding rice plants were screened out.

[0028] Example 2: Gene-editing breeding method for wheat rust resistance

[0029] This embodiment uses Jimai 44 wheat as the parent, and directionally edits the TaWRKY70 gene to breed a new wheat variety resistant to wheat rust. The specific steps are as follows:

[0030] (1) Selection of breeding parents: Select healthy, disease-free, and agronomically excellent Jimai 44 wheat plants as candidate parents. Use PCR technology to detect the TaWRKY70 gene (rust resistance-related gene) and select high-quality parents with complete gene sequences and no mutations. The parent plants were found to be 75-85cm tall, with 12-16 tillers per plant, and free from rust infection.

[0031] (2) Gene editing vector construction: The TaWRKY70 gene was used as the target gene for editing. A specific sgRNA was designed with a matching degree of 98.5%. The sgRNA was linked to the SaCas9 protein-coding gene and inserted into the pCas9-sgRNA vector to construct a recombinant gene editing vector. Sequencing was used to verify that the vector was constructed correctly.

[0032] (3) Gene editing transformation: The recombinant vector was introduced into Agrobacterium GV3101, and Agrobacterium culture was obtained. The OD600 was adjusted to 0.5. Wheat spikelets of high-quality parental line were selected and infected with Agrobacterium-mediated transformation for 10 min. They were co-cultured in the dark at 22℃ for 2 days to obtain gene-edited wheat plants.

[0033] (4) Screening of positive plants: Transformed plants were transplanted into a screening medium containing 40 mg / L kanamycin. After two weeks of resistance screening, the TaWRKY70 editing fragment was amplified by PCR technology and sequenced to confirm that the target gene had been edited as expected, and gene-edited positive wheat plants were obtained. Plants susceptible to rust disease and those with poor growth were initially screened out.

[0034] (5) Trait stabilization cultivation: Positive plants were transplanted into a cultivation substrate for gradient environmental acclimatization. Adaptation period: Conventional cultivation conditions (temperature 22℃, light intensity 3800 lux, light duration 12h / d, air humidity 68%), treatment for 3 days; Enhancement period: Simulated rust stress environment (inoculation with rust fungus), treatment for 5 days; Stabilization period: Return to conventional cultivation conditions, treatment for 6 days. During the cultivation period, plant growth regulators (gibberellin 60mg / L, salicylic acid 20mg / L) were sprayed every 4 days, with each spraying amount being 10mL / plant, until the wheat reached maturity at the heading stage.

[0035] (6) Breeding identification and screening: Mature plants were identified and screened. The target trait identification showed that the wheat after editing had significantly improved resistance to rust, with an incidence rate of ≤5%, which was more than 30% lower than the parent (incidence rate of 35%). Agronomic trait testing showed that the plant height was 78-82cm, the number of tillers was 14-18 / plant, and the seed setting rate was ≥88%. Genetic stability analysis showed that after two consecutive generations of planting, the TaWRKY70 gene expression was stable, the rust resistance trait was stably inherited, and the genetic stability was 96%. The target breeding wheat plants resistant to rust were screened out.

[0036] Example 3: Gene Editing Breeding Method for Drought Resistance in Maize

[0037] This embodiment uses Zhengdan 958 maize as the parent, and directionally edits the DREB2A gene to breed a new drought-resistant maize variety. The specific steps are as follows:

[0038] (1) Selection of breeding parents: Zhengdan 958 maize plants with strong growth, no diseases and pests, and strong drought resistance were selected as candidate parents. The DREB2A gene (drought resistance related gene) was detected by PCR technology. High-quality parents with complete gene sequence and no mutation were selected. The parents were found to be 170-180cm tall, 18-22cm long, and moderately drought resistant.

[0039] (2) Gene editing vector construction: The DREB2A gene was used as the target gene for editing. A specific sgRNA with a matching degree of 99% was designed. The sgRNA was linked to the SpCas9 protein-coding gene and inserted into the pCas9-sgRNA vector to construct a recombinant gene editing vector. Sequencing was used to verify that the vector was constructed correctly.

[0040] (3) Gene editing transformation: The recombinant vector was introduced into Agrobacterium EHA105, and Agrobacterium culture was obtained. The OD600 was adjusted to 0.7. High-quality maize leaves were selected and infected with Agrobacterium-mediated transformation for 20 min. The maize plants were co-cultured in the dark at 25℃ for 3 days to obtain gene-edited transformed maize plants.

[0041] (4) Screening of positive plants: Transformed plants were transplanted into a screening medium containing 50 mg / L hygromycin. After two weeks of resistance screening, the DREB2A editing fragment was amplified by PCR technology and sequenced to confirm that the target gene had been edited as expected, and gene-edited positive maize plants were obtained. Plants that were drought-sensitive and had wilted leaves were initially screened out.

[0042] (5) Trait stabilization cultivation: Positive plants were transplanted into the cultivation substrate for gradient environmental acclimatization. Adaptation period: Conventional cultivation conditions (temperature 26℃, light intensity 4200 lux, light duration 14h / d, air humidity 55%), treatment for 5 days; Enhancement period: Moderate drought stress (soil moisture content 12%), treatment for 7 days; Stabilization period: Return to conventional cultivation conditions, treatment for 8 days. During the cultivation period, plant growth regulators (gibberellin 90mg / L, salicylic acid 30mg / L) were sprayed every 3 days, with each spraying amount being 15mL / plant, until the corn reached maturity at the grain-filling stage.

[0043] (6) Breeding identification and screening: Mature plants were identified and screened. Target trait identification showed that the proline content of the edited maize was 45% higher than that of the parents, the relative water content of the leaves was 20% higher, and the survival rate under drought stress was ≥90%, which was more than 35% higher than that of the parents (survival rate 55%). Agronomic trait detection showed that the plant height was 175-185cm, the ear length was 20-24cm, and the yield was 18% higher than that of the parents. Genetic stability analysis showed that after three consecutive generations of planting, the expression of the DREB2A gene was stable, the drought resistance trait did not segregate, and the genetic stability was 97%. Drought-resistant target breeding maize plants were screened.

[0044] Example 4: Gene Editing Breeding Method for Superior Traits in Soybeans

[0045] This embodiment uses Zhonghuang 13 soybean as the parent, and selectively edits the GmFAD2 gene to cultivate a new high-oleic soybean variety. The specific steps are as follows:

[0046] (1) Selection of breeding parents: Select robust, disease-free, and plump Zhonghuang 13 soybean plants as candidate parents. Use PCR technology to detect the GmFAD2 gene (oleic acid synthesis related gene) and select high-quality parents with complete gene sequences and no mutations. The parent plants are 60-70cm tall, have 35-40 pods per plant, and have an oleic acid content of 22%-25%.

[0047] (2) Gene editing vector construction: The GmFAD2 gene was used as the target gene for editing. A specific sgRNA was designed with a matching degree of 98.8%. The sgRNA was linked to the SpCas9 protein-coding gene and inserted into the pCas9-sgRNA vector to construct a recombinant gene editing vector. Sequencing was used to verify that the vector was constructed correctly.

[0048] (3) Gene editing transformation: The recombinant vector was introduced into Agrobacterium GV3101, and Agrobacterium culture was obtained. The OD600 was adjusted to 0.65. Cotyledons of high-quality parent soybeans were selected and infected with Agrobacterium-mediated transformation for 16 min. The plants were co-cultured in the dark at 24℃ for 2.5 days to obtain gene-edited transformed soybean plants.

[0049] (4) Screening of positive plants: Transformed plants were transplanted into a screening medium containing 45 mg / L kanamycin. After two weeks of resistance screening, the GmFAD2 editing fragment was amplified by PCR technology and sequenced to confirm that the target gene had been edited as expected, and gene-edited positive soybean plants were obtained. Plants with deformed grains and slow growth were initially screened out.

[0050] (5) Trait stabilization cultivation: Positive plants were transplanted into a cultivation substrate for gradient environmental acclimatization. Adaptation period: Conventional cultivation conditions (temperature 24℃, light intensity 4000 lux, light duration 13h / d, air humidity 62%), treatment for 4 days; Enhancement period: Conventional cultivation conditions, supplemented with appropriate amounts of phosphorus and potassium fertilizer, treatment for 6 days; Stabilization period: Return to conventional cultivation conditions, treatment for 7 days. During the cultivation period, plant growth regulators (gibberellin 70mg / L, salicylic acid 23mg / L) were sprayed every 4 days, with each spraying amount being 13mL / plant, until soybean maturity.

[0051] (6) Breeding identification and screening: Mature plants were identified and screened. The target trait identification showed that the oleic acid content of the edited soybean increased to 55%-60%, which was more than 30% higher than that of the parent, and the quality was significantly improved. Agronomic trait testing showed that the plant height was 65-75cm, the number of pods per plant was 40-45, the grains were full, and there were no diseases or pests. Genetic stability analysis showed that after two consecutive generations of planting, the expression of the GmFAD2 gene was stable, the high oleic acid trait was stably inherited, and the genetic stability was 95%. High oleic acid target breeding soybean plants were screened out.

[0052] Example 5: Gene Editing Breeding Method for Salt Tolerance in Tomatoes

[0053] This embodiment uses the Zhongza 105 tomato as the parent, and selectively edits the SlNHX1 gene to cultivate a new salt-tolerant tomato variety. The specific steps are as follows:

[0054] (1) Selection of breeding parents: Select robust, disease-free, and fruit-bearing Zhongza 105 tomato plants as candidate parents. Use PCR technology to detect the SlNHX1 gene (salt resistance-related gene) and screen out high-quality parents with complete gene sequences and no mutations. The parent plants are 80-90cm tall, with 25-30 fruits per plant and moderate salt tolerance.

[0055] (2) Gene editing vector construction: Using the SlNHX1 gene as the target gene for editing, a specific sgRNA was designed with a matching degree of 99%. The sgRNA was linked to the SaCas9 protein-coding gene and inserted into the pCas9-sgRNA vector to construct a recombinant gene editing vector. Sequencing was used to verify that the vector was constructed correctly.

[0056] (3) Gene editing transformation: The recombinant vector was introduced into Agrobacterium EHA105, and Agrobacterium culture was obtained. The OD600 was adjusted to 0.6. Tomato leaves of high-quality parent plants were selected and infected with Agrobacterium-mediated transformation for 14 min. The plants were co-cultured in the dark at 23℃ for 2 days to obtain gene-edited tomato plants.

[0057] (4) Screening of positive plants: Transformed plants were transplanted into a screening medium containing 50 mg / L hygromycin. After two weeks of resistance screening, the SlNHX1 editing fragment was amplified by PCR technology and sequenced to confirm that the target gene had been edited as expected, and gene-edited positive tomato plants were obtained. Plants that were salt-sensitive or had scorched leaves were initially screened out.

[0058] (5) Trait stabilization cultivation: Positive plants were transplanted into the cultivation substrate for gradient environmental acclimatization. Adaptation period: Conventional cultivation conditions (temperature 23℃, light intensity 3600 lux, light duration 12.5 h / d, air humidity 66%), treatment for 4 days; Enhancement period: Mild salt stress (irrigation with 150 mM NaCl solution), treatment for 6 days; Stabilization period: return to conventional cultivation conditions, treatment for 7 days. During the cultivation period, plant growth regulators (gibberellin 65 mg / L, salicylic acid 22 mg / L) were sprayed every 3.5 days, with each spraying amount being 11 mL / plant, until the tomato fruit matured.

[0059] (6) Breeding identification and screening: Mature plants were identified and screened. Target trait identification showed that the relative electrical conductivity of the leaves of the edited tomatoes decreased by 35% compared with the parents, the proline content increased by 42%, and the survival rate under salt stress was ≥88%, which was more than 40% higher than the parents (survival rate 48%). Agronomic trait detection showed that the plant height was 85-95cm, the number of fruits per plant was 30-35, the fruit size was uniform, and the quality was excellent. Genetic stability analysis showed that after two consecutive generations of planting, the expression of the SlNHX1 gene was stable, the salt resistance trait was stably inherited, and the genetic stability was 96%. Salt-resistant target breeding tomato plants were screened out.

[0060] In summary, the plant gene editing breeding method provided by this invention precisely solves the technical problems of low breeding efficiency, unstable target traits, poor heritability, and unclear screening criteria in existing plant gene editing breeding methods through the coordinated efforts of parental selection, gene editing vector construction, gene editing transformation, positive plant selection, trait stabilization cultivation, and breeding identification screening. This achieves targeted and efficient improvement of target plant traits. The method is highly targeted, easy to operate, and highly reproducible, suitable for various plants such as rice, wheat, corn, soybean, and tomato. It can rapidly cultivate new plant varieties with excellent traits and stable genetics, effectively shortening the breeding cycle and improving breeding quality. It provides high-quality varieties to support agricultural production and has significant theoretical and practical value for promoting agricultural industrial upgrading, ensuring food security, and improving agricultural product quality, with broad application prospects.

Claims

1. A breeding method for plant gene editing, characterized in that, Includes the following steps: (1) Selection of breeding parents: Select robust plants with excellent traits and free from pests and diseases as parents, detect the genes related to the target traits of the parents, and select high-quality parents with complete gene sequences and no mutations; (2) Gene editing vector construction: Based on the target breeding traits, specific sgRNAs were designed, and the sgRNAs were linked to the Cas9 protein-coding gene to construct gene editing vectors; (3) Gene editing transformation: Gene editing vectors were introduced into the callus tissue or young organs of parent plants using Agrobacterium-mediated transformation to obtain gene-edited transformed plants; (4) Screening of positive plants: PCR detection, sequencing verification and preliminary screening of target traits were performed on the transformed plants to obtain gene-edited positive plants; (5) Stable trait cultivation: positive plants are subjected to gradient environmental acclimatization and field cultivation, and cultivation conditions are controlled to promote stable expression of target traits; (6) Breeding identification and screening: The target traits, agronomic traits and genetic stability of the cultivated plants are identified and screened to select target breeding plants with excellent traits and stable genetics.

2. The breeding method according to claim 1, characterized in that, The target traits mentioned in step (1) include one or more of stress resistance, yield, quality and disease resistance; the plants include one or more of rice, wheat, corn, soybean, tomato and cotton; the parental screening also includes agronomic trait survey, and the survey indicators include one or more of plant height, number of tillers, seed setting rate and fruit size.

3. The breeding method according to claim 1, characterized in that, The gene editing vector described in step (2) is the pCas9-sgRNA vector. The design of sgRNA is based on the coding region or regulatory region of the target gene, specifically matching the target gene sequence with a matching degree of ≥98%. The Cas9 protein encoding gene is the SpCas9 gene or the SaCas9 gene.

4. The breeding method according to claim 1, characterized in that, The transformation conditions of Agrobacterium-mediated transformation in step (3) are: Agrobacterium bacterial suspension OD600 = 0.5-0.7, infection time 10-20 min, co-culture temperature 22-25℃, co-culture time 2-3 days, and maintaining a dark environment during co-culture; the young organs include one of the following: leaves, young spikes, and cotyledons.

5. The breeding method according to claim 1, characterized in that, The PCR detection described in step (4) uses specific primers to amplify the target editing fragment, and sequencing verifies that the target gene has undergone the expected editing; the preliminary screening criteria are: the target trait is better than the parent and there is no obvious abnormal growth.

6. The breeding method according to claim 1, characterized in that, The gradient environment acclimatization described in step (5) is divided into three stages: adaptation period, reinforcement period, and stabilization period. During the adaptation period, the environmental parameters are controlled under conventional cultivation conditions for 3-5 days. During the reinforcement period, the field adverse environment is simulated for 5-7 days. During the stabilization period, the conventional cultivation conditions are restored for 6-8 days. The cultivation conditions are: light intensity 3000-4500 lux, light duration 12-14 h / d, temperature 20-28℃, air humidity 55%-70%, soil pH 6.0-7.5, and soil organic matter content ≥2.5%.

7. The breeding method according to claim 1, characterized in that, The target trait identification described in step (6) includes physiological index detection and phenotypic identification. Genetic stability analysis is performed by planting for 2-3 consecutive generations to detect the expression level of the target gene and the performance of the target trait. The screening criteria are: the target trait is significantly better than the parent, the agronomic traits are excellent, and the genetic stability is ≥95%.

8. The breeding method according to claim 1, characterized in that, During the cultivation period in step (5), a plant growth regulator is sprayed every 3-4 days. The plant growth regulator is a mixture of gibberellin and salicylic acid in a concentration ratio of 3:1, with a total concentration of 50-100 mg / L. The amount sprayed each time is 10-15 mL per plant.