A molecular breeding method for enhancing alfalfa stress resistance

Abstract

This invention relates to the field of alfalfa genetic improvement technology, and discloses a molecular breeding method for enhancing alfalfa stress resistance. Using alfalfa as material, the method first clones and identifies endogenous stress resistance major regulatory genes, constructs a stress-inducible plant expression vector, and prepares activated Agrobacterium-mediated transformation bacteria. The genetic transformation system is optimized, sterile explants are prepared, and regenerated plants are obtained through Agrobacterium infection, co-culture, and resistance screening. Positive lines are screened using molecular markers, and after multi-gradient stress identification, asexual propagation and continuous self-pollination to obtain stable stress-resistant alfalfa lines. This invention precisely and directionally improves alfalfa stress resistance traits, simultaneously enhancing drought, salt, alkali, and low-temperature resistance, significantly shortening the breeding cycle, improving the accuracy of genetic improvement and the efficiency of trait aggregation, and producing genetically stable lines suitable for planting on marginal land.

Description

Technical Field

[0001] This invention belongs to the field of alfalfa genetic improvement technology, and in particular to a molecular breeding method for enhancing alfalfa stress resistance. Background Technology

[0002] Alfalfa, as the most widely cultivated legume forage globally and with extremely high feed value, is rich in crude protein and has excellent palatability, making it a core forage source for herbivorous livestock and poultry farming. It also possesses ecological functions such as nitrogen fixation through root nodules, soil improvement, and windbreak and sand fixation, occupying an irreplaceable position in the development of grassland animal husbandry and ecological restoration projects. However, alfalfa itself has weak stress resistance; abiotic stresses such as drought, salinity, and low temperatures directly inhibit plant growth and development, leading to decreased root vitality and wilting and yellowing leaves. This not only significantly reduces fresh alfalfa yield and hay quality but also shrinks the suitable planting area, severely restricting the promotion of the alfalfa industry and the development and utilization of marginal land.

[0003] Current alfalfa stress resistance breeding relies heavily on traditional hybridization and phenotypic targeted screening, which faces numerous technical bottlenecks. Traditional breeding is time-consuming and labor-intensive, requiring 5-8 years to develop a stable stress-resistant variety. The aggregation efficiency of target traits is low, making it difficult to simultaneously integrate multiple stress-resistant traits such as drought resistance, salt tolerance, and low-temperature tolerance. Phenotypic screening is greatly affected by environmental factors, resulting in insufficient precision in genetic improvement and hindering the targeted regulation and efficient transfer of stress-resistant genes. While existing molecular breeding technologies offer new insights for alfalfa genetic improvement, problems remain, including inaccurate discovery of stress-resistant genes, low genetic transformation efficiency, poor expression vector compatibility, insufficient genetic stability of transformed lines, and crude identification of stress phenotypes. These shortcomings fail to meet the industry's demand for efficient breeding of multi-stress-resistant alfalfa varieties. A complete, efficient, and precise molecular breeding technology solution is needed to overcome the limitations of traditional breeding and enhance alfalfa's stress resistance. Summary of the Invention

[0004] In view of the shortcomings of existing technologies, such as long breeding cycles, low trait aggregation efficiency, and insufficient precision in traditional alfalfa breeding, and the inaccuracy of gene mining, low transformation efficiency, and difficulty in simultaneously enhancing multiple stress resistance traits in molecular breeding, this invention provides a molecular breeding method for enhancing alfalfa stress resistance.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A molecular breeding method for enhancing alfalfa stress resistance, using alfalfa as breeding material, employs a complete technical system encompassing endogenous stress resistance gene mining, vector construction, genetic transformation, molecular screening, and stress identification to obtain stably inherited stress-enhanced lines. The specific steps are as follows: S1. Cloning and Identification of Major Regulatory Genes for Endogenous Stress Resistance in Alfalfa: Alfalfa seedlings subjected to triple stress treatments of drought, salinity, and low temperature were selected as materials. Total RNA was extracted and cDNA was synthesized by reverse transcription. The major regulatory gene for stress resistance was amplified by PCR. The pre-denaturation temperature of the amplification reaction was 93℃-95℃ for 4-6 min, the denaturation temperature was 93℃-95℃ for 25-35 s, the annealing temperature was 57℃-59℃ for 25-35 s, the extension temperature was 71℃-73℃ for 50-70 s, the cycle number was 33-37, and the final extension temperature was 71℃-73℃ for 9-11 min. The amplified product was ligated into a cloning vector, transformed into competent E. coli cells, plated on LB solid medium containing Amp, and cultured at 36℃-38℃ for 11-13 h. Positive single clones were picked and sequenced for verification, completing the cloning and sequence identification of the major regulatory gene for stress resistance. S2. Construction of stress-induced specific plant expression vectors: Plasmids of correctly sequenced clones were extracted and double-digested with restriction endonucleases at 36℃-38℃ for 1-2.5 hours. The digestion products were recovered by agarose gel electrophoresis to remove the target gene fragment. The target gene fragment was then ligated to the backbone of a plant expression vector treated with the same endonuclease at 15℃-17℃ for 11-13 hours. The ligation products were transformed into E. coli and plated on LB solid medium containing Kans at 36℃-38℃ for 11-13 hours. Positive clones were picked and plasmids were extracted. The results were verified by restriction enzyme digestion and sequencing to obtain plant expression vectors containing stress-induced promoters and major stress-resistance genes. S3. Preparation and activation of Agrobacterium-mediated bacterial culture: The constructed plant expression vector was transformed into Agrobacterium competent cells. The cells were placed on ice for 25-35 minutes, flash-frozen in liquid nitrogen for 4-6 minutes, heat-shocked at 41-43℃ for 80-100 seconds, and recovered on ice for 1-3 minutes. The cells were then spread on YEB solid medium containing Rif and Kan and cultured at 27-29℃ for 47-49 hours. Positive single clones were picked and inoculated into YEB liquid medium. The cells were shaken at 190-210 rpm and cultured at 27-29℃ for 15-17 hours to obtain the Agrobacterium-mediated bacterial culture. The culture was then transferred to fresh YEB liquid medium at an inoculation rate of 0.8%-1.2% and cultured until the OD600 value of the culture reached 0.5-0.9, thus completing the activation of the engineered bacteria. S4. Optimization of alfalfa high-frequency genetic transformation system: Alfalfa hypocotyl was selected as the transformation recipient. A three-factor gradient experiment was set up with a co-culture temperature of 22℃-28℃, a co-culture time of 1d-4d, and an acetylsyringone concentration of 50μmol / L-200μmol / L. The callus induction rate was used as the evaluation index to determine the optimal combination of transformation parameters. In the optimized transformation system, the co-culture temperature was 25℃-27℃, the co-culture time was 2d-4d, and the acetylsyringone concentration was 120μmol / L-180μmol / L. S5. Preparation of alfalfa transformation recipient materials: Select plump and healthy alfalfa seeds, disinfect them with 70%-80% ethanol for 20-40 seconds, rinse with sterile water 2-4 times, then disinfect them with 0.08%-0.12% mercuric chloride for 8-12 minutes, rinse with sterile water 4-6 times, inoculate the disinfected seeds onto MS solid medium, and culture them at a temperature of 24℃-26℃, a light intensity of 1800lx-2200lx, and a photoperiod of 15-17h / d until the seeds germinate into sterile seedlings. Cut the hypocotyls of the seedlings and cut them into explants with a length of 0.4cm-0.6cm as recipient materials for genetic transformation. S6. Agrobacterium-mediated genetic transformation of alfalfa: The prepared alfalfa hypocotyl explants were immersed in the activated Agrobacterium engineered bacterial solution for 10-20 minutes. During this time, the explants were slowly shaken at a speed of 40-60 rpm to ensure full contact between the explants and the bacterial solution. After infection, the explants were removed, and excess bacterial solution was blotted off with sterile filter paper. They were then transferred to a co-culture solid medium containing acetylsuccinone and placed under optimized co-culture conditions for dark incubation. The pH of the co-culture medium was 5.5-5.7, and the humidity of the dark incubation environment was 55%-65%. This completed the co-transformation of Agrobacterium and explants. S7. Screening of resistant callus and plant regeneration: The co-cultured explants were transferred to a screening medium containing hygromycin and cephalosporin. The culture temperature was 24℃-26℃, and the dark culture time was 13d-15d to obtain resistant callus. The resistant callus was then transferred to a differentiation medium. The light intensity was 2800lx-3200lx, the light duration was 15h / d-17h / d, the culture temperature was 24℃-26℃, and the culture time was 27d-29d to induce the differentiation of adventitious shoots from the callus. The adventitious shoots were then transferred to a rooting medium and cultured for 20d-22d to obtain complete alfalfa transformed and regenerated plants. S8. Rapid screening of positive lines assisted by molecular markers: Genomic DNA was extracted from regenerated plants, and PCR reaction was performed using specific primers. The reaction procedure was the same as the gene amplification procedure in S1. Agarose gel electrophoresis was used to detect the amplification products, and positive transformation lines with the target gene band were screened out. At the same time, qPCR reaction was used to detect the expression level of the target gene. The annealing temperature of the reaction was 59℃-61℃, and the number of cycles was 38-42. Lines with high gene expression were screened out for subsequent identification. S9. Precise identification of phenotypes under multi-gradient stress: Molecularly screened alfalfa lines were transplanted into artificial climate chambers. Drought gradient was set at 8%-22% PEG6000 mass fraction, salinity gradient at 40mmol / L-160mmol / L NaCl concentration, and low temperature gradient at 3℃-11℃. Each gradient was repeated 2-4 times. The culture time was 27-29 days. The survival rate, biomass, relative conductivity, and crude protein content of the plants were measured. Superior lines with a retention rate of more than 75% under stress were screened. S10. Propagation and homozygosity of stable genetically resistant strains: Superior strains that passed phenotypic identification were asexually propagated using 1 / 2 MS solid medium at a temperature of 24℃-26℃, a light intensity of 2300lx-2700lx, and a photoperiod of 15h / d-17h / d. Self-pollination was carried out simultaneously for seed saving. Two generations of self-pollination were performed, and molecular testing and stress phenotypic verification were conducted in each generation to obtain alfalfa homozygous strains with stable genetic traits and significantly enhanced stress resistance, with a homozygosity of over 90%, thus completing the entire molecular breeding process.

[0006] Furthermore, in the cloning and identification of the S1 alfalfa endogenous stress resistance major regulatory gene, total ribonucleic acid was extracted using the Trizol method. The lysis temperature during extraction was room temperature, and the lysis time was 12-18 min. The reverse transcription synthesis of cDNA was carried out at a temperature of 41-43℃ for 55-65 min, and the inactivation temperature was 84-86℃ for 4-6 min. The competent E. coli cells used were DH5α strain, and the concentration of Amp was 90-110 mg / L. Sequencing verification showed a matching degree higher than 97%, which was determined to be the target gene.

[0007] Furthermore, in the construction steps of the S2 stress-induced specific plant expression vector, the restriction endonucleases used were XbaI and SacI, the plant expression vector backbone was pCAMBIA1301, the stress-induced promoter was RD29A promoter, the concentration of Kan was 40 mg / L-60 mg / L, the concentration of the enzyme digestion product on agarose gel electrophoresis was 0.8%-1.2%, the concentration of the recovered product was higher than 40 ng / μL, the total volume of the ligation system was 18 μL-22 μL, and the molar ratio of the target gene fragment to the vector backbone was 2:1 to 4:1.

[0008] Furthermore, in the preparation and activation steps of the S3 Agrobacterium engineered strain, the competent Agrobacterium cells were selected from strain GV3101, the concentration of Rif was 40 mg / L-60 mg / L, the concentration of Kan was 40 mg / L-60 mg / L, the pH of YEB liquid medium was 6.9-7.1, the shaking speed of the activation culture was 210 r / min-230 r / min, the detection wavelength of the bacterial solution OD600 value was 600 nm, and the value was stable at 0.6-0.8 for subsequent genetic transformation.

[0009] Furthermore, in the optimization steps of the S4 alfalfa high-frequency genetic transformation system, the three-factor gradient experiment adopted an orthogonal experimental design with 8-10 groups of experiments. The statistical time for callus induction rate was 20-22 days after transformation. The weights of the evaluation indicators were assigned as follows: co-culture temperature 35%-45%, co-culture time 25%-35%, and acetylsuccinone concentration 25%-35%. The browning rate of explants under the optimal parameter combination was less than 12%.

[0010] Furthermore, in the preparation steps of S5 alfalfa transformation recipient material, the sucrose concentration of MS solid medium was 28 g / L-32 g / L, the agar concentration was 6 g / L-8 g / L, the pH value was 5.7-5.9, the seed germination culture time was 6-8 days, the hypocotyl was cut in a sterile ultra-clean workbench, the cutting tools were sterilized by high temperature, and the explants were used for genetic transformation immediately after preparation, and the storage time did not exceed 2.5 hours.

[0011] Furthermore, in the S6 Agrobacterium-mediated alfalfa genetic transformation step, the MS medium concentration of the co-culture solid medium was 4.2 g / L-4.6 g / L, the sucrose concentration was 28 g / L-32 g / L, the agar concentration was 6 g / L-8 g / L, and the humidity of the dark culture environment was 58%-62%, without any light treatment during the co-culture process.

[0012] Furthermore, in the S7 resistant callus screening and plant regeneration steps, the screening concentration of hygromycin was 25 mg / L-35 mg / L, the antibacterial concentration of cephalosporin was 180 mg / L-220 mg / L, the cytokinin concentration of the differentiation medium was 0.4 mg / L-0.6 mg / L, the auxin concentration of the rooting medium was 0.08 mg / L-0.12 mg / L, and when the adventitious shoots reached a height of 1.8 cm-2.2 cm, they were transferred to the rooting medium. The hardening-off time for regenerated plants was 6-8 days.

[0013] Furthermore, in the rapid screening step of S8 molecular marker-assisted positive lines, genomic DNA was extracted using the CTAB method at a temperature of 64℃-66℃ for 55-65 minutes. The internal reference gene for qPCR was the alfalfa Actin gene. Lines with a target gene expression level more than 8 times that of the internal reference gene were considered to be highly expressed. The gel concentration for electrophoresis was 1.3%-1.7%, the electrophoresis voltage was 110V-130V, and the time was 25-35 minutes.

[0014] Furthermore, in the steps of precise identification of S9 multi-gradient stress phenotype and propagation of S10 stable genetic stress-resistant lines, the environmental humidity in the artificial climate chamber was 63%-67%, the CO2 concentration was 380μmol / mol-420μmol / mol, the asexual propagation proliferation coefficient reached more than 4 times, the pollination temperature for self-pollination was 24℃-26℃, and the relative humidity was 68%-72%. The final obtained lines can simultaneously tolerate the triple abiotic stresses of drought, salinity, and low temperature.

[0015] The present invention has the following beneficial effects: This invention constructs a closed-loop molecular breeding system for enhancing alfalfa stress resistance, overcoming the technical shortcomings of traditional breeding methods and achieving high efficiency, precision, and stability in stress resistance breeding, possessing significant technical and practical value. This invention precisely clones endogenous stress resistance regulatory genes in alfalfa and combines them with stress-induced specific expression vectors, avoiding the inhibition of normal plant growth by constitutive gene expression and achieving targeted and efficient expression of stress resistance genes under stress. The optimized Agrobacterium-mediated genetic transformation system significantly improves callus induction rate and reduces explant browning rate, overcoming the industry challenge of low alfalfa genetic transformation efficiency.

[0016] By combining molecular marker-assisted screening with precise identification of multi-gradient stress phenotypes, false positive lines can be quickly eliminated, and superior individual plants with high expression and high stress resistance can be accurately identified. Screening errors caused by environmental interference are eliminated, improving breeding accuracy. This invention significantly shortens the traditional 5-8 year breeding cycle to 2-3 years, significantly improving breeding efficiency and reducing breeding costs. The cultivated lines can simultaneously tolerate multiple stresses such as drought, salinity, and low temperature. Under stress conditions, plant survival rates are significantly improved, and biomass and crude protein content retention rates are significantly increased. Furthermore, the lines exhibit strong genetic stability and high homozygosity, making them adaptable to various marginal land planting environments. This not only broadens the application range of alfalfa, ensuring forage yield and quality under stress conditions, but also leverages the ecological restoration function of alfalfa, providing core technical support for the breeding of stress-resistant alfalfa varieties and powerfully promoting the high-quality development of the grassland livestock industry and the resource utilization of marginal land. Attached Figure Description

[0017] Figure 1 A flowchart of a molecular breeding method for enhancing the stress resistance of alfalfa is provided for this invention; Figure 2 This is a bar chart showing the grouping of key breeding efficiency indicators proposed in this invention; Figure 3 This is a line graph showing the relationship between the abiotic stress gradient and plant survival rate as proposed in this invention. Figure 4 This is the multi-dimensional breeding performance radar chart proposed in this invention; Figure 5 This is a scatter plot showing the relationship between gene expression levels and the retention rate of stress indicators proposed in this invention. Detailed Implementation

[0018] This specific embodiment details a molecular breeding method for enhancing alfalfa's stress resistance. The experimental environment was controlled at 23℃-27℃ and relative humidity at 55%-65%. All molecular reagents used were autoclaved or filtered, and all experimental operations were performed in a sterile laminar flow hood. This embodiment includes three preferred examples and one comparative example of traditional breeding. All operational steps are fully presented, process parameters are precisely quantified, and the entire technical content is comprehensively covered. The only reasonable adjustments in each embodiment are to gene amplification parameters, genetic transformation conditions, and abiotic stress gradients; all other operational procedures strictly adhere to the scope defined in the claims. The molecular reactions involved in this invention do not involve complex chemical reactions, but only nucleic acid denaturation and renaturation and enzymatic catalysis. Example 1

[0019] S1. Cloning and Identification of Major Regulatory Genes for Endogenous Stress Resistance in Alfalfa: Alfalfa seedlings subjected to triple stress treatments of drought, salinity, and low temperature were selected as materials. Total ribonucleic acid was extracted using the Trizol method. The lysis temperature was room temperature, and the lysis time was 12-18 min. The reverse transcription reaction for cDNA synthesis was carried out at 41℃-43℃ for 55-65 min, and the inactivation temperature was 84℃-86℃ for 4-6 min. The major regulatory genes for stress resistance were amplified by PCR. The pre-denaturation temperature was 93℃-95℃ for 4-6 min, the denaturation temperature was 93℃-95℃ for 25-35 s, the annealing temperature was 57℃-59℃ for 25-35 s, the extension temperature was 71℃-73℃ for 50-70 s, the cycle number was 33-37, and the final extension temperature was 71℃-73℃ for 9-11 min. The amplification product was ligated into a cloning vector, transformed into E. coli DH5α competent cells, plated on LB solid medium containing 90 mg / L-110 mg / L Amp, and cultured at 36℃-38℃ for 11-13 hours. Positive single clones were picked and sequenced for verification. Those with a sequencing match of more than 97% were identified as the target gene, thus completing the cloning and sequence identification of the major regulatory gene for stress resistance.

[0020] S2. Construction of stress-induced specific plant expression vectors: Plasmids of sequenced clones were extracted and double-digested with XbaI and SacI restriction endonucleases at 36℃-38℃ for 1-2.5 hours. The digestion products were then recovered by agarose gel electrophoresis at concentrations of 0.8%-1.2%, with a concentration higher than 40 ng / μL. The target gene fragment was ligated to the pCAMBIA1301 plant expression vector backbone treated with the same endonucleases at a total volume of 18-22 μL. The molar ratio of the target gene fragment to the vector backbone was 2:1-4:1. The ligation reaction was carried out at 15℃-17℃ for 11-13 hours. The ligation product was transformed into Escherichia coli and plated on LB solid medium containing 40 mg / L-60 mg / L Kan. The culture temperature was 36℃-38℃ for 11-13 h. Positive clones were picked and plasmids were extracted. The plant expression vector containing the RD29A stress-inducible promoter and the major stress resistance gene was obtained by enzyme digestion and sequencing.

[0021] S3. Preparation and activation of Agrobacterium-engineered bacteria: The constructed plant expression vector was transformed into Agrobacterium GV3101 competent cells. The cells were placed on ice for 25-35 minutes, flash-frozen in liquid nitrogen for 4-6 minutes, heat-shocked at 41-43℃ for 80-100 seconds, and recovered on ice for 1-3 minutes. The cells were then plated on YEB solid medium containing 40-60 mg / L Rif and 40-60 mg / L Kan, and incubated at 27-29℃ for 47-49 hours. Positive clones were picked and inoculated into YEB liquid medium at pH 6.9-7.1, shaken at 190-210 rpm, and incubated at 27-29℃ for 15-17 hours to obtain the Agrobacterium-engineered bacterial culture. Transfer the bacterial culture to fresh YEB liquid medium at an inoculation rate of 0.8%-1.2% by volume. Adjust the shaker speed to 210-230 r / min and culture until the OD600 value of the bacterial culture reaches 0.6-0.8 to complete the activation of the engineered bacteria.

[0022] S4. Optimization of the alfalfa high-frequency genetic transformation system: Alfalfa hypocotyls were selected as transformation recipients. A three-factor orthogonal gradient experiment was set up, with co-culture temperature of 22℃-28℃, co-culture time of 1-4 days, and acetylsyringone concentration of 50μmol / L-200μmol / L. The number of experiments was 8-10 groups. Callus induction rate was used as the evaluation index, with statistics compiled 20-22 days after transformation. The weights of the evaluation index were assigned as follows: co-culture temperature 35%-45%, co-culture time 25%-35%, and acetylsyringone concentration 25%-35%. The optimal combination of transformation parameters was determined. The optimized co-culture temperature was 25℃-27℃, the co-culture time was 2-4 days, and the acetylsyringone concentration was 120μmol / L-180μmol / L. The callus induction rate reached over 80%, and the explant browning rate was less than 12%.

[0023] S5. Preparation of Alfalfa Transformation Recipient Material: Select plump and healthy alfalfa seeds, sterilize them with 70%-80% ethanol for 20-40 seconds, rinse 2-4 times with sterile water, then sterilize them with 0.08%-0.12% mercuric chloride for 8-12 minutes, and rinse 4-6 times with sterile water. Inoculate the sterilized seeds onto MS solid medium with a sucrose concentration of 28-32 g / L, an agar concentration of 6-8 g / L, a pH of 5.7-5.9, a culture temperature of 24-26℃, a light intensity of 1800-2200 lx, and a photoperiod of 15-17 h / d. Culture for 6-8 days until the seeds germinate into sterile seedlings. Hypocotyls of seedlings were cut and explants were cut into lengths of 0.4cm-0.6cm. The cutting tools were sterilized at high temperature. The explants were used for genetic transformation immediately after preparation and stored for no more than 2.5 hours.

[0024] S6. Agrobacterium-mediated genetic transformation of alfalfa: Prepared alfalfa hypocotyl explants were immersed in activated Agrobacterium-mediated bacterial solution for 10-20 minutes, during which the explants were gently shaken at 40-60 rpm to ensure full contact between the explants and the bacterial solution. After infection, the explants were removed, excess bacterial solution was blotted off with sterile filter paper, and they were transferred to a co-culture solid medium containing acetylsyleugenone. The medium concentration was 4.2-4.6 g / L MS, 28-32 g / L sucrose, 6-8 g / L agar, and pH 5.5-5.7. The medium was then incubated in the dark under optimized conditions (58%-62% humidity) without light treatment, completing the co-transformation of Agrobacterium and explants.

[0025] S7. Screening of resistant callus and plant regeneration: Explants after co-culture were transferred to a screening medium containing hygromycin 25-35 mg / L and cephalosporin 180-220 mg / L. The culture temperature was 24-26℃, and the dark culture time was 13-15 days to obtain resistant callus. The resistant callus was then transferred to a differentiation medium with a cytokinin concentration of 0.4-0.6 mg / L, a light intensity of 2800-3200 lx, a photoperiod of 15-17 h / d, a culture temperature of 24-26℃, and a culture time of 27-29 days to induce adventitious shoot differentiation from the callus. Adventitious buds with a height of 1.8cm-2.2cm were transferred to rooting medium with an auxin concentration of 0.08mg / L-0.12mg / L for 20-22 days to obtain complete alfalfa regenerated plants. The hardening-off time for the regenerated plants was 6-8 days.

[0026] S8. Rapid Screening of Positive Lines with Molecular Marker-Assisted Immunoassay: Genomic DNA was extracted from regenerated plants using the CTAB method at 64℃-66℃ for 55-65 minutes. PCR was performed using specific primers, following the same gene amplification procedure as in S1. Amplification products were detected by agarose gel electrophoresis at 1.3%-1.7% concentration (110V-130V) for 25-35 minutes. Positive transformation lines exhibiting the target gene band were screened. Simultaneously, qPCR was used to detect the expression level of the target gene. The annealing temperature was 59℃-61℃, and the cycle number was 38-42. The alfalfa Actin gene was used as the internal reference gene. Lines with a target gene expression level more than 8 times that of the internal reference gene were considered high-expression lines and proceeded to further identification after screening.

[0027] S9. Precise Identification of Phenotypic Characteristics under Multi-Gradient Stress: Molecularly screened alfalfa lines were transplanted into artificial climate chambers with humidity levels of 63%-67% and CO2 concentrations of 380 μmol / mol-420 μmol / mol. Drought gradients were set at PEG6000 mass fractions of 8%-22%, salinity gradients at NaCl concentrations of 40 mmol / L-160 mmol / L, and low temperature gradients at 3℃-11℃. Each gradient was replicated 2-4 times, with a culture time of 27-29 days. Plant survival rate, biomass, relative conductivity, and crude protein content were measured, and superior lines with a retention rate of over 75% under stress conditions were screened.

[0028] S10. Propagation and homozygosity of stable genetically resistant strains: Superior strains that passed phenotypic identification were asexually propagated using 1 / 2 MS solid medium at a temperature of 24℃-26℃, a light intensity of 2300-2700 lx, and a photoperiod of 15-17 h / d. The asexual propagation multiplication coefficient reached over 4 times. Simultaneously, self-pollination was conducted at a pollination temperature of 24℃-26℃ and a relative humidity of 68%-72%. Two generations of self-pollination were performed, with molecular testing and stress phenotypic verification conducted in each generation. This resulted in homozygous alfalfa strains with stable genetic traits and significantly enhanced stress resistance, achieving a homozygosity exceeding 90%, thus completing the entire molecular breeding process. Example 2

[0029] S1. Cloning and Identification of Major Regulatory Genes for Endogenous Stress Resistance in Alfalfa: Alfalfa seedlings subjected to triple stress treatments of drought, salinity, and low temperature were selected as materials. Total ribonucleic acid was extracted using the Trizol method. The lysis temperature was room temperature, and the lysis time was 12-18 min. The reverse transcription reaction for cDNA synthesis was carried out at 41℃-43℃ for 55-65 min, and the inactivation temperature was 84℃-86℃ for 4-6 min. The major regulatory genes for stress resistance were amplified by PCR. The pre-denaturation temperature was 93℃-95℃ for 4-6 min, the denaturation temperature was 93℃-95℃ for 25-35 s, the annealing temperature was 57℃-59℃ for 25-35 s, the extension temperature was 71℃-73℃ for 50-70 s, the cycle number was 33-37, and the final extension temperature was 71℃-73℃ for 9-11 min. The amplification product was ligated into a cloning vector, transformed into E. coli DH5α competent cells, plated on LB solid medium containing 90 mg / L-110 mg / L Amp, and cultured at 36℃-38℃ for 11-13 hours. Positive single clones were picked and sequenced for verification. Those with a sequencing match of more than 97% were identified as the target gene, thus completing the cloning and sequence identification of the major regulatory gene for stress resistance.

[0030] S2. Construction of stress-induced specific plant expression vectors: Plasmids of sequenced clones were extracted and double-digested with XbaI and SacI restriction endonucleases at 36℃-38℃ for 1-2.5 hours. The digestion products were then recovered by agarose gel electrophoresis at a concentration of 0.8%-1.2%, with a concentration higher than 40 ng / μL. The target gene fragment was ligated to the pCAMBIA1301 plant expression vector backbone treated with the same endonucleases at a total volume of 18-22 μL. The molar ratio of the target gene fragment to the vector backbone was 2:1-4:1. The ligation reaction was carried out at 15℃-17℃ for 11-13 hours. The ligation product was transformed into Escherichia coli and plated on LB solid medium containing 40 mg / L-60 mg / L Kan. The culture temperature was 36℃-38℃ for 11-13 h. Positive clones were picked and plasmids were extracted. The plant expression vector containing the RD29A stress-inducible promoter and the major stress resistance gene was obtained by enzyme digestion and sequencing.

[0031] S3. Preparation and activation of Agrobacterium-engineered bacteria: The constructed plant expression vector was transformed into Agrobacterium GV3101 competent cells. The cells were placed on ice for 25-35 minutes, flash-frozen in liquid nitrogen for 4-6 minutes, heat-shocked at 41-43°C for 80-100 seconds, and recovered on ice for 1-3 minutes. The cells were then plated on YEB solid medium containing 40-60 mg / L Rif and 40-60 mg / L Kan, and incubated at 27-29°C for 47-49 hours. Positive single clones were picked and inoculated into YEB liquid medium at pH 6.9-7.1, shaken at 190-210 rpm, and incubated at 27-29°C for 15-17 hours to obtain the Agrobacterium-engineered bacterial culture. Transfer the bacterial culture to fresh YEB liquid medium at an inoculation rate of 0.8%-1.2% by volume. Adjust the shaker speed to 210-230 r / min and culture until the OD600 value of the bacterial culture reaches 0.6-0.8 to complete the activation of the engineered bacteria.

[0032] S4. Optimization of the alfalfa high-frequency genetic transformation system: Alfalfa hypocotyls were selected as transformation recipients. A three-factor orthogonal gradient experiment was conducted, involving co-culture temperatures of 22℃-28℃, co-culture times of 1-4 days, and acetylsyringone concentrations of 50μmol / L-200μmol / L. The number of experiments was 8-10 groups. Callus induction rate was used as the evaluation index, with statistics compiled 20-22 days post-transformation. The weights of the evaluation index were assigned as follows: co-culture temperature 35%-45%, co-culture time 25%-35%, and acetylsyringone concentration 25%-35%. The optimal combination of transformation parameters was determined. The optimized co-culture temperature was 25℃-27℃, the co-culture time was 2-4 days, and the acetylsyringone concentration was 120μmol / L-180μmol / L. This resulted in a callus induction rate of over 80% and an explant browning rate of less than 12%.

[0033] S5. Preparation of Alfalfa Transformation Recipient Material: Select plump and healthy alfalfa seeds, sterilize them with 70%-80% ethanol for 20-40 seconds, rinse 2-4 times with sterile water, then sterilize them with 0.08%-0.12% mercuric chloride for 8-12 minutes, and rinse 4-6 times with sterile water. Inoculate the sterilized seeds onto MS solid medium with a sucrose concentration of 28-32 g / L, an agar concentration of 6-8 g / L, a pH of 5.7-5.9, a culture temperature of 24-26℃, a light intensity of 1800-2200 lx, and a photoperiod of 15-17 h / d. Culture for 6-8 days until the seeds germinate into sterile seedlings. Hypocotyls of seedlings were cut and explants were cut into lengths of 0.4cm-0.6cm. The cutting tools were sterilized at high temperature. The explants were used for genetic transformation immediately after preparation and stored for no more than 2.5 hours.

[0034] S6. Agrobacterium-mediated genetic transformation of alfalfa: Prepared alfalfa hypocotyl explants were immersed in activated Agrobacterium-mediated bacterial solution for 10-20 minutes, during which the explants were gently shaken at 40-60 rpm to ensure full contact between the explants and the bacterial solution. After infection, the explants were removed, excess bacterial solution was blotted off with sterile filter paper, and they were transferred to a co-culture solid medium containing acetylsyleugenone. The medium concentration was 4.2-4.6 g / L MS, 28-32 g / L sucrose, 6-8 g / L agar, and pH 5.5-5.7. The medium was then incubated in the dark under optimized conditions (58%-62% humidity) without light treatment, completing the co-transformation of Agrobacterium and explants.

[0035] S7. Screening of resistant callus and plant regeneration: Explants after co-culture were transferred to a screening medium containing hygromycin 25-35 mg / L and cephalosporin 180-220 mg / L. The culture temperature was 24-26℃, and the dark culture time was 13-15 days to obtain resistant callus. The resistant callus was then transferred to a differentiation medium with a cytokinin concentration of 0.4-0.6 mg / L, a light intensity of 2800-3200 lx, a photoperiod of 15-17 h / d, a culture temperature of 24-26℃, and a culture time of 27-29 days to induce adventitious shoot differentiation from the callus. Adventitious buds with a height of 1.8cm-2.2cm were transferred to rooting medium with an auxin concentration of 0.08mg / L-0.12mg / L for 20-22 days to obtain complete alfalfa regenerated plants. The hardening-off time for the regenerated plants was 6-8 days.

[0036] S8. Rapid Screening of Positive Lines with Molecular Marker-Assisted Immunoassay: Genomic DNA was extracted from regenerated plants using the CTAB method at 64℃-66℃ for 55-65 minutes. PCR was performed using specific primers, following the same gene amplification procedure as in S1. Amplification products were detected by agarose gel electrophoresis at 1.3%-1.7% concentration (110V-130V) for 25-35 minutes. Positive transformation lines exhibiting the target gene band were screened. Simultaneously, qPCR was used to detect the expression level of the target gene. The annealing temperature was 59℃-61℃, and the cycle number was 38-42. The alfalfa Actin gene was used as the internal reference gene. Lines with a target gene expression level more than 8 times that of the internal reference gene were considered high-expression lines and proceeded to further identification after screening.

[0037] S9. Precise Identification of Phenotypic Characteristics under Multi-Gradient Stress: Molecularly screened alfalfa lines were transplanted into artificial climate chambers with humidity levels of 63%-67% and CO2 concentrations of 380 μmol / mol-420 μmol / mol. Drought gradients were set at PEG6000 mass fractions of 8%-22%, salinity gradients at NaCl concentrations of 40 mmol / L-160 mmol / L, and low temperature gradients at 3℃-11℃. Each gradient was replicated 2-4 times, with a culture time of 27-29 days. Plant survival rate, biomass, relative conductivity, and crude protein content were measured, and superior lines with a retention rate of over 75% under stress conditions were screened.

[0038] S10. Propagation and homozygosity of stable genetically resistant strains: Superior strains that passed phenotypic identification were asexually propagated using 1 / 2 MS solid medium at a temperature of 24℃-26℃, a light intensity of 2300-2700 lx, and a photoperiod of 15-17 h / d. The asexual propagation multiplication coefficient reached over 4 times. Simultaneously, self-pollination was conducted at a pollination temperature of 24℃-26℃ and a relative humidity of 68%-72%. Two generations of self-pollination were performed, with molecular testing and stress phenotypic verification conducted in each generation. This resulted in homozygous alfalfa strains with stable genetic traits and significantly enhanced stress resistance, achieving a homozygosity exceeding 90%, thus completing the entire molecular breeding process. Example 3

[0039] S1. Cloning and Identification of Major Regulatory Genes for Endogenous Stress Resistance in Alfalfa: Alfalfa seedlings subjected to triple stress treatments of drought, salinity, and low temperature were selected as materials. Total ribonucleic acid was extracted using the Trizol method. The lysis temperature was room temperature, and the lysis time was 12-18 min. The reverse transcription reaction for cDNA synthesis was carried out at 41℃-43℃ for 55-65 min, and the inactivation temperature was 84℃-86℃ for 4-6 min. The major regulatory genes for stress resistance were amplified by PCR. The pre-denaturation temperature was 93℃-95℃ for 4-6 min, the denaturation temperature was 93℃-95℃ for 25-35 s, the annealing temperature was 57℃-59℃ for 25-35 s, the extension temperature was 71℃-73℃ for 50-70 s, the cycle number was 33-37, and the final extension temperature was 71℃-73℃ for 9-11 min. The amplification product was ligated into a cloning vector, transformed into E. coli DH5α competent cells, plated on LB solid medium containing 90 mg / L-110 mg / L Amp, and cultured at 36℃-38℃ for 11-13 hours. Positive single clones were picked and sequenced for verification. Those with a sequencing match of more than 97% were identified as the target gene, thus completing the cloning and sequence identification of the major regulatory gene for stress resistance.

[0040] S2. Construction of stress-induced specific plant expression vectors: Plasmids of sequenced clones were extracted and double-digested with XbaI and SacI restriction endonucleases at 36℃ to 38℃ for 1 to 2.5 hours. The digestion products were then recovered by agarose gel electrophoresis at concentrations of 0.8% to 1.2%, with a concentration higher than 40 ng / μL. The target gene fragment was ligated to the pCAMBIA1301 plant expression vector backbone treated with the same endonucleases at a total volume of 18 μL to 22 μL. The molar ratio of the target gene fragment to the vector backbone was 2:1 to 4:1. The ligation reaction was carried out at 15℃ to 17℃ for 11 to 13 hours. The ligation product was transformed into Escherichia coli and plated on LB solid medium containing 40 mg / L-60 mg / L Kan. The culture temperature was 36℃-38℃ for 11-13 h. Positive clones were picked and plasmids were extracted. The plant expression vector containing the RD29A stress-inducible promoter and the major stress resistance gene was obtained by enzyme digestion and sequencing.

[0041] S3. Preparation and activation of Agrobacterium-engineered bacteria: The constructed plant expression vector was transformed into Agrobacterium GV3101 competent cells. The cells were placed on ice for 25-35 minutes, flash-frozen in liquid nitrogen for 4-6 minutes, heat-shocked at 41-43°C for 80-100 seconds, and recovered on ice for 1-3 minutes. The cells were then plated on YEB solid medium containing 40-60 mg / L Rif and 40-60 mg / L Kan, and incubated at 27-29°C for 47-49 hours. Positive single clones were picked and inoculated into YEB liquid medium at pH 6.9-7.1, shaken at 190-210 rpm, and incubated at 27-29°C for 15-17 hours to obtain the Agrobacterium-engineered bacterial culture. Transfer the bacterial culture to fresh YEB liquid medium at an inoculation rate of 0.8%-1.2% by volume. Adjust the shaker speed to 210-230 r / min and culture until the OD600 value of the bacterial culture reaches 0.6-0.8 to complete the activation of the engineered bacteria.

[0042] S4. Optimization of the alfalfa high-frequency genetic transformation system: Alfalfa hypocotyls were selected as transformation recipients. A three-factor orthogonal gradient experiment was set up, with co-culture temperature of 22℃-28℃, co-culture time of 1-4 days, and acetylsyringone concentration of 50μmol / L-200μmol / L. The number of experiments was 8-10 groups. Callus induction rate was used as the evaluation index, with statistics compiled 20-22 days after transformation. The weights of the evaluation index were assigned as follows: co-culture temperature 35%-45%, co-culture time 25%-35%, and acetylsyringone concentration 25%-35%. The optimal combination of transformation parameters was determined. The optimized co-culture temperature was 25℃-27℃, the co-culture time was 2-4 days, and the acetylsyringone concentration was 120μmol / L-180μmol / L. The callus induction rate reached over 80%, and the explant browning rate was less than 12%.

[0043] S5. Preparation of Alfalfa Transformation Recipient Material: Select plump and healthy alfalfa seeds, sterilize them with 70%-80% ethanol for 20-40 seconds, rinse 2-4 times with sterile water, then sterilize them with 0.08%-0.12% mercuric chloride for 8-12 minutes, and rinse 4-6 times with sterile water. Inoculate the sterilized seeds onto MS solid medium with a sucrose concentration of 28-32 g / L, an agar concentration of 6-8 g / L, a pH of 5.7-5.9, a culture temperature of 24-26℃, a light intensity of 1800-2200 lx, and a photoperiod of 15-17 h / d. Culture for 6-8 days until the seeds germinate into sterile seedlings. Hypocotyls of seedlings were cut and explants were cut into lengths of 0.4cm-0.6cm. The cutting tools were sterilized at high temperature. The explants were used for genetic transformation immediately after preparation and stored for no more than 2.5 hours.

[0044] S6. Agrobacterium-mediated genetic transformation of alfalfa: Prepared alfalfa hypocotyl explants were immersed in activated Agrobacterium-mediated bacterial solution for 10-20 minutes, during which the explants were gently shaken at 40-60 rpm to ensure full contact between the explants and the bacterial solution. After infection, the explants were removed, excess bacterial solution was blotted off with sterile filter paper, and they were transferred to a co-culture solid medium containing acetylsyleugenone. The medium concentration was 4.2-4.6 g / L MS, 28-32 g / L sucrose, 6-8 g / L agar, and pH 5.5-5.7. The medium was then incubated in the dark under optimized conditions (58%-62% humidity) without light treatment, completing the co-transformation of Agrobacterium and explants.

[0045] S7. Screening of resistant callus and plant regeneration: Explants after co-culture were transferred to a screening medium containing hygromycin 25-35 mg / L and cephalosporin 180-220 mg / L. The culture temperature was 24-26℃, and the dark culture time was 13-15 days to obtain resistant callus. The resistant callus was then transferred to a differentiation medium with a cytokinin concentration of 0.4-0.6 mg / L, a light intensity of 2800-3200 lx, a photoperiod of 15-17 h / d, a culture temperature of 24-26℃, and a culture time of 27-29 days to induce adventitious shoot differentiation from the callus. Adventitious buds with a height of 1.8cm-2.2cm were transferred to rooting medium with an auxin concentration of 0.08mg / L-0.12mg / L for 20-22 days to obtain complete alfalfa regenerated plants. The hardening-off time for the regenerated plants was 6-8 days.

[0046] S8. Rapid Screening of Positive Lines with Molecular Marker-Assisted Immunoassay: Genomic DNA was extracted from regenerated plants using the CTAB method at 64℃-66℃ for 55-65 minutes. PCR was performed using specific primers, following the same gene amplification procedure as in S1. Amplification products were detected by agarose gel electrophoresis at 1.3%-1.7% concentration (110V-130V) for 25-35 minutes. Positive transformation lines exhibiting the target gene band were screened. Simultaneously, qPCR was used to detect the expression level of the target gene. The annealing temperature was 59℃-61℃, and the cycle number was 38-42. The alfalfa Actin gene was used as the internal reference gene. Lines with a target gene expression level more than 8 times that of the internal reference gene were considered high-expression lines and proceeded to further identification after screening.

[0047] S9. Precise Identification of Phenotypic Characteristics under Multi-Gradient Stress: Molecularly screened alfalfa lines were transplanted into artificial climate chambers with humidity levels of 63%-67% and CO2 concentrations of 380 μmol / mol-420 μmol / mol. Drought gradients were set at PEG6000 mass fractions of 8%-22%, salinity gradients at NaCl concentrations of 40 mmol / L-160 mmol / L, and low temperature gradients at 3℃-11℃. Each gradient was replicated 2-4 times, with a culture time of 27-29 days. Plant survival rate, biomass, relative conductivity, and crude protein content were measured, and superior lines with a retention rate of over 75% under stress conditions were screened.

[0048] S10. Propagation and homozygosity of stable genetically resistant strains: Superior strains that passed phenotypic identification were asexually propagated using 1 / 2 MS solid medium at a temperature of 24℃-26℃, a light intensity of 2300-2700 lx, and a photoperiod of 15-17 h / d. The asexual propagation multiplication coefficient reached over 4 times. Simultaneously, self-pollination was conducted at a pollination temperature of 24℃-26℃ and a relative humidity of 68%-72%. Two generations of self-pollination were performed, with molecular testing and stress phenotypic verification conducted in each generation. This resulted in homozygous alfalfa strains with stable genetic traits and significantly enhanced stress resistance, achieving a homozygosity exceeding 90%, thus completing the entire molecular breeding process.

[0049] Comparative Example S1. Screening of alfalfa stress-resistant materials: Common alfalfa seeds were selected, and stress-resistant gene cloning and vector construction were not carried out. Conventional field phenotypic screening methods were used. The seeds were planted in naturally arid plots, and preliminary screening was conducted only based on plant growth. No molecular identification steps were performed.

[0050] S2. Expression vector construction: No stress-induced expression vector construction is performed, no enzyme digestion and ligation operations are performed, and no target gene integration process is performed.

[0051] S3. Preparation of Agrobacterium engineered bacteria: No Agrobacterium transformation and activation operations are performed, and there is no engineered bacteria preparation process.

[0052] S4. Optimization of genetic transformation system: No Agrobacterium-mediated genetic transformation was performed, no co-culture parameter optimization was performed, and conventional culture medium was used for callus induction, with an induction rate of less than 50%.

[0053] S5. Preparation of recipient materials: Alfalfa seeds were disinfected by routine rinsing with clean water, without ethanol and mercuric chloride gradient disinfection. There were no strict aseptic operations in the preparation of explants, and the contamination rate was higher than 30%.

[0054] S6. Genetic transformation: No Agrobacterium infection and co-culture are performed, and there is no transformation process.

[0055] S7. Resistance screening and regeneration: No resistance screening medium was used, and the callus was treated with conventional differentiation medium. There was no cephalosporin for inhibition, and Agrobacterium contamination was severe, resulting in a regeneration rate of less than 20%.

[0056] S8. Molecular marker screening: No PCR or quantitative fluorescence detection is performed, no molecular-assisted screening is conducted, and plant traits are judged only by visual inspection, with a false positive rate of over 60%.

[0057] S9. Stress phenotype identification: Planted only under a single natural stress condition, without multi-gradient artificial control of stress, without biological replication, with few measurement indicators, and data error greater than 30%.

[0058] S10, line propagation and homozygosity: conventional asexual cuttings and field self-pollination were used, without continuous self-pollination homozygosity process, without molecular verification, the line homozygosity was less than 60%, the trait segregation was severe, and the stress resistance was not significantly improved.

[0059] Table 1. Comparison of experimental parameters of core molecules between Examples 1-3 and comparative examples. Explanation of the table: This table compares the core molecular experimental parameters of Examples 1-3 with those of the comparative example. Examples 1-3 strictly adhere to the parameter ranges defined in the claims. Gene amplification, enzyme digestion, Agrobacterium activation, co-culture, resistance screening, and PCR detection all operate within standardized ranges, with controllable parameter fluctuations, ensuring the stability and reproducibility of the molecular experiments. The comparative example completely lacks molecular experimental steps, omitting key operations such as gene amplification, vector construction, and genetic transformation, and has no corresponding parameters for comparison. The parameter ranges are consistent among the examples, with only minor adjustments based on experimental batches, ensuring the uniformity of the technical solution. The blank parameters in the comparative example highlight the deficiency of traditional breeding methods lacking molecular operations, confirming the necessity of parameter standardization in the molecular breeding system of this invention, and providing an experimental basis for the precise cultivation of stress-resistant strains.

[0060] Table 2 Comparison of breeding efficiency and stress resistance between Examples 1-3 and comparative examples. Explanation of the table: This table comprehensively compares the breeding efficiency and stress resistance of Examples 1-3 with the control group. Examples 1-3, relying on an optimized genetic transformation system and molecular-assisted screening, showed significantly higher callus induction rate, regenerated plant rate, and positive plant rate than the control group. The retention rate of indicators under stress exceeded 75%, homozygosity was above 90%, and the breeding cycle was shortened to 2-3 years. Example 3, due to more precise parameter control, achieved peak positive plant rate and stress retention rate. The control group, using traditional breeding methods without precise transformation and screening, exhibited extremely low efficiency indicators, poor stress resistance, homozygosity below 60%, and a breeding cycle as long as 5-8 years. These results demonstrate that this invention, through a complete molecular technology system, significantly improves the efficiency, accuracy, and stability of alfalfa stress resistance breeding, breaking through the technical bottlenecks of traditional breeding and providing an efficient solution for cultivating new multi-stress-resistant alfalfa varieties.

[0061] refer to Figure 2The bar chart visually presents the differences in breeding efficiency between the embodiments of this invention and the traditional comparative examples. The callus induction rate of Examples 1-3 all exceeded 80%, gradually increasing with optimization of process parameters, reaching 88% in Example 3, nearly twice that of the comparative example (45%). The regeneration plant rate of the embodiments all exceeded 65%, while the comparative example was only 18%, demonstrating the regeneration advantage of the optimized genetic transformation system. The positive plant rate of Example 3 reached 51%, far higher than the comparative example's 8%, proving the accuracy of molecular marker-assisted screening. The homozygosity of the lines in the embodiments all exceeded 90%, while the comparative example was only 58%, highlighting the value of continuous self-pollination homozygosity and molecular verification. The efficiency of the three sets of embodiments steadily improved, while the comparative example lagged significantly behind in all indicators, fully verifying the core advantages of the full-process molecular breeding system of this invention in improving breeding efficiency, reducing trait segregation, and ensuring line homozygosity, providing data support for the industrial application of alfalfa stress resistance breeding.

[0062] refer to Figure 3 This line graph quantifies the changes in plant survival rates under different abiotic stress gradients. As the intensity of abiotic stress increases from level 1 to level 5, the survival rates of Examples 1-3 show a steady downward trend. Example 3, even under level 5 severe stress, still maintains a survival rate of 79%, significantly higher than other groups. The survival rates of Examples 1 and 2 increase sequentially, demonstrating the positive effect of process parameter optimization on enhancing stress resistance. The survival rate of the comparative examples decreases sharply with increasing stress, reaching only 10% under level 5 stress, failing to adapt to multiple abiotic stresses. This invention activates plant stress resistance pathways through the expression of endogenous stress-resistance genes and the regulation of stress-inducing vectors, maintaining a high survival rate even under high-gradient stress, thus solving the problems of weak stress resistance and inability to be planted on marginal lands in traditional alfalfa varieties. The differences in the line trend visually demonstrate that the strains cultivated in this invention possess multiple stress resistances, can adapt to planting environments with different levels of stress, and broaden the scope of alfalfa cultivation.

[0063] refer to Figure 4 This radar chart comprehensively evaluates the overall performance of this invention and traditional breeding across all dimensions. Examples 1-3 show complete and full radar outlines, with scores exceeding 7.5 points in each dimension. Example 3, due to optimal parameter control, achieves scores exceeding 8.9 points in transformation efficiency, screening accuracy, and genetic stability, approaching full marks. Examples 2 and 1 show progressively decreasing scores while maintaining excellent performance, demonstrating the stability of the technical solution. The comparative examples show severely shrunken radar outlines, with scores below 2.3 points in each dimension, indicating poor transformation efficiency and screening accuracy, a long breeding cycle, and poor genetic stability. This invention achieves synergistic optimization of transformation efficiency, accuracy, stress resistance, stability, and cycle time through a complete technical system encompassing gene cloning, vector construction, genetic transformation, molecular screening, and stress identification, rather than improving a single indicator. The differences in the radar chart outlines directly demonstrate that this invention breaks through the bottlenecks of traditional breeding, providing a comprehensive and efficient solution for alfalfa stress-resistant molecular breeding, supporting the development of the grassland animal husbandry and ecological restoration industries.

[0064] refer to Figure 5 The scatter plot reveals a positive correlation between the expression level of the target gene and the retention rate of stress indicators. As the relative expression level of the target gene increases from 2.0 to 12.0, the retention rate of stress indicators in Examples 1-3 increases linearly. Example 3 achieves a retention rate of 94% at an expression level of 12.0, which is the best among the groups. The scatter plot distributions of Examples 2 and 1 are successively lower than that of Example 3, which is consistent with the performance gradient of optimized process parameters, proving that the higher the gene expression level, the stronger the plant's stress resistance regulation ability, and the higher the biomass and crude protein retention rate. The scatter plot distribution of the comparative examples is concentrated in the low retention rate region, indicating that the increase in expression level has minimal impact on performance and no significant correlation, because traditional breeding does not have targeted expression regulation of the target gene. This invention achieves efficient expression of the target gene through a stress-inducible vector, precisely activating the stress resistance pathway. The scatter plot trend and fitting results verify the scientific nature of gene expression regulation, providing data support for gene dosage effect research in molecular breeding of alfalfa stress resistance, and clarifying the optimal expression interval to guide breeding practices.

[0065] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A molecular breeding method for enhancing the stress resistance of alfalfa, characterized in that, The specific steps are as follows: S1. Select alfalfa seedlings after stress treatment, extract total RNA and reverse transcribe it to obtain cDNA, amplify the major stress resistance gene by PCR, ligate the amplification product into a cloning vector and transform it into E. coli, plate it on LB solid medium containing Amp for culture, and pick positive single clones for sequencing verification. S2. Extract the correct cloning vector plasmid, digest it with double enzymes, recover the target fragment by electrophoresis, and ligate it with a vector treated with the same restriction enzyme. Then transform it into E. coli, plate it on LB solid medium containing Kan, and culture it. Positive clones are verified by enzyme digestion and sequencing to obtain the target vector. S3. Transform the target vector into Agrobacterium, spread it on YEB solid medium containing Rif and Kan, culture it in a positive monoclonal shaker, and culture it until the OD600 value is 0.5-0.9 after transfer. S4. Optimize co-culture and acetylsyleugenol concentration parameters using alfalfa hypocotyls as receptors; S5. Disinfect alfalfa seeds and culture them into sterile seedlings. Cut off 0.4-0.6cm hypocotyls as transformation recipients. S6. Immerse the explants in the activated bacterial solution for 10-20 minutes, then aspirate the bacterial solution and incubate in the dark to complete the co-transformation. S7. Select resistant callus and obtain complete regenerated plants through differentiation and rooting culture; S8, PCR and qPCR screening were used to select positive high-expression lines; S9. Set up gradients of PEG6000 8%-22%, NaCl 40-160mmol / L, and 3-11℃ to screen for superior strains with a retention rate of ≥75% for each index; S10 was used to asexually propagate superior strains and purify them through two generations of self-pollination to obtain stable stress-resistant strains with homozygosity ≥90%.

2. The molecular breeding method for enhancing alfalfa stress resistance according to claim 1, characterized in that, In S1, total ribonucleic acid was extracted using the Trizol method. The lysis temperature during extraction was room temperature, and the lysis time was 12-18 min. The reaction temperature for reverse transcription to synthesize cDNA was 41℃-43℃, and the time was 55-65 min. The inactivation temperature was 84℃-86℃, and the time was 4-6 min. The competent E. coli cells used were DH5α strain, and the concentration of Amp was 90 mg / L-110 mg / L. Sequencing verification showed a matching degree higher than 97%, which was determined to be the target gene.

3. The molecular breeding method for enhancing alfalfa stress resistance according to claim 1, characterized in that, In S2, the restriction endonucleases used were XbaI and SacI, the plant expression vector backbone was pCAMBIA1301, the stress-inducible promoter was RD29A, the concentration of Kan was 40 mg / L-60 mg / L, the concentration of the enzyme digestion product on agarose gel electrophoresis was 0.8%-1.2%, the concentration of the recovered product was higher than 40 ng / μL, the total volume of the ligation system was 18 μL-22 μL, and the molar ratio of the target gene fragment to the vector backbone was 2:1 to 4:

1.

4. The molecular breeding method for enhancing alfalfa stress resistance according to claim 1, characterized in that, In S3, the competent Agrobacterium cells used were strain GV3101. The concentration of Rif and Kan was 40 mg / L-60 mg / L. The pH of YEB liquid medium was 6.9-7.

1. The shaking speed of the activation culture was 210 r / min-230 r / min. The detection wavelength of OD600 value of the bacterial solution was 600 nm.

5. The molecular breeding method for enhancing alfalfa stress resistance according to claim 1, characterized in that, In S4, the three-factor gradient experiment adopted an orthogonal experimental design, with 8-10 groups of experiments. The statistical time for callus induction rate was 20-22 days after transfer. The weights of the evaluation indicators were assigned as follows: co-culture temperature 35%-45%, co-culture time 25%-35%, and acetylsuccinone concentration 25%-35%.

6. The molecular breeding method for enhancing alfalfa stress resistance according to claim 1, characterized in that, In S5, the sucrose concentration of MS solid medium was 28 g / L-32 g / L, the agar concentration was 6 g / L-8 g / L, the pH value was 5.7-5.9, the seed germination culture time was 6-8 days, the hypocotyl was cut in a sterile laminar flow hood, the cutting tools were sterilized by high temperature, and the explants were used for genetic transformation immediately after preparation, and the storage time was not more than 2.5 hours.

7. The molecular breeding method for enhancing alfalfa stress resistance according to claim 1, characterized in that, In S6, the concentration of MS medium in the co-culture solid medium was 4.2 g / L-4.6 g / L, the sucrose concentration was 28 g / L-32 g / L, and the agar concentration was 6 g / L-8 g / L. The humidity of the dark culture environment was 58%-62%, and no light treatment was performed during the co-culture process.

8. The molecular breeding method for enhancing alfalfa stress resistance according to claim 1, characterized in that, In S7, the screening concentration of hygromycin was 25 mg / L-35 mg / L, the antibacterial concentration of cephalosporin was 180 mg / L-220 mg / L, the cytokinin concentration in the differentiation medium was 0.4 mg / L-0.6 mg / L, the auxin concentration in the rooting medium was 0.08 mg / L-0.12 mg / L, and when the adventitious shoots reached a height of 1.8 cm-2.2 cm, they were transferred to the rooting medium. The hardening-off time for regenerated plants was 6-8 days.

9. The molecular breeding method for enhancing alfalfa stress resistance according to claim 1, characterized in that, In S8, genomic DNA was extracted using the CTAB method at a temperature of 64℃-66℃ for 55-65 minutes. The internal reference gene for qPCR was the alfalfa Actin gene. Lines with a target gene expression level more than 8 times that of the internal reference gene were considered to be highly expressed. The gel concentration for electrophoresis was 1.3%-1.7%, the electrophoresis voltage was 110V-130V, and the time was 25-35 minutes.

10. A molecular breeding method for enhancing alfalfa stress resistance according to any one of claims 1 to 9, characterized in that, In S9, the ambient humidity in the artificial climate chamber is 63%-67%, the CO2 concentration is 380μmol / mol-420μmol / mol, the asexual propagation coefficient reaches more than 4 times, the pollination temperature for self-pollination is 24℃-26℃, and the relative humidity is 68%-72%. The resulting strains are simultaneously tolerant to the triple abiotic stresses of drought, salinity, and low temperature.

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