Method for analyzing alfalfa stress resistance gene function based on metabonomics and breeding method

Abstract

The invention relates to the technical field of plant breeding, and particularly discloses a metabonomics-based alfalfa stress resistance gene analysis function and a breeding method. The core of the method is that screening is carried out by combining a tissue culture technology with metabolic intervention; an alfalfa explant is inoculated into an induction culture medium containing a specific metabolic pathway regulator for metabolic intervention culture; then transferring the obtained callus to a verification culture medium containing the metabolism regulator and an abiotic adversity stress factor at the same time, and carrying out metabolism mark verification and screening; and finally regenerating the screened cell line into a complete plant. According to the method, metabolic pressure is actively applied to the cellular level, and dual verification screening is implemented, so that early-stage, efficient and accurate breeding of the stress-resistant alfalfa material is realized, and the technical problems of long period, low efficiency and insufficient utilization of metabolic information in traditional breeding are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant breeding, more particularly, the present application relates to a method for analyzing alfalfa stress resistance gene function and breeding based on metabolomics. BACKGROUND

[0002] Alfalfa is an important legume forage, its yield and quality are often severely restricted by abiotic stress such as drought, salinity, etc. Therefore, breeding new alfalfa varieties with strong stress resistance is of great significance to guarantee forage supply and sustainable agricultural development.

[0003] Traditional stress resistance breeding mainly relies on phenotypic selection of mature plants under natural or simulated stress in the field, which is a long cycle, usually takes several years, is greatly affected by environmental factors, and has low and unstable screening efficiency. With the development of molecular biology, molecular marker assisted selection technology has been applied, but its effectiveness is highly dependent on known stress-related gene loci; while alfalfa stress traits are controlled by multiple gene networks, and the functions of many key genes are unknown, which limits the application of this technology in actual breeding. In recent years, metabolomics technology has been used to analyze plant stress response mechanisms, which can efficiently identify key metabolites and pathways related to stress resistance. However, existing technologies mostly use metabolomics analysis to explain the mechanism of known stress-resistant materials or as a post-validation tool, and have not yet been deeply integrated into the early breeding cycle to form an efficient breeding strategy that actively uses metabolic information to directly guide and accelerate stress resistance trait screening and fixation.

[0004] Therefore, the method for analyzing alfalfa stress resistance gene function and breeding based on metabolomics is proposed to solve the problems of long screening cycle, dependence on prior genetic knowledge, and ineffective use of metabolic information to drive the breeding process in existing alfalfa stress resistance breeding methods. SUMMARY

[0005] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present application provide a method for analyzing alfalfa stress resistance gene function and breeding based on metabolomics to solve the problems raised in the background art.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solution: a method for analyzing alfalfa stress resistance gene function and breeding based on metabolomics, which is screened and cultivated by tissue culture technology combined with metabolic intervention, including the following steps: S1: metabolic intervention culture: inoculate alfalfa explants into an induction medium containing plant growth regulators and at least one metabolic regulator for induction and first-stage proliferation culture of callus; the metabolic regulator can specifically inhibit or interfere with a pre-set metabolic pathway related to stress response in alfalfa cells; S2: Metabolic signature verification and selection: the first-stage callus obtained in S1 is transferred to a verification medium containing both the metabolic regulator and an abiotic stress factor for a second-stage culture; callus cell lines that can maintain sustained proliferation ability in the verification medium are selected and retained; S3: Plant regeneration: the callus cell lines selected in S2 are regenerated into intact alfalfa plants.

[0007] Preferably, the pre-set metabolic pathway is proline synthesis pathway; and the metabolic regulator is at least one proline analog or competitive inhibitor selected from Azetidine-2-carboxylic acid (A2C) or 3,4-dehydro-DL-proline.

[0008] Preferably, the pre-set metabolic pathway is polyamine synthesis pathway; and the metabolic regulator is at least one polyamine synthesis inhibitor or substrate analog selected from D-arginine, α-difluoromethylornithine (DFMO).

[0009] Preferably, in S1, the concentration of the metabolic regulator in the induction medium is 5 μM to 100 μM, which is configured to reduce the growth rate of callus by 15% to 50% compared to the control medium without the regulator.

[0010] Preferably, in S1, the first-stage proliferation culture lasts for at least 2 subculture cycles, each subculture cycle being 21 days to 28 days.

[0011] Preferably, in S2, the abiotic stress factor is mannitol for simulating drought stress, at a concentration of 50 mM to 150 mM in the verification medium; or sodium chloride for simulating salt stress, at a concentration of 50 mM to 100 mM in the verification medium.

[0012] Preferably, in S2, the concentration of the metabolic regulator in the verification medium is 50% to 100% of the concentration in the induction medium of S1.

[0013] Preferably, in S2, the selection criterion for "being able to maintain sustained proliferation ability" is that the callus increases in fresh weight or dry weight by no less than 70% of the increase in the control callus cultured in the same generation number of regular medium after at least 2 rounds of subculture in the verification medium.

[0014] Preferably, in S3, the step of regenerating into whole plants specifically comprises: transferring the screened callus into a differentiation medium containing cytokinin and auxin but free of the metabolic regulator and the abiotic stress factor to induce shoot formation, and then transferring the resulting shoots into a rooting medium containing auxin to induce root formation.

[0015] Preferably, the method further comprises a step S0 before S1: determining metabolic intervention targets based on metabolomics analysis; the step S0 comprises: performing metabolomics analysis on the alfalfa material treated by abiotic stress, screening target metabolites with changed content and mapping to their biochemical pathways to analyze core metabolic pathway targets, and selecting the metabolic regulator used in S1 accordingly.

[0016] Technical effects and advantages of the present application: Compared with existing screening techniques relying on end-of-field phenotypes or known molecular markers, the present application artificially creates a controllable metabolic stress environment by adding a regulator capable of specifically inhibiting a pre-set stress-resistant metabolic pathway (such as proline synthesis pathway) to the culture medium during the callus induction and proliferation stage of alfalfa tissue culture. This step forces callus cells to start or strengthen alternative compensatory metabolic mechanisms to adapt to this environment, thereby actively shaping a new and more stable metabolic homeostasis at the cellular level. Subsequently, secondary culture in a verification medium containing both the metabolic regulator and the actual stress factor (such as sodium chloride) can efficiently identify cell lines that have successfully restructured their metabolic networks and truly possess strong stress-resistant potential. This active intervention-stress verification process enables early simulation, strengthening, and screening of stress-resistant traits in tissue culture bottles, improves the reliability of breeding decisions based on metabolic phenotypes, and provides a method for breeding complex stress-resistant traits controlled by multiple genes without relying on prior genetic site information. BRIEF DESCRIPTION OF DRAWINGS

[0017] Fig. 1 The overall workflow diagram of the method of the present application is shown in FIG.

[0018] Fig. 2 The core processing and data analysis flowchart of the present application is shown in FIG.

[0019] Fig. 3 The verification and screening decision flowchart of the present application is shown in FIG. DETAILED DESCRIPTION

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 As attached Figs. 1 to 3 The method for analyzing alfalfa stress-resistance gene function and breeding based on metabolomics, as shown, begins with the identification of key metabolic pathways in the salt stress response; specific chemical interventions targeting these pathways are applied at the callus stage; quantitative screening is then conducted under the dual pressure of superimposed chemical interventions and ion stress; and finally, stable and tolerant cell lines are regenerated into plants. This approach shifts trait selection from the field individual level to the in vitro cell level, directly enriching cells with the target metabolic adaptation capabilities through actively designed stress environments, thereby shortening the breeding cycle and improving directionality.

[0022] Detailed Example: Salt Tolerance Improvement of Alfalfa 'Zhongmu No. 1' Step 1: Metabolic Target Analysis and Standardization of Experimental Materials 1. Plant material culture: Alfalfa 'Zhongmu No. 1' seeds were surface-sterilized with 5% sodium hypochlorite solution for 15 minutes, rinsed with sterile water, and sown on hormone-free MS solid medium for germination. After 7 days of culture, seedlings with uniform growth were selected and transferred to tissue culture bottles containing half-strength Hoagland solution for hydroponics in a light-controlled culture room.

[0023] The culture chamber conditions were set as follows: day / night temperature 25 / 22°C, light intensity 120 μmol m⁻ 2 s⁻ 1 The photoperiod is 16 / 8 hours, and the relative humidity is 60%. The hydroponic nutrient solution is changed every 5 days, and ventilation is continuous.

[0024] 2. Stress Treatment and Sample Collection: After 14 days of hydroponic cultivation, treatment and control groups were established, with at least 30 seedlings in each group. The nutrient solution in the treatment group was replaced with a half-strength Hoagland solution supplemented with 100 mmol / L sodium chloride; the control group maintained the original nutrient solution. The stress treatment lasted for 168 hours (7 days).

[0025] Four hours after the treatment, the second and third true leaves from the top of each of the two seedling groups were collected and immediately flash-frozen in liquid nitrogen, then transferred to an ultra-low temperature freezer at -80°C for storage. This sampling time point was intended to capture a relatively stable metabolic state adapted to stress, avoiding interference from diurnal metabolic fluctuations.

[0026] 3. Metabolomics analysis: Sample extraction: About 100 mg of frozen leaf blade tissue was quickly ground into fine powder in a mortar pre-cooled with liquid nitrogen. The powder was transferred to a 2 mL centrifuge tube, and 1.2 mL of pre-cooled extraction solvent (methanol:acetonitrile:water = 2:2:1, v / v / v, containing 0.1% formic acid) was accurately added.

[0027] Vortexed for 2 min, extracted by ultrasonic for 30 min in ice bath, and then placed at -20 °C for 1 h. Centrifuged at 4 °C, 13000 rpm for 15 min, and 1 mL of supernatant was taken, filtered through a 0.22 μm microporous filter, and then transferred to an injection vial for LC-MS analysis.

[0028] Instrument analysis: An ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry system was used. The chromatographic column was a C18 column, and the column temperature was 40 °C. The mobile phase A was water containing 0.1% formic acid, and the mobile phase B was acetonitrile containing 0.1% formic acid. The gradient elution program was as follows: 0-2 min, 2% B; 2-15 min, B linearly increased to 98%; 15-18 min, maintained at 98% B; 18-18.1 min, decreased to 2% B; 18.1-20 min, balanced. The mass spectrometry used an electrospray ionization source, and the positive and negative ion modes were scanned respectively, with a scanning range of m / z 50-1500.

[0029] Data processing and target determination: The instrument's built-in software was used to convert the raw data into a data matrix containing mass-to-charge ratio, retention time, and peak area. After missing value filtering and normalization, multivariate statistical analysis was performed. The orthogonal partial least squares discriminant analysis model verified that all sample points were within the 95% confidence interval, and the model was effective. According to the standard of VIP value > 1.5 and p value < 0.01, 15 significantly different metabolites such as proline, betaine, and fructose-6-phosphate were screened out. Through KEGG pathway enrichment analysis, it was found that the "arginine and proline metabolism" pathway was the most significantly enriched. The proline synthesis pathway was determined as the key target. For the subsequent tissue culture, another batch of hydroponic seedlings was pretreated with 50 mmol / L NaCl for 72 h, and the middle section of the epicotyl was cut into small pieces about 1.0 cm long for use.

[0030] Step two: metabolic intervention culture 1. Medium preparation: The basal medium was MS medium supplemented with 30 g / L sucrose and 7 g / L agar powder, pH adjusted to 5.8 ± 0.1 with 1 mol / L KOH or HC1. The high-pressure steam sterilization condition was 121 °C for 20 min. After sterilization, the medium was allowed to cool to about 55 °C, and the hormone and metabolic modulator stock solutions, which were sterilized by filtration through a 0.22 μm filter, were added aseptically in sequence in a clean bench. Finally, the hormone composition of the induction medium was: 1.0 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L kinetin. The final concentration of the metabolic modulator Azetidine-2-carboxylic acid was 20 μmol / L. Another medium without Azetidine-2-carboxylic acid, but with the same other components, was prepared as a control.

[0031] 2. Callus induction and primary intervention: The pretreated stem segments were placed in a clean bench, immersed in 70% ethanol for 30 s, then in 2% sodium hypochlorite solution (with 2 drops of Tween-20 added) for 15 min, and rinsed with sterile water for 5 times. After the surface moisture was absorbed with sterile filter paper, the explants were cut off about 2 mm at both ends, and placed flat on the induction medium with 5 segments per dish. After sealing with parafilm, they were placed in a constant temperature incubator at 25 ± 1 °C, and cultured in complete darkness. After 28 days of culture, the explants swelled at both ends, and formed yellowish-white, loose-structured primary calli.

[0032] 3. Continuous intervention and subculture: In a clean bench, the primary calli were peeled off from the explants with sterile forceps and surgical knives, and cut into small pieces with a diameter of about 3-4 mm and a weight of about 30-50 mg. These callus pieces were transferred to fresh induction medium containing the same concentration (20 μmol / L) of Azetidine-2-carboxylic acid, and subjected to the first round of subculture under the same conditions.

[0033] After 28 days, the callus pieces were observed to have increased in volume, and some of them became more compact. This subculture was repeated once, and the second round of subculture was completed. By this time, the calli had been cultured in the presence of the metabolic modulator for a total of 84 days, and had undergone multiple cell division cycles. During this process, some calli grew slowly or browned, while some maintained their proliferative ability adaptively, and forced the cell population to adaptively evolve or select in the direction of enhancing precursor supply, activating alternative pathways, or improving metabolic efficiency, etc. under the "metabolic crisis" by continuously suppressing the synthesis of proline osmotic regulators.

[0034] Step three: verification and quantitative screening of metabolic imprint 1. Verification medium preparation and screening initiation: The verification medium is identical to the induction medium on MS basal components, but contains double stress components: Azetidine-2-carboxylic acid at 10 μmol / L (50% of the induction phase concentration), and sodium chloride at 80 mmol / L. Sodium chloride is autoclaved together with the other medium components. From the intervention cultures that have been subcultured twice and are in good growth condition, select the yellowish in color and dense in texture callus pieces, and weigh 10 pieces as a group using an analytical balance (precision 0.1 mg). Record the initial total fresh weight (denoted as W_initial). Inoculate this group of callus pieces into the same dish of verification medium, and set 3 biological replicates for each treatment.

[0035] 2. Two rounds of verification culture and data recording: The inoculated dishes are cultured under standard light conditions for 28 days (first round of verification). At the end of the culture, remove all callus pieces, rinse them gently with sterile water to remove the surface medium, and carefully blot excess water with filter paper, and immediately weigh the total fresh weight, and record the number of surviving pieces. Subsequently, transfer all surviving callus pieces (regardless of size) into new verification medium, and start the second round of culture for 28 days. At the end of the culture, weigh again, and record the final total fresh weight (denoted as W_final_ver). At the same time, set up a parallel control: inoculate ordinary callus that has not been subjected to any metabolic intervention into standard MS medium without any additives, in the same grouping and initial weight (W_initial_ctrl), and culture synchronously for two rounds for a total of 56 days, and record the final total fresh weight (denoted as W_final_ctrl).

[0036] 3. Execution of quantitative screening criteria: The screening decision is based on the screening ratio calculated by the following formula: SR = (W_final_ver - W_initial) / (W_final_ctrl - W_initial_ctrl) where SR represents the screening ratio. This formula quantifies the biomass accumulation efficiency of the test material under double stress relative to the control material grown under ideal conditions. Set when SR ≥ 0.70, the callus population (i.e., a cell line) is considered to pass the verification.

[0037] For example, a candidate line has W_initial of 0.512 g, and W_final_ver of 0.789 g; its control has W_initial_ctrl of 0.505 g, and W_final_ctrl of 1.201 g.

[0038] Then, the SR = (0.789 - 0.512) / (1.201 - 0.505) = 0.277 / 0.696 = 0.398 is calculated.

[0039] This value is lower than 0.70, so the candidate line is eliminated. Conversely, if the calculated SR value is 0.75, it is retained. Through this mathematical criterion, it is possible to objectively identify, from a large number of candidate materials, those elite cell lines that can maintain a relatively high relative growth rate under severe combined stress. Through the calculation in this example, three cell lines (Nos. T-1, T-2, and T-3) are finally determined to meet the standard.

[0040] Step four: plant regeneration and transplant acclimation 1. Differentiation induction: The callus blocks of the T-1, T-2, and T-3 cell lines that passed the verification are transferred to the differentiation medium. The medium does not contain Azetidine-2-carboxylic acid and sodium chloride, and the hormone composition is MS + 0.5 mg / L 6-benzylaminopurine + 0.05 mg / L naphthaleneacetic acid. The culture conditions are adjusted to a photoperiod of 16 / 8 hours. After 21-28 days of culture, green bud points appear on the surface of the callus and gradually develop into clustered buds.

[0041] 2. Rooting and seedling raising: When the adventitious buds grow to 2-3 cm high, they are cut from the base with a sterile scalpel and inserted into the rooting medium. The rooting medium is 1 / 2 MS + 0.2 mg / L indole-3-butyric acid. After about 14 days, most of the bud seedlings form adventitious roots with a length of more than 1 cm at the base. The well-rooted tissue culture seedlings are opened with a bottle cap and acclimated in the culture room for 3 days, then the seedlings are carefully washed of the root agar and transplanted into a substrate mixed from sterilized grass carbon, vermiculite, and perlite at a volume ratio of 3:1:1.

[0042] 3. Post-transplant management and trait observation: The first week after transplanting, the plants are covered with a transparent plastic film to maintain moisture, and the environmental humidity is maintained above 85%, then the film is gradually removed to adapt to the greenhouse environment. After the plants recover and grow more than 3 new leaves, preliminary observation of salt tolerance can be carried out.

[0043] For example, the regenerated plants and control plants are subjected to quantitative irrigation treatment with Hoagland nutrient solution containing 150 mmol / L NaCl, and at different time points after treatment, phenotypic analysis is carried out by measuring physiological indicators such as leaf electrolyte permeability and photosynthetic parameters. This analysis aims to provide evidence at the individual level that the metabolic adaptation potential obtained through directional screening at the cellular level has been successfully integrated and expressed in the overall physiological system of the regenerated plants, thereby providing a trait-excellent basis material for subsequent variety comparison tests and genetic stability evaluation.

[0044] Finally, it should be noted that: first, in the description of the present application, it should be noted that, unless otherwise specified and limited, the terms "mounting", "connected", "connected" should be broadly understood, which can be mechanical connection or electrical connection, or the internal communication of two elements, or direct connection, "up", "down", "left", "right" and the like are only used to indicate relative positional relationship, when the absolute position of the described object changes, the relative positional relationship may change; Secondly: the drawings of the disclosed embodiments of the present application only involve the structures involved in the disclosed embodiments, other structures can refer to the usual design, and in the case of no conflict, the same embodiment and different embodiments of the present application can be combined with each other; Finally: the above only describes the preferred embodiments of the present application and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for analyzing the function of alfalfa stress-resistance genes and developing breeding techniques based on metabolomics, characterized in that... Screening and cultivation using tissue culture techniques combined with metabolic intervention includes the following steps: S1: Metabolic intervention culture: Alfalfa explants were inoculated into an induction medium containing plant growth regulators and at least one metabolic regulator to induce callus and carry out the first stage of proliferation culture; the metabolic regulators can specifically inhibit or interfere with a pre-defined metabolic pathway in alfalfa cells that is related to stress response. S2: Metabolic imprint verification and screening: The first-stage callus obtained in S1 is transferred to a verification medium containing the metabolic regulator and an abiotic stress factor for a second-stage culture; callus cell lines that can maintain continuous proliferation in the verification medium are screened and retained. S3: Plant regeneration: The callus cell lines obtained in S2 are regenerated into complete alfalfa plants.

2. The method for analyzing the function of alfalfa stress-resistance genes and breeding based on metabolomics according to claim 1, characterized in that, The preset metabolic pathway is the proline synthesis pathway; the metabolic regulator is at least one proline analog or competitive inhibitor selected from Azetidine-2-carboxylic acid (A2C) or 3,4-dehydro-DL-proline.

3. The method for analyzing the function of alfalfa stress-resistance genes and breeding based on metabolomics according to claim 1, characterized in that, The preset metabolic pathway is a polyamine synthesis pathway; the metabolic regulator is at least one polyamine synthesis inhibitor or substrate analog selected from D-arginine, α-difluoromethylornithine (DFMO).

4. The method for analyzing the function of alfalfa stress-resistance genes and breeding based on metabolomics according to claim 1, characterized in that, In S1, the concentration of the metabolic regulator in the induction medium is from 5 μM to 100 μM, and this concentration range is configured to reduce the growth rate of callus tissue by 15% to 50% compared to the control medium without the regulator.

5. The method for analyzing the function of alfalfa stress-resistance genes and breeding based on metabolomics according to claim 1, characterized in that, In S1, the proliferation culture in the first stage lasts for at least two subculture cycles, each lasting 21 to 28 days.

6. The method for analyzing the function of alfalfa stress-resistance genes and breeding based on metabolomics according to claim 1, characterized in that, In S2, the abiotic stress factor is mannitol used to simulate drought stress, with a concentration of 50 mM to 150 mM in the validation medium; or sodium chloride used to simulate salt stress, with a concentration of 50 mM to 100 mM in the validation medium.

7. The method for analyzing the function of alfalfa stress-resistance genes and breeding based on metabolomics according to claim 1 or 6, characterized in that, In S2, the concentration of the metabolic regulator in the verification medium is 50% to 100% of the concentration in the induction medium in S1.

8. The method for analyzing the function of alfalfa stress-resistance genes and breeding based on metabolomics according to claim 1, characterized in that, In S2, the screening criterion for "being able to maintain continuous proliferation capacity" is: after the callus has been cultured in the validation medium for at least 2 rounds, the increase in its fresh weight or dry weight is not less than 70% of the increase in the control callus cultured in the same generation of conventional medium.

9. The method for analyzing the function of alfalfa stress-resistance genes and breeding based on metabolomics according to claim 1, characterized in that, In S3, the step of regenerating into a complete plant specifically includes: transferring the screened callus tissue to a differentiation medium that does not contain the metabolic regulator and the abiotic stress factor, but contains cytokinins and auxins to induce shoot growth, and then transferring the resulting cluster shoots to a rooting medium containing auxins to induce root growth.

10. The method for analyzing the function of alfalfa stress-resistance genes and breeding based on metabolomics according to claim 1, characterized in that, The method further includes a step S0 before step S1: determining metabolic intervention targets based on metabolomics analysis; step S0 includes: performing metabolomics analysis on alfalfa materials treated with abiotic stress, screening target metabolites with changes in content and mapping them to their biochemical pathways to resolve core metabolic pathway targets, and selecting the metabolic regulators used in S1 accordingly.

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