Rapid screening method for salt-tolerant bamboo reed germplasm

Abstract

The invention belongs to the technical field of bamboo reed germplasm screening, and discloses a salt-tolerant bamboo reed germplasm rapid screening method which comprises the following steps: S1, preparing a culture medium; s2, preparing test materials; s3, implementing stress treatment; s4, standardizing data acquisition; s5, a salt tolerance evaluation system; the culture medium preparation comprises substrate screening and pretreatment, high-temperature and high-pressure sterilization treatment, secondary dry heat sterilization and substrate tray loading preparation, the test material preparation comprises tissue culture seedling pretreatment, seedling recovery treatment and disinfection before transplanting, and the data acquisition specification comprises survival rate determination and biomass determination. The salt tolerance evaluation system comprises unidirectional index calculation and grading standards. According to the invention, the key window period of salt-tolerant character expression is accurately locked. A standardized seedling pretreatment and dynamic index evaluation system is adopted, so that the screening efficiency of germplasm with different batches and different genetic backgrounds is remarkably improved, and reliable provenance guarantee is provided for large-scale saline-alkali soil treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of Phragmites australis germplasm screening, and specifically relates to a method for rapid screening of salt-tolerant Phragmites australis germplasm. Background Art

[0002] Phragmites australis is a perennial C3 plant in the Poaceae family and genus Phragmites. It has well-developed rhizomes and an exceptionally high photosynthetic rate, resulting in rapid growth and a high biomass, making it a promising energy plant. Saline-alkali soils are widespread in my country, severely hindering regional economic development and ecological conservation. Accelerating the management of saline-alkali soils is urgent. Planting Phragmites australis on saline-alkali lands not only improves saline-alkali soil management but also allows for the utilization of marginal land in extensive saline-alkali regions as raw material for plant energy. Phragmites australis has excellent tolerance to various abiotic stresses and can survive on degraded and marginal lands. However, saline-alkali soils can inhibit its growth. Screening for saline-alkali-tolerant Phragmites australis germplasm is an effective method for ensuring the success of Phragmites australis cultivation on saline-alkali lands. Therefore, identifying Phragmites australis ecotypes that can withstand high salinity while maintaining high biomass is crucial.

[0003] There is a method for inducing and acclimating salt-tolerant Phragmites australis seedlings (authorization publication number CN103749302B). The present invention discloses a method for inducing and acclimating salt-tolerant Phragmites australis seedlings, wherein NaCl (50 mmol / l)-MS+6-BA (6 mg / l)+IBA (0.4 mg / l) is used to induce and culture Phragmites australis axillary buds to obtain sterile young stem segments, which are then induced to dedifferentiate and form callus tissue by NaCl (50-100 mmol / l)-MS+2,4-D (4 mg / l)+KT (0.05 mg / l). The NaCl concentration gradient is controlled to adjust the temperature of the stem segments. l (50-300mmol / l) for continuous subculture, then select callus tissue blocks with good salt tolerance (above 200mmol / lNaCl), and carry out differentiation culture, rooting and seedling hardening and transplanting under 200mmol / lNaCl stress with 200mmol / lNaCl-MS+CPPU (1.5mg / l)+NAA (0.01mg / l), thereby obtaining Phragmites australis seedlings with high salt tolerance. This method takes several months from explant extraction and sterilization to the final maintenance of salt-tolerant seedlings, and the experimental cycle is long. The induction and acclimation process requires a sterile operating environment, which is complicated and difficult to screen and evaluate large quantities of germplasm.

[0004] There is currently a method for quickly evaluating the salt tolerance of reeds using plant functional traits (application publication number CN116660461 A), which includes the following steps: detecting the plant functional traits of reeds, including plant height, leaf width, relative water content of leaves, and aboveground biomass of reeds; calculating the reed salt tolerance evaluation index S = (S1+S2+S3+S4) / 4 based on the plant functional traits; wherein S1 is the leaf width evaluation index, S2 is the relative water content evaluation index, S3 is the plant height evaluation index, and S4 is the aboveground biomass evaluation index; and using the salt tolerance evaluation index to evaluate the salt tolerance of reeds. The method provided by the present invention does not require long-term control and salt-added planting, and can directly measure and evaluate growing reeds; this method can directly measure wild reeds, which is more time-saving than existing methods. This method can directly measure wild reeds, but reeds in different regions may be in different growth stages and cannot be compared; the field measurement data is large and inconvenient to operate; and it is impossible to evaluate the salt tolerance of collected germplasm. Summary of the Invention

[0005] The object of the present invention is to provide a method for rapid screening of salt-tolerant Phragmites australis germplasm to solve the problems raised in the above background technology.

[0006] In order to achieve the above object, the present invention provides the following technical solution: a method for rapid screening of salt-tolerant Phragmites australis germplasm, comprising the following steps:

[0007] S1: Culture substrate preparation;

[0008] S2: Preparation of experimental materials;

[0009] S3: implementation of coercive treatment;

[0010] S4: Data collection specifications;

[0011] S5: Salt tolerance evaluation system;

[0012] The culture matrix preparation includes matrix screening and pretreatment, high-temperature and high-pressure sterilization, secondary dry heat sterilization, and matrix tray preparation. The test material preparation includes tissue culture seedling pretreatment, seedling hardening treatment, and disinfection before transplanting. The data collection specifications include survival rate determination and biomass determination. The salt tolerance evaluation system includes one-way index calculation and grading standards.

[0013] As a further technical solution of the present invention, the matrix screening and pretreatment includes passing the river sand through a 2mm aperture sieve to remove large particles of impurities, repeatedly rinsing with tap water 3-5 times until the water is clear to remove mud and dust, and completely immersing the washed river sand in a 1‰ carbendazim solution, and standing for disinfection for 30 minutes. The high temperature and high pressure sterilization treatment includes placing the sterilized river sand into a stainless steel frame (capacity ≤ 2 / 3 of the volume of the autoclave), and sterilizing it in a high pressure autoclave (121°C, 0.12MPa, maintained for 30 minutes). After sterilization, the matrix is ​​transferred to an oven and dried at 100°C until there is no visible moisture.

[0014] As a further technical solution of the present invention, the secondary dry heat sterilization includes evenly spreading the dried matrix on the sterilization tray, adjusting the oven temperature to 180°C, sterilizing for 2-4 hours, turning the matrix every 30 minutes to ensure that all parts are heated evenly, and the matrix tray preparation includes taking a 72-hole tray (hole diameter 5 cm), laying a 1cm×1cm non-woven fabric on the bottom, wetting the non-woven fabric with deionized water to saturation, and filling the sterilized matrix into the tray, with the filling volume accounting for 90% of the hole volume.

[0015] As a further technical solution of the present invention, the tissue culture seedling pretreatment includes selecting Phragmites australis tissue culture rooting seedlings with consistent plant height (12±1 cm) and root system (≥3 roots / plant), gently rinsing the roots with deionized water 3 times, removing residual agar culture medium, and uniformly trimming the above-ground part to 5 cm with sterile scissors, retaining 3 cm of the root system. The seedling hardening treatment includes placing the treated seedlings vertically in a 500 ml beaker, adding deionized water to immerse the roots, setting the light incubator to a temperature of 25±0.5°C, a humidity of 70%±5%, and a light intensity of 2000±100 Lux, and continuing the seedling hardening for 24 hours, during which the culture water is replaced every 4 hours.

[0016] As a further technical solution of the present invention, the disinfection before transplanting includes preparing a 1g / L carbendazim solution, completely immersing the roots of the Phragmites australis seedlings, disinfecting them for 30 minutes, and rinsing them with sterile water three times to remove the drug residue.

[0017] As a further technical solution of the present invention, the seedling acclimatization management includes watering with 1 / 2MS nutrient solution (pH 5.8±0.2) after transplanting, maintaining the culture environment with a day / night temperature of 25±0.5℃ / 22±0.5℃ and a relative humidity of 70%±5%, observing and recording the seedling acclimatization situation daily, and screening surviving plants on the 7th-10th day. The salt stress treatment includes preparing the treatment solution of the experimental group, 1 / 2MS+250mmol / L NaCl (conductivity 28.5±0.5mS / cm), and maintaining the control group with 1 / 2MS nutrient solution culture. The treatment solution is replaced regularly on Mondays, Wednesdays, and Fridays to keep the liquid level level with the substrate surface. The stress period is 21 days, and the phenotypic changes of the plants are recorded daily.

[0018] As a further technical solution of the present invention, the survival rate determination includes counting the surviving plants after the stress ends (plants with stretched leaves and upright stems are considered alive), survival rate = number of surviving plants / initial number of plants × 100%, and biomass determination includes fresh weight measurement, cutting the aboveground part, weighing it with a 0.0001g precision balance, dry weight measurement, placing the sample in an 80°C oven to dry to a constant weight (the difference between two consecutive weighings is <0.5%), and plant height measurement, using a digital caliper to measure the vertical distance from the base of the stem to the tip of the highest leaf.

[0019] As a further technical solution of the present invention, the calculation of the individual index includes S_i=1-(T_i / C_i) (i=1-4 corresponds to survival rate, fresh weight, plant height, and dry weight, respectively), the total salt tolerance index S=(S1+S2+S3+S4) / 4, and the grading standards include high tolerance (S≥0.80), medium tolerance (0.60≤S<0.80), and low tolerance (S<0.60). All experiments require 3 biological replicates, and data statistics are analyzed by ANOVA using SPSS 26.0 (p<0.05).

[0020] The beneficial effects of the present invention are as follows:

[0021] (1) This invention effectively eliminates the interference of substrate contamination and external environmental fluctuations on salt tolerance assessment by constructing a standardized sterile culture system and a precise environmental control system, ensuring the authenticity and comparability of phenotypic data. Gradient sterilization and substrate structure optimization technology are used to completely inactivate pathogens while maintaining the physical support and ion exchange capacity required for root development, providing a stable experimental carrier for the study of salt stress response mechanisms. The dynamic stress loading system is combined with multi-dimensional phenotypic tracking to fully capture the full cycle response characteristics of plants from physiological adaptation to morphological reconstruction, accurately locking in the key window period for the expression of salt tolerance traits. The standardized seedling pretreatment and dynamic indicator evaluation system significantly improves the screening efficiency of germplasms from different batches and different genetic backgrounds, providing a reliable seed source guarantee for large-scale saline-alkali land management.

[0022] (2) The present invention integrates the dual evaluation dimensions of morphological adaptability and material accumulation efficiency, breaks through the limitation of traditional methods that rely on a single phenotypic indicator, and realizes multi-level analysis of salt tolerance. The composite salt tolerance index model is combined with biostatistical verification to objectively quantify the survival strategy and energy distribution law of plants under salt stress, accurately distinguish the boundary between genetic resistance and physiological adaptation, and effectively shorten the screening cycle and reduce the dependence on the experimental environment by combining standardized operation of the whole process with the synergistic effect of the controllable stress field, making it possible to conduct large-scale parallel evaluation of germplasm resources. The established dynamic grading standard provides a transferable evaluation framework for the screening of germplasm suitable for saline-alkali land, simultaneously supports the mining of salt-resistant genes and field application verification, and promotes the coordinated development of energy plant breeding and marginal land management. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a flow chart of the method experiment of the present invention;

[0024] Figure 2 Schematic diagram of the survival rate, fresh weight, plant height and dry weight of different Phragmites australis germplasms of the present invention;

[0025] Figure 3 Schematic diagram of the salt tolerance evaluation index of different germplasms of the present invention;

[0026] Figure 4 A schematic diagram of the overall process of the present invention;

[0027] Figure 5 Schematic diagram of the process for preparing the culture matrix of the present invention;

[0028] Figure 6 Schematic diagram of the process for preparing the test materials of the present invention;

[0029] Figure 7 Schematic diagram of the process for implementing the stress treatment of the present invention;

[0030] Figure 8 Schematic diagram of the data collection process of the present invention;

[0031] Figure 9 Schematic diagram of the process of the salt tolerance evaluation system of the present invention. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] like Figures 1 to 9 As shown in the embodiment of the present invention, a method for rapid screening of salt-tolerant Phragmites australis germplasm comprises the following steps:

[0034] S1: Culture substrate preparation;

[0035] S2: Preparation of experimental materials;

[0036] S3: implementation of coercive treatment;

[0037] S4: Data collection specifications;

[0038] S5: Salt tolerance evaluation system;

[0039] Culture matrix preparation includes matrix screening and pretreatment, high-temperature and high-pressure sterilization, secondary dry heat sterilization, and matrix tray preparation. Experimental material preparation includes tissue culture seedling pretreatment, seedling hardening treatment, and disinfection before transplanting. Data collection specifications include survival rate determination and biomass determination. The salt tolerance evaluation system includes one-way index calculation and grading standards.

[0040] By establishing a standardized sterile culture system and a precise environmental control mechanism, the stability and repeatability of salt tolerance screening are significantly improved. It integrates the dynamic observation of morphological and physiological indicators to form a multi-dimensional comprehensive evaluation network, effectively overcoming the problem of weak correlation between phenotype and resistance in traditional screening. Through gradient stress response analysis and key threshold definition, objective grading of salt tolerance is achieved and subjective judgment errors are reduced. Systematic disinfection processes and matrix optimization strategies can eliminate interference from non-salt factors and ensure data authenticity. The established dynamic evaluation model can quickly lock in core indicators related to salt tolerance and provide a phenotypic verification basis for the mining of stress-resistant genes. Standardized management of the entire process effectively shortens the screening cycle, simultaneously completes the assessment of plant adaptability and resilience, and significantly improves the predictive value of screening results for salinized habitats.

[0041] like Figure 4 As shown in the figure, the substrate screening and pretreatment include passing the river sand through a 2mm pore size screen to remove large particles of impurities, repeatedly rinsing with tap water 3-5 times until the water is clear to remove mud and dust, and completely immersing the washed river sand in a 1‰ carbendazim solution and standing for disinfection for 30 minutes. The high temperature and high pressure sterilization treatment includes placing the disinfected river sand into a stainless steel iron frame (capacity ≤ 2 / 3 of the volume of the sterilizer) and sterilizing it in a high pressure sterilizer (121℃, 0.12MPa, for 30 minutes). The matrix was transferred to an oven and dried at 100°C until no visible moisture was present. Secondary dry heat sterilization included spreading the dried matrix evenly on a sterilization tray, adjusting the oven temperature to 180°C, and sterilizing for 2-4 hours. The matrix was turned over every 30 minutes to ensure uniform heating of all parts. The matrix tray preparation included taking a 72-hole tray (hole diameter 5 cm), laying a 1cm×1cm non-woven fabric on the bottom, moistening the non-woven fabric with deionized water to saturation, and filling the tray with sterilized matrix, with the filling volume accounting for 90% of the hole volume.

[0042] Through multiple physical purification and gradient sterilization processes, the biological and non-biological interference factors in the matrix are completely eliminated to ensure the absolute cleanliness of the root development environment. Phased thermal sterilization and synergistic chemical disinfection can accurately destroy the active structure of pathogens, forming a continuous antibacterial barrier, while maintaining the biocompatibility of the matrix pore structure. The matrix layered loading technology constructs a stable rhizosphere micro-domain environment, which not only achieves efficient transfer of water and nutrients, but also avoids the risk of physical damage. The entire treatment system significantly improves the water-holding and air-permeability balance of the matrix, effectively blocks the penetration of external pollutants, and ensures that the plants show true salt-tolerant response characteristics under undisturbed conditions. Through microstructure optimization and surface activity regulation, a carrier interface conducive to the formation of salt ion gradients is created, providing an ideal medium environment for the selective absorption of ions by the root system.

[0043] like Figure 5 As shown, tissue culture seedling pretreatment includes selecting Phragmites australis tissue culture rooted seedlings with consistent plant height (12±1cm) and root system (≥3 roots / plant), gently rinsing the roots with deionized water three times, removing residual agar culture medium, and uniformly trimming the aboveground part to 5cm with sterile scissors, retaining 3cm of roots. Seedling hardening treatment includes placing the treated seedlings vertically in a 500ml beaker, adding deionized water to immerse the roots, and setting the light incubator at a temperature of 25±0.5℃, a humidity of 70%±5%, and a light intensity of 2000±100Lux. The seedling hardening treatment continues for 24 hours, during which the culture water is replaced every 4 hours. Disinfection before transplanting includes preparing a 1g / L carbendazim solution, completely immersing the roots of the Phragmites australis seedlings, disinfecting them for 30 minutes, and rinsing them with sterile water three times to remove the residual agent.

[0044] Through standardized material selection and adaptive repair and regulation, the inhibitory effect of tissue culture residues on root physiological activities is effectively eliminated, and the precise reconstruction of water metabolic balance in the body is achieved. Progressive decontamination combined with a gradient repair strategy can simultaneously repair the oxidative stress state caused by in vitro damage and promote the recovery of transporter protein activity. Fine environmental regulation promotes the rapid coupling of photosynthetic-respiratory metabolic pathways, forming a stable biological rhythm coordination mechanism. Targeted morphological pruning optimizes the assimilate distribution pattern, enhances the efficiency of transmembrane transport of substances, and improves the plant's response sensitivity to environmental control signals. The deep disinfection system establishes a dynamic protective barrier, maintaining the membrane lipid integrity of root cells during detoxification treatment, and ensuring the normal expression of osmotic regulation function. Overall regulation effectively activates the plant's defense response preparation state, laying a homogenized physiological foundation for subsequent systemic salt tolerance phenotype analysis.

[0045] like Figure 6As shown, management during the seedling acclimatization period included watering with 1 / 2MS nutrient solution (pH 5.8±0.2) after transplanting, maintaining a culture environment with a day / night temperature of 25±0.5℃ / 22±0.5℃ and a relative humidity of 70%±5%, observing and recording the seedling acclimatization situation daily, and screening surviving plants on the 7th-10th day. Salt stress treatment included the preparation of the experimental group treatment solution, 1 / 2MS+250mmol / L NaCl (conductivity 28.5±0.5mS / cm), and the control group maintained 1 / 2MS nutrient solution culture. The treatment solution was replaced regularly on Mondays, Wednesdays, and Fridays to keep the liquid level flush with the substrate surface. The stress period was 21 days, and plant phenotypic changes were recorded daily.

[0046] Through precise control of environmental parameters and gradual progression of stress factors, a microenvironment for root function remodeling and ion homeostasis regulation is constructed. Circadian rhythm simulation coordinates the dynamic balance of nutrient solution, activates the temporal expression of the plant's endogenous stress resistance signal network, and promotes the efficient synthesis of osmotic regulatory substances. The sustained and stable stress field effectively induces the specificity of salt ion transmembrane transport and simultaneously inhibits the energy consumption of non-target metabolic pathways. Systematic phenotypic tracking fully reveals the dynamic response trajectory of the plant from physiological adaptation to morphological reconstruction, and accurately locates the key window period for the expression of salt-tolerant phenotypes. The double-group control system eliminates the interference of matrix-solution interactions, ensuring the independence and traceability of salt stress responses. This model significantly improves the plant's ability to break through the threshold of salt damage through the directional guidance of metabolic flows and the targeted strengthening of defense mechanisms, providing an environmental guarantee for the stable expression of germplasm stress resistance.

[0047] like Figure 7 As shown in the figure, the survival rate determination included counting the surviving plants after the stress ended (plants with stretched leaves and upright stems were considered alive), survival rate = number of surviving plants / initial number of plants × 100%. Biomass determination included fresh weight measurement, which was achieved by cutting the aboveground parts and weighing them with a 0.0001g precision balance, and dry weight measurement, which was achieved by drying the samples in an 80℃ oven to a constant weight (the difference between two consecutive weighings was <0.5%). Plant height was measured by measuring the vertical distance from the stem base to the highest leaf tip with a digital caliper.

[0048] Through the coordinated analysis of morphological structure and material accumulation, the temporal and spatial expression differences of plant stress resistance can be objectively quantified. The precise survival judgment criteria fully capture the dynamic physiological response of the cell membrane system under salt stress and effectively distinguish the adaptive threshold of the survival strategy. The multi-dimensional biomass detection technology simultaneously reveals the coupling mechanism between the distribution of photosynthetic products and organ development, and accurately locates key salt-sensitive targets. The constant weight drying method eliminates the interference of water fluctuations and accurately analyzes the accumulation characteristics of secondary metabolites. The vertical growth detection reflects the activity state of the apical meristem and reveals the gradient inhibitory effect of salt ions on cell division. The entire set of evaluation indicators forms a phenotypic-physiological closed-loop verification network, providing multi-scale data support for the analysis of salt resistance mechanisms and ensuring the reliability of the correspondence between salt-tolerant phenotypes and genetic backgrounds.

[0049] like Figure 8 As shown in the figure, the calculation of individual indexes includes S_i=1-(T_i / C_i) (i=1-4 corresponds to survival rate, fresh weight, plant height, and dry weight, respectively), the total salt tolerance index S=(S1+S2+S3+S4) / 4, and the grading standards include high tolerance (S≥0.80), medium tolerance (0.60≤S<0.80), and low tolerance (S<0.60). All experiments required three biological replicates, and data statistics were analyzed by ANOVA using SPSS 26.0 (p<0.05).

[0050] Through the dynamic weight integration of multi-dimensional resistance indicators, a correlation mapping network between phenotypic adaptability and physiological stress resistance was constructed. The composite index algorithm effectively balances the differences in sensitivity of individual indicators and objectively quantifies the overall response intensity of plants under salt stress. The grading mechanism is based on the morphological-physiological coordinated damage threshold to accurately divide the ecological adaptation boundaries of different salt-tolerant germplasms. Strict repeated design and statistical verification eliminate the interference of environmental heterogeneity and ensure the causal certainty of the association between salt-tolerant phenotype and genotype. The standardized evaluation system simultaneously analyzes the resistance maintenance mechanism and damage repair ability, revealing the functional modular characteristics of salt adaptation strategies. This model provides a transferable evaluation framework for the targeted screening of germplasm resources, and guides the targeted optimization of salt-resistant breeding strategies through the analysis of the contribution of key factors.

[0051] Take three accessions of Phragmites australis as an example to illustrate. Figure 2 The survival rate, fresh weight, plant height and dry weight of the three germplasm experimental groups and the control group were counted. Three replicates were performed on each germplasm, with 5 plants in each replicate for each germplasm experimental group and control group. Figure 3 The survival rate evaluation index, fresh weight evaluation index, plant height evaluation index and dry weight evaluation index of different germplasms were calculated, and finally the salt tolerance evaluation index of different germplasms was obtained. As can be seen from Table 1, at a NaCl concentration of 250 mmol / L, there are large differences in the survival rate of different germplasms, and the differences between indicators such as fresh weight, plant height and dry weight are relatively small. The smaller the salt tolerance evaluation index value, the better the salt tolerance. The salt tolerance evaluation index of the germplasm numbered cd1 is better than that of cd2 and cd3. Compared with the existing technology, the present invention can quickly measure the salt tolerance of existing germplasms, screen out salt-tolerant germplasms of Phragmites australis, and can be used for large-scale experiments.

[0052] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for rapid screening of salt-tolerant Phragmites australis germplasm, characterized by: The following steps are involved: S1: Culture substrate preparation; S2: Preparation of experimental materials; S3: implementation of coercive treatment; S4: Data collection specifications; S5: Salt tolerance evaluation system; The culture matrix preparation includes matrix screening and pretreatment, high-temperature and high-pressure sterilization, secondary dry heat sterilization, and matrix tray preparation. The test material preparation includes tissue culture seedling pretreatment, seedling hardening treatment, and disinfection before transplanting. The data collection specifications include survival rate determination and biomass determination. The salt tolerance evaluation system includes one-way index calculation and grading standards.

2. A rapid screening method for salt-tolerant Phragmites australis germplasm according to claim 1, characterized in that: The matrix screening and pretreatment includes passing the river sand through a 2mm aperture sieve to remove large particles of impurities, repeatedly rinsing with tap water 3-5 times until the water is clear to remove mud and dust, completely immersing the washed river sand in a 1‰ carbendazim solution, and standing for disinfection for 30 minutes. The high temperature and high pressure sterilization treatment includes placing the disinfected river sand into a stainless steel frame (capacity ≤ 2 / 3 of the volume of the sterilizer), and sterilizing it in a high pressure sterilizer (121°C, 0.12MPa, maintained for 30 minutes). After sterilization, the matrix is ​​transferred to an oven and dried at 100°C until there is no visible moisture.

3. A rapid screening method for salt-tolerant Phragmites australis germplasm according to claim 1, characterized in that: The secondary dry heat sterilization includes evenly spreading the dried matrix on a sterilization tray, adjusting the oven temperature to 180°C, sterilizing for 2-4 hours, and turning the matrix every 30 minutes to ensure that all parts are evenly heated. The matrix tray preparation includes taking a 72-hole tray (hole diameter 5 cm), laying a 1cm×1cm non-woven fabric on the bottom, wetting the non-woven fabric with deionized water to saturation, and filling the tray with the sterilized matrix, with the filling volume accounting for 90% of the hole volume.

4. A method for rapid screening of salt-tolerant Phragmites australis germplasm according to claim 1, characterized in that: The tissue culture seedling pretreatment includes selecting Phragmites australis tissue culture rooting seedlings with consistent plant height (12±1 cm) and root system (≥3 roots / plant), gently rinsing the roots with deionized water three times, removing residual agar culture medium, and uniformly trimming the above-ground part to 5 cm with sterile scissors, leaving 3 cm of roots. The seedling hardening treatment includes placing the treated seedlings vertically in a 500 ml beaker, adding deionized water to immerse the roots, setting the light incubator to a temperature of 25±0.5°C, a humidity of 70%±5%, and a light intensity of 2000±100 Lux, and continuing the seedling hardening for 24 hours, during which the culture water is replaced every 4 hours.

5. A rapid screening method for salt-tolerant Phragmites australis germplasm according to claim 1, characterized in that: The disinfection before transplanting includes preparing a 1g / L carbendazim solution, completely immersing the roots of the Phragmites australis seedlings, disinfecting for 30 minutes, and rinsing with sterile water for 3 times to remove the drug residue.

6. A rapid screening method for salt-tolerant Phragmites australis germplasm according to claim 1, characterized in that: The seedling acclimatization management includes watering with 1 / 2MS nutrient solution (pH 5.8±0.2) after transplanting, maintaining a culture environment with a day / night temperature of 25±0.5℃ / 22±0.5℃ and a relative humidity of 70%±5%, observing and recording the seedling acclimatization situation daily, and screening surviving plants on the 7th-10th day. The salt stress treatment includes preparing the treatment solution of the experimental group, 1 / 2MS+250mmol / L NaCl (conductivity 28.5±0.5mS / cm), and maintaining the control group with 1 / 2MS nutrient solution culture. The treatment solution is replaced regularly on Mondays, Wednesdays, and Fridays to keep the liquid level level with the substrate surface. The stress period is 21 days, and the phenotypic changes of the plants are recorded daily.

7. The method for rapid screening of salt-tolerant Phragmites australis germplasm according to claim 1, wherein: The survival rate determination includes counting the surviving plants after the stress ends (plants with stretched leaves and upright stems are considered alive), survival rate = number of surviving plants / initial number of plants × 100%. The biomass determination includes fresh weight measurement, cutting the aboveground part and weighing it with a 0.0001g precision balance, dry weight measurement, and placing the sample in an 80°C oven to dry to a constant weight (the difference between two consecutive weighings is less than 0.5%). Plant height measurement, using a digital caliper to measure the vertical distance from the stem base to the highest leaf tip.

8. The method for rapid screening of salt-tolerant Phragmites australis germplasm according to claim 1, wherein: The calculation of the individual index includes S_i=1-(T_i / C_i) (i=1-4 corresponds to survival rate, fresh weight, plant height, and dry weight, respectively), the total salt tolerance index S=(S1+S2+S3+S4) / 4, and the grading standards include high tolerance (S≥0.80), medium tolerance (0.60≤S<0.80), and low tolerance (S<0.60). All experiments required three biological replicates, and data statistics were analyzed by ANOVA using SPSS 26.0 (p<0.05).

Images