CN113170625A

CN113170625A - A method for determining the effect of roxithromycin on wheat germination

Info

Publication number: CN113170625A
Application number: CN202110462801.XA
Authority: CN
Inventors: 龚镇洪; 余彬彬; 吕磊; 张天莹; 钱晓晴
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2021-04-28
Filing date: 2021-04-28
Publication date: 2021-07-27

Abstract

A method for determining the effect of roxithromycin on wheat germination, belonging to the technical field of ecotoxicology, including preparing a nutrient solution, preparing roxithromycin with different concentration gradients, measuring the germination status of wheat seeds, and measuring the biomass of wheat seedlings. step. By observing the germination status of three kinds of wheat under different roxithromycin concentrations, the effect of stress on seed germination can be systematically observed. It provides a scientific basis for evaluating the ecological impact of antibiotic pollution, and provides theoretical support for the selection and breeding of different genotypes of wheat and the rational use of biogas slurry containing antibiotics, ensuring the safety of agricultural products.

Description

Method for determining influence of roxithromycin on wheat germination

Technical Field

The invention belongs to the technical field of ecotoxicology, relates to a method for determining influence of roxithromycin on wheat germination, and particularly relates to a determination method for determining influence of antibiotic residue on wheat seed germination and providing scientific basis for production application.

Background

Wheat is one of main grain crops in China, the planting area is wide, organic fertilizer is often used to promote the growth and development of the wheat in the planting process, the wheat is also an important model organism for ecological toxicological diagnosis of soil pollution, and researches show that the early growth of wheat in higher plants is very sensitive to the response of soil organic pollution. The existing research finds that the antibiotic has little influence on the germination period of the plant, but has certain influence on the germination process of the plant, namely promotion at low concentration and inhibition at high concentration. Seed germination and seedling morphogenesis are critical periods of crop growth and are one of the most sensitive important stages of plants to external environmental factors. Currently, the antibiotics studied are mainly focused on tetracyclines, sulfonamides and quinolones, and macrolide antibiotics have less influence on the germination of vegetative species.

Disclosure of Invention

The invention aims to overcome the defects that the research on the aspect of the germination of plant seeds influenced by antibiotics is less, the growth of crops is not facilitated and the like at present, provides a method for determining the influence of roxithromycin on the germination of wheat, and determines the influence of roxithromycin with different concentrations on the growth and development of different types of wheat by determining the germination rates of three types of wheat seeds, the inhibition rates of root length and bud length and the biomass of wheat seedlings under different concentrations, thereby providing a new method for evaluating the ecological influence of the antibiotics on the crops such as the wheat.

The technical scheme of the invention is as follows: a method for determining the influence of roxithromycin on wheat germination is characterized by comprising the following steps:

(1) preparing a nutrient solution: ca (NO)₃)₂·4H₂O 1mM、KNO₃ 1.25mM、NH₄NO₃ 0.25mM、KH₂PO₄ 0.25 mM、MgSO₄·7H₂O 0.5 mM、Fe-EDTA 0.125 mM、KI 6.25×10^-6 mM、HBO₃ 1.25×10^-4 mM、MnSO₄1.85×10^-4 mM、ZnSO₄ 1.25×10^-5 mM、Na₂MoO₄ 2.5×10^-7 mM、CuSO₄ 4×10^-8 mM、CoCl₂·6H₂O 2.5×10^-8 mM；

(2) Preparing a roxithromycin solution: setting macrolides antibiotic roxithromycin with concentration gradient of 0, 0.1, 0.5, 1, 5, 10 mg.L^-1Respectively fixing the volume by using nutrient solution;

(3) after wheat seeds are disinfected, adding roxithromycin solution to a part of the sterilized wheat seeds for determining the germination rate, and using deionized water to accelerate germination of the rest of the sterilized wheat seeds;

(4) selecting a part of wheat seeds exposed to white after accelerating germination for 1 day, putting the part of wheat seeds into a culture dish, adding a roxithromycin solution for culture, measuring the influence of roxithromycin on the root length and the bud length of the wheat seeds after 4 days, and continuously culturing the rest of wheat seeds by using deionized water;

(5) pouring the prepared roxithromycin solution into a water culture box, inserting the wheat seed root system which sprouts out roots after 3 days of deionized water culture into the water culture box, fixing the wheat seeds, and putting the water culture box into an illumination incubator for continuous culture for 8 days;

(6) determining the growth and development conditions of the wheat seedlings: and (4) after 8 days of wheat cultivation, measuring the fresh weight, the dry weight, the length of the overground part and the length of the underground part of the wheat seedling, and determining the biomass size of the wheat seedling.

The specific method for disinfection and pregermination in the step (3) is as follows: selecting full wheat seeds with the same size, soaking the wheat seeds in 20% sodium hypochlorite solution for disinfection for 20min, repeatedly washing the wheat seeds with deionized water for several times, placing three layers of filter paper in a glass culture dish with the diameter of 9cm, and adding roxithromycin with the concentration gradient of 0, 0.1, 0.5, 1, 5, 10 mg.L^-1The nutrient solution (6 mL) is added with 10 wheat seeds with uniform size, the rest wheat seeds are uniformly placed in an aluminum tray fully paved with filter paper, deionized water is added to immerse the bottoms of the wheat seeds, the wetted filter paper is covered, and the wheat seeds are placed in a constant-temperature incubator at 24-26 ℃ for germination acceleration.

The specific method of culturing in step (4) is as follows: placing in a 9cm diameter glass culture dishThree layers of filter paper are added with roxithromycin with the concentration gradient of 0, 0.1, 0.5, 1, 5 and 10 mg.L^-1The method comprises the following steps of uniformly placing 10 exposed and white wheat seeds in an aluminum plate in a glass culture dish, keeping the tail end and the growth direction of the radicle of the wheat seeds to be linear, covering the glass culture dish, placing the glass culture dish in a constant-temperature incubator at 24-26 ℃ in a dark place for 96 hours, setting a contrast treatment, and setting 3 parallels for each treatment.

And (5) putting the water culture box into an illumination incubator for continuous culture for 8 days, replacing the nutrient solution in the water culture box every two days during the culture, and culturing for 13 hours at the temperature of 24-26 ℃ and under the illumination intensity of 6000lx in the illumination incubator and culturing for 11 hours at the temperature of 19-21 ℃ without illumination.

In the step (6), the root length and the partial length of the root of the wheat seedling are measured by a ruler; respectively weighing the fresh weight of the wheat seedlings by using a balance, marking, putting the wheat seedlings with the weighed fresh weight into a 75 ℃ oven, drying to constant weight, and weighing the dry weight.

The invention has the beneficial effects that: the invention provides a method for determining the influence of roxithromycin on wheat germination, which mainly comprises the steps of preparing a nutrient solution, preparing roxithromycin with different concentration gradients, determining the germination condition of wheat seeds and determining the biomass of wheat seedlings. By observing the germination conditions of the three types of wheat under different roxithromycin concentrations, the influence of adversity stress on seed germination can be observed systematically. Provides scientific basis for evaluating the ecological influence of antibiotic pollution, provides theoretical support for breeding wheat with different genotypes and reasonably using antibiotic-containing biogas slurry, and ensures the safety of agricultural products.

Drawings

FIG. 1 shows the effect of different roxithromycin concentrations on the root length of three wheat species.

FIG. 2 shows the effect of different roxithromycin concentrations on the length of three wheat malt.

FIG. 3 shows the effect of three kinds of wheat in different roxithromycin concentrations on the length of overground part of wheat after 8 days of culture.

FIG. 4 shows the effect of three kinds of wheat with different roxithromycin concentrations on the underground part length of wheat after 8 days of culture.

FIG. 5 shows the effect of three kinds of wheat with different roxithromycin concentrations on the fresh weight of wheat after 8 days of culture.

FIG. 6 is a graph showing the effect on dry weight of wheat after 8 days of culture of three types of wheat at different roxithromycin concentrations.

FIG. 7 shows the effect of three kinds of wheat with different roxithromycin concentrations on the germination rate of wheat after 8 days of culture.

FIG. 8 is a flowchart of the method for determining the effect of roxithromycin with different concentrations on the development of different wheat species according to the present invention.

Detailed Description

The invention is further illustrated by the following examples in conjunction with the accompanying drawings:

the wheat related to the invention is Xumai 33 variety wheat (Triticumaestivum L16, 20 varieties of wheat, Yangmai, purchased from a seed station of the agricultural college of Yangzhou university, Jiangsu province, Liyunkong City, Okinawa, et al. The specific implementation steps are shown in fig. 8:

(1) selecting full wheat seeds with the same size, soaking the wheat seeds in 20% sodium hypochlorite solution for disinfection for 20min, repeatedly washing the wheat seeds with deionized water for several times, placing three layers of filter paper in a glass culture dish with the diameter of 9cm, and adding roxithromycin with the concentration gradient of 0, 0.1, 0.5, 1, 5, 10 mg.L^-1The nutrient solution (6 mL) is added with 10 wheat seeds with uniform size, the rest wheat seeds are uniformly placed in an aluminum tray fully paved with filter paper, deionized water is added to immerse the bottoms of the wheat seeds, the wetted filter paper is covered, and the wheat seeds are placed in a constant-temperature incubator at 24-26 ℃ for germination acceleration. Uniformly placing 10 exposed wheat seeds in the aluminum plate in a glass culture dish, and keeping the tail end of the radicle of the wheat seeds and the growth direction in a straight line. Covering a glass culture dish, and placing in a constant temperature incubator at 24-26 ℃ in the dark for culturing for 72 h. One control treatment was set simultaneously, and 3 replicates were set for each treatment.

The root and bud length of the wheat seeds are measured by a 20cm ruler.

Root length inhibition (%) = (control-treated)/control × 100%

Bud length inhibition (%) = (control-treated)/control × 100%

Calculating the half Inhibition Concentration (IC) of the antibiotic to the wheat seed germination through the root length and bud length inhibition rate₅₀)；

(2) Pouring the prepared nutrient solution into a water culture box, inserting the wheat root system with the germinated root into the hole and fixing the seeds. Placing the water culture box into an illumination incubator for continuous culture for 8 days, replacing nutrient solution in the water culture box every two days during culture, and culturing for 13h at the temperature of 24-26 ℃ and under 6000lx illumination intensity in the illumination incubator and culturing for 11h at the temperature of 19-21 ℃ without illumination.

(3) Measuring the root length and the partial length of the root of the wheat seedling by using a ruler; respectively weighing the fresh weight of the wheat seedlings by using a balance, marking, putting the wheat seedlings with the weighed fresh weight into a 75 ℃ oven, drying to constant weight, and weighing the dry weight.

Example 1

Setting the concentration gradient of macrolides antibiotic Roxithromycin (ROX) as 0, 0.1, 0.5, 1, 5, 10 mg.L^-1And determining the influence of each concentration ROX on the germination of Yangmai No. 16 seeds and each physiological and biochemical index of wheat seedlings.

Example 2

Setting the concentration gradient of macrolides antibiotic Roxithromycin (ROX) as 0, 0.1, 0.5, 1, 5, 10 mg.L^-1And determining the influence of each concentration ROX on the germination of Yangmai No. 20 seeds and each physiological and biochemical index of wheat seedlings.

Example 3

Setting the concentration gradient of macrolides antibiotic Roxithromycin (ROX) as 0, 0.1, 0.5, 1, 5, 10 mg.L^-1And determining the influence of each concentration of ROX on the germination of Xumai No. 33 seeds and each physiological and biochemical index of wheat seedlings.

FIGS. 1-2 show the bud length and root length trends of three wheat species at different roxithromycin concentrations; FIGS. 3 to 6 show the variation trend of the length, fresh weight and dry weight of the ground and underground parts of three kinds of wheat cultured for 8 days under different roxithromycin concentrations; FIG. 7 shows the germination rate trends of three wheat species at different roxithromycin concentrations.

Claims

1. a method for measuring the influence of roxithromycin on wheat germination, is characterized in that, comprises the following steps:

(1) Preparation of nutrient solution: Ca(NO ₃ ) ₂ ·4H ₂ O 1 mM, KNO ₃ 1.25 mM, NH ₄ NO ₃ 0.25 mM, KH ₂ PO ₄ 0.25 mM, MgSO ₄ ·7H ₂ O 0.5 mM, Fe-EDTA 0.125 mM, KI 6.25× ^10-6 mM, _HBO3 1.25× ^10-4 mM, _MnSO4 1.85× ^10-4 mM, _ZnSO4 1.25× ^10-5 mM, _Na2MoO4 _2.5 × ^10-7 mM, CuSO ₄ 4 x 10 ^-8 mM, CoCl ₂ ·6H ₂ O 2.5 x 10 ^-8 mM;

(2) Preparation of roxithromycin solution: set the macrolide antibiotic roxithromycin with a concentration gradient of 0, 0.1, 0.5, 1, 5, and 10 mg·L ^-1 , and make up the volume with nutrient solution respectively;

(3) after the wheat seeds are sterilized, a part of the roxithromycin solution is added to measure the germination rate, and the rest use deionized water to accelerate the germination of the wheat seeds;

(4) select a part of the wheat seeds that are exposed to germination after 1 day and put them into the petri dish, add roxithromycin solution to cultivate, measure the influence of roxithromycin on the root length and bud length of wheat seeds after 4 days, and the rest of the wheat seeds continue to be used deionized water culture;

(5) pour the preparation roxithromycin solution into the hydroponic box, insert the root system of the wheat seeds that germinate after 3 days of deionized water culture into the hydroponic box and fix the wheat seeds, and the hydroponic box is put into the light incubator cultured continuously for 8 days;

(6) Determination of the growth and development of wheat seedlings: After culturing wheat for 8 days, the fresh weight, dry weight, aerial length and underground length of the wheat seedlings were measured to determine the biomass of the wheat seedlings.

2. a method for measuring the influence of roxithromycin on wheat germination, is characterized in that, the concrete method of sterilization described in step (3), germination promotion are as follows: choose the wheat seed that particle is full, the same size, with 20% sodium hypochlorite After soaking the wheat seeds in the solution for 20 minutes for disinfection, and then repeatedly washing the wheat seeds with deionized water for several times, put three layers of filter paper in a glass petri dish with a diameter of 9 cm, and add roxithromycin with a concentration gradient of 0, 0.1, 0.5, 1, 5 , 6mL of nutrient solution of 10 mg·L ^-1 , put 10 wheat seeds of uniform size, and put the rest of the wheat seeds evenly in an aluminum pan covered with filter paper, add deionized water to immerse the bottom of the wheat seeds, cover with moisturizing Wet filter paper and germinate in a constant temperature incubator at 24-26°C.

3. a method for measuring the influence of Roxithromycin on wheat germination, is characterized in that, the concrete method of cultivating described in step (4) is as follows: put three layers of filter paper in the glass petri dish of 9cm diameter, add Roxithromycin 6 mL of a nutrient solution with a concentration gradient of 0, 0.1, 0.5, 1, 5, and 10 mg·L ^-1 was used to place 10 whitened wheat seeds in an aluminum dish evenly in a glass petri dish to keep the radicle end of the wheat seed. Be in a straight line with the growth direction, cover the glass petri dish, and place it in a constant temperature incubator at 24-26 °C for 96 h in the dark.

4. a method for measuring the influence of roxithromycin on wheat germination is characterized in that: described in step (5), the hydroponic box is put into the light incubator for continuous cultivation for 8 days, and the hydroponic culture is replaced every two days during the cultivation period. The nutrient solution in the box, the culture conditions in the light incubator are 24-26°C temperature, 6000lx light intensity for 13 hours, and 19-21°C temperature for 11 hours without light.

5. a method for measuring the influence of roxithromycin on wheat germination, is characterized in that: measuring method described in step (6) is to measure wheat seedling root length and root part length with ruler; The fresh weight of the seedlings was marked, and the fresh weight of the wheat seedlings was put into a 75°C oven to dry to constant weight, and then the dry weight was weighed.