Combined bacteriostatic agent and application thereof in culture medium for tissue culture

Abstract

The invention provides a combined bacteriostatic agent and application thereof in a culture medium for tissue culture. The combined bacteriostatic agent is prepared from the following components: ethylicin, carbendazim and metasine, and the mass ratio of ethylicin to carbendazim to metasine is 1: 4: 4 in sequence. According to the method, the combined bacteriostatic agent containing ethylicin, carbendazim and metasine components is adopted for doubling and seedling formation of the corn immature embryos, the pollution rate is reduced, the economic loss is reduced, the plant tissue culture cost is reduced, and the haploid breeding production engineering level is improved.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial agent technology, specifically relating to a combined antibacterial agent and its application in tissue culture media. Background Technology

[0002] As one of the world's three major staple foods, maize is not only an important component of the human diet but also occupies a strategic position in livestock breeding, industrial raw material supply, and bioenergy development, playing a crucial supporting role in food security and sustainable socio-economic development. As the crop with the most mature application of heterosis theory, maize hybrids have become the main cultivation type in modern maize production systems due to their significant yield advantages, quality stability, and stress resistance. The core of hybrid breeding lies in the selection of homozygous inbred lines, which requires continuous purification and screening over multiple generations to ultimately obtain superior pure lines, thereby creating hybrid combinations with uniform traits and outstanding advantages. Conventional breeding typically requires 6-8 generations of continuous self-pollination for pure line selection, while doubled haploid breeding technology, through haploid induction and chromosome doubling, can obtain pure lines within two generations with 100% purity, greatly shortening the breeding cycle and representing a key technology for germplasm innovation and improved breeding efficiency.

[0003] The creation of DH lines involves three core technical steps: haploid induction, identification, and doubling. Haploid induction refers to pollinating other maize materials with pollen from a maize inducible line to obtain maternal haploids. In the haploid identification stage, the key technology lies in utilizing the R1-nj dominant marker gene carried by the paternal inducible line: when the inducible line pollen is crossed with the maternal parent, diploid kernels, inheriting the paternal marker gene, exhibit purple endosperm and embryo tissue; while haploids are formed only through parthenogenesis in the maternal parent, hence the endosperm is purple but the embryo is colorless. This allows for efficient phenotypic screening. Currently, commonly used haploid doubling methods include root soaking, bud soaking, seed soaking, injection, and tissue culture doubling. Among these, tissue culture doubling is the most efficient, with good pollen shedding and seed setting rates. However, in the tissue culture stage, contamination often occurs, preventing maize haploid embryos from germinating, resulting in significant economic losses and wasted human and material resources. Currently, commonly used measures for preventing contamination in plant tissue culture mainly include pretreatment of explant materials, standardized operation, and adding antibacterial agents to the culture medium. Among these, adding antibacterial agents to the culture medium can achieve long-term antibacterial effects and increase the survival rate of tissue culture seedlings. Therefore, developing an antibacterial agent is of great significance for the prevention and control of contamination in maize tissue culture seedlings. Summary of the Invention

[0004] The technical problem to be solved by this invention is: to develop an antibacterial agent to prevent contamination of maize haploid embryo doubling culture medium.

[0005] To address the aforementioned technical problems, this invention provides a combined antibacterial agent and its preparation method.

[0006] The combined antibacterial agent provided by this invention is made from the following components: allicin, carbendazim, and termethin. The mass ratio of ethoxysulfuron to carbendazim and termethin can be 1:2-4:2-4, or specifically 1:4:4.

[0007] The present invention also provides an antibacterial culture medium containing the above-mentioned combined antibacterial agents and a method for preparing the same.

[0008] The antibacterial culture medium provided by this invention is prepared by a method comprising the following steps: 1) Preparation of stock solution of single-component antibacterial agent Prepare stock solutions of allicin, carbendazim, and termethin respectively; 2) Preparation of antibacterial culture medium Take a certain volume of the stock solution of allicin, carbendazim, and termethin respectively, add them to the culture medium, mix them, and obtain the antibacterial culture medium. Furthermore, in step 1) of the above method, the concentration of the stock solution for each antibacterial agent is 5 mg / L; Furthermore, in step 1) of the above method, a stock solution of the antibacterial agent is prepared using a solvent and an auxiliary solvent, wherein the solvent is water and dimethyl sulfoxide (DMSO), and the auxiliary solvent is Tween 80. 80); Furthermore, the volume ratio of water, dimethyl sulfoxide, and Tween 80 is 45~46:1~3:0.5~1, specifically 46:3:1; Furthermore, in step 2) of the above method, the concentrations of allicin, carbendazim, and termethin in the antibacterial culture medium are 5 mg / L, 10-20 mg / L, and 10-20 mg / L, respectively, specifically 5 mg / L, 20 mg / L, and 20 mg / L.

[0009] Furthermore, the culture medium can be a seedling culture medium.

[0010] The seedling culture medium has the following formula: MS salt 2.37 g / L, sucrose 30 g / L, agar (as a solidifying agent) 6.5 g / L, and the remainder is water, with a pH of 5.8.

[0011] The application of the above-mentioned combined antibacterial agent and antibacterial culture medium in the doubling of corn embryos into seedlings also falls within the scope of protection of this invention.

[0012] The present invention also provides a corn antibacterial seedling culture medium.

[0013] The corn antibacterial seedling culture medium provided by this invention has the following formula: MS salt 2.37 g / L, sucrose 30 g / L, agar (as a solidifying agent) 6.5 g / L, allicin 5 mg / L, carbendazim 20 mg / L, termethin 20 mg / L, and the remainder is water, with a pH of 5.8.

[0014] The present invention has the following beneficial effects: This invention utilizes a combination of antibacterial agents containing allicin, carbendazim, and termethin to double the seedling size of maize embryos, thereby reducing contamination rates, economic losses, and plant tissue culture costs, and is expected to improve the engineering level of DH series production. Attached Figure Description

[0015] Figure 1 The antibacterial effect of the combined antibacterial agent of this invention on day 10 in a natural colony-forming experiment. Figure 1 In the diagram, A represents the blank group, B represents the control group, and C represents the experimental group.

[0016] Figure 2 This shows the growth of maize diploid embryos after 10 days in a seedling culture medium.

[0017] Figure 3 This shows the growth of maize haploid embryos after 10 days in a seedling culture medium. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0020] The corn in the following implementation case ( Zea mays L.) The haploid doubling material is Zhengdan 958, which is a publicly available material. It was disclosed in the literature "Wu Fenglan. New maize variety Zhengdan 958[J]. Crop Journal, 2001, (04):40." The public can obtain it from the applicant. This material is only used to repeat the relevant experiments of this invention and cannot be used for other purposes.

[0021] The corn in the following examples ( Zea maysThe haploid inducer CHOI4 (L.) was provided by Professor Chen Shaojiang's research group at China Agricultural University and has been disclosed in the literature "Chenxu Liu, et al. (2022). Development of high-oilmaize haploid inducer with a novel phenotyping strategy. The Crop Journal 10(2): 524-531." The public can obtain this material from the applicant. This material is only for repeating the relevant experiments of this invention and cannot be used for other purposes.

[0022] In the following examples, the MS salt is a Solarbio product, catalog number M8521, also known as MS medium (agar and sucrose-free).

[0023] In the following embodiments, the sucrose was purchased from Beijing Solarbio Technology Co., Ltd., with product number S8271.

[0024] In the following examples, the dimethyl sulfoxide was purchased from Beijing Solarbio Technology Co., Ltd., product number D8370.

[0025] In the following examples, the agar was purchased from Beijing Coolplay Technology Co., Ltd., product number CA1331.

[0026] In the following examples, the allicin was purchased from Sichuan Weikeqi Biotechnology Co., Ltd., product number WKQ4359.

[0027] In the following embodiments, the Temitin was purchased from Beijing Solarbio Technology Co., Ltd., with product number T8660.

[0028] In the following examples, the carbendazim was purchased from Shanghai E-En Chemical Technology Co., Ltd., with product number R093925.

[0029] In the following examples, the amphotericin B was purchased from Shanghai Beyotime Biotechnology Co., Ltd., with product number A800428.

[0030] In the following examples, the strong chlorine was purchased from Shanghai E. En Chemical Technology Co., Ltd., with product number R095832.

[0031] In the following examples, the culture flask (made of glass) has a size of 240 mL, a diameter of 62 mm, and a height of 90 mm.

[0032] The following examples use GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation. The t-test is used, and P < 0.05 (*) indicates a significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0033] This invention first conducted individual experiments on each component, and then combined them after determining the maximum usable concentration of each antibacterial agent. The combinations were then screened. The following are detailed experiments and data: Allicin was administered at concentrations of 5, 10, 20, 30, and 50 mg / L; carbendazim at 20, 30, and 40 mg / L; termethin at 20, 50, 100, and 200 mg / L; amphotericin B at 10 and 20 mg / L; and chlorhexidine at 2.5, 5, and 10 mg / L. A blank control (MS medium without any fungicide) was also included for each fungicide to investigate the effects of these three fungicides, when used alone, on maize seedling emergence.

[0034] Preparation of medium containing a single antimicrobial agent: Mix 2.37 g / L MS salt, 30 g / L sucrose, and 6.5 g / L agar, add ddH2O to 1 L, adjust the pH to 5.8, and then autoclave at 121℃ for 20 min to obtain MS medium. Then, add each of the above-set antimicrobial agent concentrations to the MS medium to obtain MS medium containing a single antimicrobial agent. Five replicates were set for each concentration, and 5-6 immature embryos were inoculated per bottle. After inoculation, the immature embryos were cultured in a culture room for 10 days under the following conditions: 25℃, 16 h light / 8 h dark, and relative humidity 60%-70%. Phenotypic data such as plant height, root length, root number, and dry weight of maize tissue culture seedlings were investigated and analyzed using Excel 2019 and GraphPad Prism 9.5. The experimental results are as follows: Table 1. Experimental results of culture media containing a single antibacterial agent

[0035] In summary, the results show that only 5 mg / L allicin, 20 mg / L carbendazim, 20 mg / L termethin, 20 mg / L amphotericin B, and 5 mg / L chlorhexidine showed no significant difference from the control (CK) at different indicators; all other concentrations inhibited corn seedling emergence.

[0036] The following are screening experiments on different combinations of antibacterial agents. The formulations of each combination of antibacterial agents are as follows: Table 2. Formulations of each combination of antibacterial agents

[0037] Preparation of culture media containing combined antimicrobial agents: Mix 2.37 g / L MS salt, 30 g / L sucrose, and 6.5 g / L agar, add ddH2O to 1 L, adjust the pH to 5.8, and then autoclave at 121℃ for 20 min to obtain 1 / 2 MS medium. Then, add each of the above-mentioned combined antimicrobial agents to the 1 / 2 MS medium to achieve the corresponding concentration, thus obtaining 1 / 2 MS medium containing different combinations of antimicrobial agents. 1 / 2 MS medium without any added antimicrobial agents serves as a blank control. Each treatment is set up in 5 replicates, with 5-6 immature embryos inoculated per bottle. After inoculation, the immature embryos are cultured in a culture room for 10 days under the following conditions: 25℃, 16h light / 8h dark, and relative humidity 60%-70%. Phenotypic data such as plant height, root length, root number, and dry weight of maize tissue culture seedlings are investigated and analyzed using Excel 2019 and GraphPad Prism 9.5. The following are the experimental results: Table 3. Experimental results of culture medium containing combined antibacterial agents

[0038] Based on the above results, only the antibacterial agent combination MIX-3 showed no significant difference in plant height, root length, root number, and dry weight compared to the control CK. Therefore, the antibacterial agent combination MIX-3 was selected as the optimal formulation for the subsequent experiments.

[0039] Example 1: Preparation of antibacterial culture medium containing combined antibacterial agents The steps are as follows: 1. Weigh 250 mg of allicin, carbendazim and termethin respectively, prepare stock solutions of allicin, carbendazim and termethin, add 3 mL of DMSO and 1 mL of Tween 80, and finally add ddH2O to 50 mL. Shake well to prepare a 5 mg / mL antibacterial agent stock solution. 2. Sterilize the above antibacterial agent stock solution with ultraviolet light in the ultra-clean workbench for 15 minutes. Then, filter the stock solution using a 0.22μm filter to obtain a sterile antibacterial agent stock solution. 3. Mix 2.37 g / L MS salt, 30 g / L sucrose, and 6.5 g / L agar, add ddH2O to 1 L, adjust the pH to 5.8, and then autoclave at 121℃ for 20 min to obtain 1 / 2 MS medium. 4. When the 1 / 2MS medium cools to 65℃, add 1mL of allicin stock solution, 4mL of carbendazim stock solution, and 4mL of termethin stock solution to it in a clean bench. After mixing, the antibacterial medium containing the combined antibacterial agents is obtained (the concentration of allicin is 5mg / mL, the concentration of carbendazim is 20mg / mL, and the concentration of termethin is 20mg / mL).

[0040] Example 2: Application of combined antibacterial agents in the doubling of corn embryo seedlings The steps are as follows: 1. Induction of maize haploid embryos Using Zhengdan 958 as the female parent and the inducing line CHOI4 as the male parent, the materials were sown in Beijing in May 2024. The male parent was sown at a staggered time to ensure that the flowering periods of the parents coincided. In July, the female parent was bagged and the filaments were cut. Overpollination was performed using the male parent inducing line, and the pollination date and the pollination parent were recorded. The female parent ears were harvested 12-15 days after pollination. At this time, the embryos were about 5 mm in size. The embryos were aseptically removed from the ears for in vitro embryo culture.

[0041] 2. Preparation of doubled culture medium and seedling culture medium The components of the doubled culture medium are as follows: MS salt 2.37 g / L, sucrose 30 g / L, agar 6.5 g / L, colchicine 0.1 g / L, and the remainder is water, with a pH of 5.8.

[0042] The seedling culture medium consisted of the following components: MS salt 2.37 g / L, sucrose 30 g / L, agar 6.5 g / L, allicin 5 mg / L, carbendazim 20 mg / L, termethin 20 mg / L, and the remainder was water, with a pH of 5.8.

[0043] 3. Inoculation doubling and seedling emergence Maize embryos were extracted and placed flat on a doubling culture medium. The date and time were recorded, and the embryos were subjected to a doubling treatment for 24 hours. The paternal induction line material had an R1-nj marker. After 24 hours of light culture, the scutellum of heterozygous diploid maize embryos showed a distinct purplish-red color, while the scutellum of haploid maize embryos did not show this color. Embryos without the purplish-red marker were haploid. Therefore, haploid maize embryos were identified and selected based on the color of the scutellum. The haploid embryos were transferred to a seedling culture medium with the radicle facing down, and 1 / 3 to 1 / 2 of the embryo was inserted upright into the medium. 7 to 8 embryos were inoculated into each culture flask. The culture conditions were: a 16-hour light cycle followed by 8 hours of darkness, a temperature of 26°C to 28°C, and a humidity of 30%. Culture was carried out for 8-10 days.

[0044] Example 3: Natural colony formation experiment The specific steps are as follows: 1. Materials and Methods Based on the experimental procedures of Example 1, 1 / 2 MS medium was used as the substrate. After being autoclaved at 121°C and cooled to 65°C, a combination of antimicrobial agents was added. 1 / 2 MS medium without any antimicrobial agents was used as the blank group, and sealed, sterile 1 / 2 MS medium was used as the control group. After the medium solidified, no sealing treatment was performed, and it was left to stand at room temperature (25°C) for 24 hours. Ten replicates were set for each combination. The formation of contaminating bacteria was observed daily, and the initial contamination time point was recorded. The contamination rate for each treatment was calculated after 10 days.

[0045] Contamination rate (%) = (Total number of contaminated dishes / Total number of inoculated dishes) × 100%.

[0046] Control group: 1 / 2 MS medium (MS salt 2.37 g / L, sucrose 30 g / L, agar 6.5 g / L, pH 5.8); Control group: Blank culture medium Experimental group: blank culture medium + combination antibacterial agent of Example 1 (5 mg / L allicin + 20 mg / L carbendazim + 20 mg / L termethin); 2. Results and Analysis As can be seen from the results in Table 4 below, compared with the control group, the experimental group showed a later onset of contamination, and the contamination rate on day 10 was lower in the experimental group than in the control group. Therefore, the combined antibacterial agent obtained in Example 1 of this invention has a good antibacterial effect. A schematic diagram of the experiment is shown below. Figure 1 .

[0047] Table 4 Results of natural colony formation experiments under different treatments

[0048] Example 4: Effects of combined antibacterial agents on seedling emergence of normal diploid maize embryos The specific steps are as follows: 1. Materials and Methods This experiment was conducted in a tissue culture laboratory, and the diploid maize embryos used were taken from ears of Zhengdan 958 12-15 days after self-pollination.

[0049] Based on the experimental procedures in Example 2, a seedling culture medium was prepared, with 1 / 2MS medium without any added antibacterial agents serving as the control group.

[0050] Freshly harvested Zhengdan 958 fruit ears were surface-sterilized in a pre-sterilized ultraviolet-sterilized laminar flow hood, followed by 10 minutes of 75% alcohol disinfection. After the alcohol evaporated, the embryos were peeled and inoculated onto the seedling culture medium. The control and experimental groups used the same complete fruit ear. Each treatment was replicated in 5 groups, with 7-8 embryos inoculated per bottle. The embryos were cultured in a culture room for 10 days after inoculation. (Data can generally be recorded on day 8, but some embryos grew slowly during the experiment, so to ensure uniform embryo growth, data was recorded on day 10 in some experiments (the culture time was the same for replicates of the same formula)). The culture conditions were: 16h light and 8h darkness, temperature 26℃ to 28℃, and humidity 30%. Ten days after inoculation, the plant height, root length, root number, and dry weight of the tissue-cultured seedlings were statistically analyzed.

[0051] The survey method is as follows: (1) Measurement of plant height of tissue culture seedlings Lay the tissue culture seedlings flat on the experimental table and use a ruler (with an accuracy of 0.1 mm) to measure the horizontal length from the base of the stem to the tip of the longest leaf. Repeat the measurement three times and take the average value.

[0052] (2) Measurement of root length of tissue culture seedlings Lay the tissue culture seedlings horizontally on the experimental table, and use a ruler (with an accuracy of 0.1 mm) to measure the length from the base of the main root to the root tip. Repeat the measurement three times and take the average value.

[0053] (3) Measurement of root number of tissue culture seedlings The total number of roots is defined by counting the number of lateral roots with a length ≥ 1 mm by visual inspection, and then adding the number of principal roots to the number of lateral roots.

[0054] (4) Measurement of dry weight of tissue culture seedlings After measuring the plant height, root length, and number of roots, the tissue culture seedlings were dried in an 80℃ oven until constant weight (about 24 h). Immediately after drying, they were weighed using an analytical balance (accuracy 0.1 mg) and the dry weight was recorded.

[0055] The data above was analyzed and plotted using Excel 2019 and GraphPad Prism 9.5.

[0056] 2. Results and Analysis As shown in Table 5, when the seedling culture medium prepared using the combined antibacterial agent obtained in Example 1 of this invention was used to culture maize diploid embryos, the plant height, root length, root number, and dry weight were not significantly different from the control group. Therefore, this combined antibacterial agent has no toxic effect on maize diploid embryos, and the embryos can grow normally in the culture medium. See the maize embryo seedling diagram from this experiment for reference. Figure 2 .

[0057] Table 5. Effects of combined antibacterial agents on seedling emergence of normal diploid maize embryos.

[0058] Note: The sample size in the table represents the number of embryos.

[0059] Example 5: Effect of combined antibacterial agents on doubling of haploid seedlings in maize. The specific steps are as follows: 1. Materials and Methods This experiment was conducted in a tissue culture laboratory, and the maize haploid embryos used were taken from ears of maize 12-15 days after cross-pollination of Zhengdan 958×CHOI4.

[0060] Based on the experimental procedures in Example 2, doubled culture medium and seedling culture medium were prepared, with 1 / 2MS culture medium without any added antibacterial agents serving as the control group.

[0061] Freshly harvested hybrid fruit ears were surface-sterilized in a pre-sterilized ultraviolet-sterilized laminar flow hood, followed by 8-10 minutes of 75% alcohol disinfection. After the alcohol evaporated, the embryos were peeled and inoculated onto the seedling culture medium. The same complete fruit ear was used for the blank group, control group, and experimental group. Each treatment was replicated in 5 places, with 7-8 embryos inoculated per bottle. The embryos were cultured in a culture room for 10 days under the following conditions: 16h light and 8h dark, temperature 26℃-28℃, and humidity 30%. Ten days after inoculation, the plant height, root length, root number, and dry weight of the tissue-cultured seedlings were statistically analyzed. The methods for investigating these four indicators were the same as in Experiment 2. Excel 2019 and GraphPad Prism 9.5 were used to analyze and plot the data.

[0062] 2. Results and Analysis As shown in Table 6 below, when seedling culture medium prepared with the combined antibacterial agent obtained in Example 1 of this invention was used to culture doubled maize haploid embryos, the plant height, root length, root number, and dry weight were not significantly different from the blank group and the control group. Therefore, this combined antibacterial agent has no toxic effect on maize haploid embryos, and the embryos can grow normally in the culture medium. See the maize embryo seedling diagram for reference. Figure 3 .

[0063] Table 6. Effects of combined antibacterial agents on doubling seedling emergence in maize haploid immature embryos.

[0064] Note: The sample size in the table represents the number of embryos.

[0065] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A combination antibacterial agent, comprising the following components: ethoxysulfuron, carbendazim, and termethin, wherein, The mass ratio of ethoxysulfuron to carbendazim and termethin is 1:2~4:2~4 respectively.

2. The combined antibacterial agent according to claim 1, characterized in that, The mass ratio of allicin to carbendazim and termethin is 1:4:

4.

3. An antibacterial culture medium, said antibacterial culture medium being prepared by a method comprising the following steps: 1) Preparation of stock solution of single-component antibacterial agent Prepare stock solutions of allicin, carbendazim, and termethin respectively; 2) Prepare antibacterial culture medium Take a certain volume of the stock solution of allicin, carbendazim, and termethin respectively, add them to the culture medium, mix them, and obtain the antibacterial culture medium.

4. The antibacterial culture medium according to claim 3, characterized in that, In step 1), the antibacterial agent stock solution is prepared using a solvent and an auxiliary solvent, wherein the solvent is water and dimethyl sulfoxide, and the auxiliary solvent is Tween 80; The volume ratio of water, dimethyl sulfoxide, and Tween 80 is 45~46:1~3:0.5~1.

5. The antibacterial culture medium according to claim 3, characterized in that, In step 2), the concentrations of ethoxysulfuron, carbendazim, and termethin in the antibacterial culture medium are 5 mg / L, 10-20 mg / L, and 10-20 mg / L, respectively.

6. The antibacterial culture medium according to claim 3, characterized in that, The culture medium is a seedling culture medium with the following formula: MS salt 2.37 g / L, sucrose 30 g / L, agar 6.5 g / L, the remainder being water, and pH 5.

8.

7. The use of the combined antibacterial agent according to claim 1 or 2 or the antibacterial culture medium according to any one of claims 3-6 in the doubling of maize embryo seedlings or the generation of transgenic seedlings.

8. A corn antibacterial seedling culture medium, the formula of which is: MS salt 2.37 g / L, sucrose 30 g / L, agar 6.5 g / L, allicin 5 mg / L, carbendazim 20 mg / L, termethin 20 mg / L, the remainder being water, and the pH is 5.8.

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