A compound antibacterial agent and its application in open tissue culture of plants

Abstract

This invention relates to a compound antibacterial agent and its application in open plant tissue culture. The active ingredients of the compound antibacterial agent include rifampin, kasugamycin, ticarcillin sodium clavulanic acid, and tetrachloroisophthalonitrile. The compound antibacterial agent can synergistically inhibit Agrobacterium rhizogenes or Rhodotorula glutinis, contaminating bacteria that are easily generated during plant tissue culture.

Description

Technical Field

[0001] This invention relates to the field of antibacterial agents, and particularly to a compound antibacterial agent and its application in open tissue culture of plants. Background Technology

[0002] The culture process of plant tissues and organs mainly includes plant virus removal and in vitro rapid propagation. Plant virus removal involves removing viruses carried by plants through methods such as high-temperature treatment or shoot tip tissue culture to obtain healthy propagation material. In vitro rapid propagation involves culturing explants in a tissue culture environment to accelerate the growth rate of individual propagation materials and increase the propagation coefficient. The culture medium used in plant tissue culture has high humidity and contains abundant carbon and nitrogen sources, making it highly susceptible to bacterial and fungal growth. Therefore, the entire operation must be strictly sterilized. The preliminary preparation steps for conventional tissue culture are quite complex. For example, the sterile operating room must be thoroughly disinfected or sterilized before the experiment. This requires not only regular disinfection of the operating room and laminar flow hood using ultraviolet lamps and disinfectants, but also sterilization of experimental instruments such as petri dishes, forceps, scissors, and culture media, as well as the prepared substrate, using an autoclave before the experiment. During the experimental operation, the entire operation must be carried out in a sterilized or disinfected laminar flow hood with ventilation. The cleanliness requirements for the inoculation environment are extremely strict. Summary of the Invention

[0003] One aspect of the present invention provides a compound antibacterial agent comprising active ingredients, said active ingredients including rifampin, kasugamycin, ticarcillin sodium clavulanic acid and tetrachloroisophthalonitrile.

[0004] In one specific embodiment, the mass ratio of rifampin, kasugamycin, ticarcillin sodium clavulanic acid, and tetrachloroisophthalonitrile is 3:3:2:1.

[0005] In one specific embodiment, the compound antibacterial agent further includes a plant tissue culture-acceptable carrier.

[0006] In one specific embodiment, the plant tissue culture-acceptable carrier is water and / or dimethyl sulfoxide.

[0007] The second invention provides a compound antibacterial agent according to any one of the inventions for inhibiting Agrobacterium rhizogenes (…). Agrobacterium rhizogenes ) and / or red yeast ( Rhodotorula mucilaginosa Applications in ).

[0008] The third aspect of the present invention provides the application of the compound antibacterial agent according to any one of the present inventions in open tissue culture of plants.

[0009] In one specific embodiment, the compound antibacterial agent is used to inhibit Agrobacterium rhizogenes (Agrobacterium tumefaciens). Agrobacterium rhizogenes ) and / or red yeast ( Rhodotorula mucilaginosa ).

[0010] In one specific embodiment, the plant is Eucalyptus grandis (Giant Eucalyptus). Eucalyptus grandis Eucalyptus urophylla (), Eucalyptus urophylla ) and the giant tail antler euphoria ( Eucalyptus grandis × urophylla At least one of the following.

[0011] In one specific embodiment, the final concentration of the active ingredient in the plant tissue culture medium is 200 to 1400 mg / L.

[0012] In one specific embodiment, the final concentration of the active ingredient in the plant tissue culture medium is 400 to 1400 mg / L.

[0013] In one specific embodiment, the final concentration of the active ingredient in the plant tissue culture medium is 600 to 1400 mg / L.

[0014] The beneficial effects of this invention are as follows: This invention discovers that the combination of rifampin, kasugamycin, ticarcillin sodium clavulanic acid, and tetrachloroisophthalonitrile, particularly in a mass ratio of 3:3:2:1, can synergistically inhibit Agrobacterium rhizogenes or Rhodotorula glutinis, which are easily contaminating bacteria during plant tissue culture. Therefore, after using the antibacterial agent of this invention, plant tissue culture operations do not need to be performed in a laminar flow hood, but only in a clean, sterile room. Furthermore, the culture medium does not need to be autoclaved; conventional boiling is sufficient. Experimental tools also do not need to be autoclaved; they only need to be immersed in anhydrous ethanol for a few seconds and then sterilized by incineration. Detailed Implementation

[0015] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0016] Unless otherwise specified, the reagents and other materials used in the embodiments of this invention can be purchased commercially.

[0017] Rifampin was dissolved in dimethyl sulfoxide (DMSO) and filtered through a 0.22-micron disposable membrane (Millipore, catalog number: SLGPR33RB) to prepare a 200 mg / ml rifampin solution.

[0018] Kasugamycin was dissolved in sterilized ultrapure water and filtered through a 0.22-micron disposable filter membrane to prepare a 200 mg / ml kasugamycin solution.

[0019] Ticarcillin sodium clavulanate was dissolved in sterile ultrapure water and filtered through a 0.22-micron disposable filter membrane to prepare a 200 mg / ml ticarcillin sodium clavulanate solution.

[0020] Tetrachloroisophthalonitrile was dissolved in DMSO and filtered through a 0.22-micron disposable filter membrane to prepare a 200 mg / ml tetrachloroisophthalonitrile solution.

[0021] Cefotaxime sodium was dissolved in sterilized ultrapure water and filtered through a 0.22-micron disposable filter membrane to prepare a 200 mg / ml cefotaxime sodium solution.

[0022] Take 3 ml of rifampicin solution and 3 ml of kasugamycin solution respectively, mix them, and prepare a compound solution TW-1 with a total concentration of 200 mg / ml.

[0023] Take 3 ml of rifampicin solution and 2 ml of ticarcillin sodium clavulanic acid solution respectively, mix them, and prepare a composite solution TW-2 with a total concentration of 200 mg / ml.

[0024] Take 3 ml of rifampicin solution and 1 ml of tetrachloroisophthalonitrile solution respectively, mix them, and prepare a composite solution TW-3 with a total concentration of 200 mg / ml.

[0025] Take 3 ml of kasugamycin solution and 2 ml of ticarcillin sodium clavulanic acid solution respectively, mix them, and prepare a compound solution TW-4 with a total concentration of 200 mg / ml.

[0026] Take 3 ml of kasugamycin solution and 1 ml of tetrachloroisophthalonitrile solution respectively, mix them, and prepare a composite solution TW-5 with a total concentration of 200 mg / ml.

[0027] Take 2 ml of ticarcillin sodium clavulanic acid solution and 1 ml of tetrachloroisophthalonitrile solution, mix them, and prepare a composite solution TW-6 with a total concentration of 200 mg / ml.

[0028] Take 3 ml of rifampicin solution, 3 ml of kasugamycin solution and 2 ml of ticarcillin sodium clavulanic acid solution respectively, mix them, and prepare a compound solution TH-1 with a total concentration of 200 mg / ml.

[0029] Take 3 ml of rifampicin solution, 3 ml of kasugamycin solution and 1 ml of tetrachloroisophthalonitrile solution respectively, mix them, and prepare a composite solution TH-2 with a total concentration of 200 mg / ml.

[0030] Take 3 ml of rifampicin solution, 2 ml of ticarcillin sodium clavulanic acid solution and 1 ml of tetrachloroisophthalonitrile solution respectively, mix them, and prepare a composite solution TH-3 with a total concentration of 200 mg / ml.

[0031] Take 3 ml of kasugamycin solution, 2 ml of ticarcillin sodium clavulanic acid solution and 1 ml of tetrachloroisophthalonitrile solution respectively, mix them, and prepare a composite solution TH-4 with a total concentration of 200 mg / ml.

[0032] Take 3 ml of rifampicin solution, 3 ml of kasugamycin solution, 2 ml of ticarcillin sodium clavulanic acid solution and 1 ml of tetrachloroisophthalonitrile solution respectively, mix them, and prepare a composite solution F-1 with a total concentration of 200 mg / ml.

[0033] LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, deionized water to a final volume of 1 L, pH=7.0, sterilized by moist heat at 121 degrees Celsius for 20 min.

[0034] LB solid medium: Add 15 g / L of agar powder to LB liquid medium and sterilize by moist heat at 121 degrees Celsius for 20 min.

[0035] YPD liquid culture medium: 20 g peptone, 10 g yeast extract, 20 g glucose, deionized water to a final volume of 1 L, sterilized by moist heat at 121 degrees Celsius for 20 min.

[0036] YPD solid medium: Add 15 g / L of agar powder to YPD liquid medium and sterilize by moist heat at 121 degrees Celsius for 20 min.

[0037] Agrobacterium rhizogenes ( Agrobacterium rhizogenes K599 was provided by the Tropical Forestry Research Institute of the Chinese Academy of Forestry.

[0038] Red yeast rice ( Rhodotorula mucilaginosa (Provided by Zhanjiang Experimental Station of Tropical Science Research Institute)

[0039] Tail Giant Eucommia ( Eucalyptus grandis × urophylla ) is a huge eucalyptus ( Eucalyptus grandis ) is the male parent, Eucalyptus urophylla ( Eucalyptus urophylla Hybrid varieties with the female parent as the parent. Example 1

[0040] Agrobacterium rhizogenes and Rhodotorula glutinis are bacteria and fungi that are prone to contamination in tissue cultures of Eucalyptus grandis.

[0041] Agrobacterium rhizogenes was activated with LB liquid medium, and then the activated strain was inoculated into fresh LB liquid medium and cultured at 28°C with shaking at 200 rpm for about 20 hours until OD (Organic Dry Index) was reached. 600 =0.8, and the Agrobacterium rhizogenes culture medium was obtained.

[0042] 200 μL of Agrobacterium rhizogenes culture medium was taken in a clean bench and spread on the surface of LB solid medium in a square petri dish, so that the Agrobacterium rhizogenes culture medium evenly covered the entire petri dish. After ventilation and drying, Agrobacterium rhizogenes plate was obtained.

[0043] Take 8 μL of rifampicin solution and add it dropwise to a 6 mm diameter blank antibiotic susceptibility testing disc (Shandong Baishi Microbial Co., Ltd.). Place the disc on a sterile surface (e.g., an empty disposable sterile petri dish) in a laminar flow hood to allow for full absorption. Then, using sterile forceps, place three antibiotic susceptibility testing discs onto the surface of the Agrobacterium rhizogenes agar plate, following the graduated grid. Ensure the discs are centered within the grid and press them firmly to ensure close contact with the culture medium. This prepares the rifampicin-treated sample. One disc constitutes one replicate, for a total of three replicates.

[0044] The rifampicin solution was replaced with kasugamycin solution, ticarcillin sodium clavulanic acid solution, tetrachloroisophthalonitrile solution, cefotaxime sodium solution, TW-1, TW-2, TW-3, TW-4, TW-5, TW-6, TH-1, TH-2, TH-3, TH-4, F-1, sterile water, and dimethyl sulfoxide, respectively. All other treatments were the same as those for the rifampicin treatment.

[0045] Each petri dish was inverted and incubated at 28°C for 5 consecutive days. After incubation, the petri dishes were removed, and the formation of inhibition zones around the filter paper was observed. The diameter of the inhibition zone was measured with calipers, centered on the center of the filter paper. If no inhibition zone was formed, it was recorded as 0. Specific operating procedures and measurement methods followed the Kirby-Bauer method. Results were interpreted according to the CLSIM 100 standard specified by the Clinical and Laboratory Standards Institute (CLSI). The susceptibility of bacteria to various antimicrobial agents was assessed based on the interpretation criteria. The results are shown in Table 1. Example 2

[0046] In Example 1, Agrobacterium rhizogenes was replaced with Rhodotorula glutinis, and LB medium was replaced with YPD medium. All other operations were the same as in Example 1.

[0047] The results are shown in Table 1.

[0048] Table 1

[0049] Note: The different lowercase letters following the diameter of the inhibition zone in each column indicate that the differences were statistically significant at the P<0.05 level according to one-way ANOVA using SPSS (IBM SPSS Statistics 19).

[0050] According to the results in Table 1, for Agrobacterium rhizogenes, the TW-1 treatment was comparable to the rifampicin and kasugamycin treatments; the TW-2 treatment was comparable to the ticarcillin sodium clavulanic acid and tetrachloroisophthalonitrile treatments; the TW-3 treatment was comparable to the rifampicin and kasugamycin treatments; the TW-4 treatment was comparable to the ticarcillin sodium clavulanic acid and tetrachloroisophthalonitrile treatments; the TW-5 treatment was comparable to the rifampicin and kasugamycin treatments; and the TW-6 treatment was comparable to the ticarcillin sodium clavulanic acid and tetrachloroisophthalonitrile treatments. The TH-1 treatment was comparable to ticarcillin sodium clavulanate and tetrachloroisophthalonitrile treatments; the TH-2 treatment was comparable to rifampin and kasugamycin treatments; the TH-3 treatment was comparable to ticarcillin sodium clavulanate and tetrachloroisophthalonitrile treatments; and the TH-4 treatment was significantly superior to rifampin, kasugamycin, ticarcillin sodium clavulanate, and tetrachloroisophthalonitrile treatments. The F-1 treatment was significantly superior to rifampin, kasugamycin, ticarcillin sodium clavulanate, tetrachloroisophthalonitrile, and TH-4 treatments. These results indicate that for Agrobacterium rhizogenes, the components in the TH-4 and F-1 treatments produced a synergistic effect, achieving unexpected technical results.

[0051] As shown in Table 1, for *Agrobacterium tumefaciens*, the TW-1 treatment was comparable to rifampicin and kasugamycin treatments; the TW-2 treatment was comparable to rifampicin treatment; and the TW-3 to TW-6 treatments were comparable to kasugamycin and tetrachloroisophthalonitrile treatments. The TH-1 to TH-3 treatments were comparable to kasugamycin and tetrachloroisophthalonitrile treatments, and the TH-4 treatment was comparable to tetrachloroisophthalonitrile treatment. The F-1 treatment was significantly superior to rifampicin, kasugamycin, ticarcillin sodium clavulanate, and tetrachloroisophthalonitrile treatments. These results indicate that for *Agrobacterium rhizogenes*, the components in the F-1 treatment produced a synergistic effect, achieving unexpected technical results. Example 3

[0052] Culture medium for Eucalyptus macrocarpa tissue culture: MS medium was used as the basal medium, to which cysteine, carrageenan, sucrose, 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), and 1 mg / ml 6-benzylaminopurine (6-BA) were added to a final concentration of 0.5 mg / L, pH = 5.8. Antibacterial compound solution F-1 was added to the medium after boiling and cooling to 50°C. The final concentration gradients of the compound bacterial agent (rifampin, kasugamycin, ticarcillin sodium clavulanic acid, and tetrachloroisophthalonitrile) in the medium were 200, 400, 600, 800, 1000, 1200, and 1400 mg / L, respectively. A medium containing the same volume of DMSO and water (4:5 volume ratio) as 1400 mg / L F-1 was used as a negative control.

[0053] The inoculation room was sprayed with 70% alcohol to reduce dust, and then the experimental table surface inside the inoculation room was wiped with 70% alcohol. Afterwards, the sealed inoculation room was disinfected by irradiation with ultraviolet light for 2 hours. After disinfection, the operator disinfected their hands by spraying the latex gloves with 70% alcohol. Healthy Eucalyptus grandiflora tissue culture seedlings, subcultured for 22±2 days and with a height of 4±0.5 cm, were placed in a sterile metal dish. Stem segments at least 2 cm long with apical buds were cut and quickly transferred to culture flasks containing the aforementioned Eucalyptus grandiflora tissue culture medium containing various concentrations of F-1 and negative controls. Inoculation tools were sterilized by immersing them in anhydrous ethanol for a few seconds after use and then inserted into the inoculation tool sterilizer for later use. Before each transfer, the tools must be fully cooled before contacting the plant material. Ten stem segments were inoculated into each culture flask. After inoculation, the culture flask was capped. One culture flask constituted one replicate, and four replicates were set up.

[0054] The tissue culture environment was set at a light intensity of 2000–2500 lux, a light duration of 12 h, a temperature of 26°C, and a humidity of 80%–90%. All culture media were kept closed and incubated in the tissue culture room throughout the process. Contamination was observed every 7 days, and the contamination status was statistically analyzed after 14 days. Statistical indicators included: contamination rate (%) = number of seedlings exposed to contaminated colonies × 100 / number of inoculated seedlings; survival rate (%) = number of surviving seedlings × 100 / total number of inoculated seedlings; rooting rate (%) = number of rooted seedlings × 100 / number of inoculated seedlings. The results are shown in Table 2.

[0055] Table 2

[0056] Note: Different lowercase letters in each column indicate significant differences at the P<0.05 level in one-way ANOVA using SPSS (IBM SPSS Statistics 19); different uppercase letters in each column indicate significant differences at the P<0.01 level in one-way ANOVA using SPSS (IBM SPSS Statistics 19).

[0057] The results showed that after 7 days of incubation, contaminating colonies began to appear in the negative control group (concentration of 0). The contamination rate of each treatment group was significantly lower than that of the negative control group (p<0.01). 200 mg / L of the antibacterial agent was sufficient to significantly inhibit microbial proliferation, and no colony growth was detected at concentrations ≥400 mg / L, achieving complete antibacterial effect.

[0058] The above results indicate that the antibacterial agent can exert a significant antibacterial effect at 200 mg / L, and the antibacterial effect remains stable when the concentration is further increased. Moreover, the antibacterial agent has no obvious growth inhibition or toxicity to explants, and has no significant promoting or inhibiting effect on the rooting ability of tissue culture seedlings, indicating good biosafety. Example 4

[0059] The difference from Example 3 is that the culture bottle was opened during cultivation; otherwise, the operation was the same as in Example 3. Contamination was observed on day 7, and the contamination rate and survival rate were statistically analyzed. The results are shown in Table 3.

[0060] Table 3

[0061] Note: Different lowercase letters in each column indicate significant differences at the P<0.05 level in one-way ANOVA using SPSS (IBM SPSS Statistics 19); different uppercase letters in each column indicate significant differences at the P<0.01 level in one-way ANOVA using SPSS (IBM SPSS Statistics 19).

[0062] The results showed that by day 7 of cultivation, the negative control group was completely contaminated. At a concentration of 200 mg / L of the antimicrobial agent, microbial proliferation was significantly inhibited. Compared to the abundant colony growth in the negative control group, the size and thickness of colonies in the experimental group with the added antimicrobial agent were significantly reduced. When the antimicrobial agent concentration reached or exceeded 200 mg / L, the survival rate was significantly higher than that of the negative control group. When the concentration increased to 1400 mg / L, the survival rate showed a statistically significant decrease, indicating that this concentration had approached or reached the tolerance threshold of the plant material, potentially producing mild toxic side effects. Example 5

[0063] The difference from Example 3 is that the culture flasks were opened during cultivation, and the cultivation environment was outdoors, with natural light (approximately 6:00 am to 7:00 pm from sunrise to sunset), a temperature of 26 to 33 degrees Celsius, and humidity of 70% to 100%. All other procedures were the same as in Example 3. Contamination was observed after day 7, and the contamination rate and survival rate were statistically analyzed. The results are shown in Table 4.

[0064] Table 4

[0065] Note: Different lowercase letters in each column indicate significant differences at the P<0.05 level in one-way ANOVA using SPSS (IBM SPSS Statistics 19); different uppercase letters in each column indicate significant differences at the P<0.01 level in one-way ANOVA using SPSS (IBM SPSS Statistics 19).

[0066] The results showed that after 7 days of outdoor exposure, all samples in the negative control group (concentration 0) were contaminated with microorganisms. Treatment with a 400 mg / L antimicrobial agent significantly inhibited microbial proliferation (p<0.01). When the antimicrobial agent concentration was increased to 600 mg / L or higher, no visible colonies were detected in any of the treatment groups, even under complex outdoor conditions, and the explant survival rate reached over 50%, indicating that this concentration could achieve a stable antimicrobial effect. However, when the concentration increased to 1400 mg / L, the survival rate showed a statistically significant decrease, indicating that this concentration had approached or reached the tolerance threshold of the plant material, potentially producing mild toxic side effects.

Claims

1. A compound antibacterial agent comprising active ingredients, said active ingredients including rifampin, kasugamycin, ticarcillin sodium clavulanic acid and tetrachloroisophthalonitrile.

2. The composite antibacterial agent according to claim 1, characterized in that, The mass ratio of rifampin, kasugamycin, ticarcillin sodium clavulanic acid, and tetrachloroisophthalonitrile is 3:3:2:

1.

3. The composite antibacterial agent according to claim 1, characterized in that, The compound antibacterial agent also includes a carrier acceptable for plant tissue culture.

4. The composite antibacterial agent according to claim 1, characterized in that, The plant tissue culture-acceptable carriers are water and / or dimethyl sulfoxide.

5. The compound antibacterial agent according to any one of claims 1 to 4, used to inhibit Agrobacterium rhizogenes (… Agrobacterium rhizogenes ) and / or red yeast ( Rhodotorula mucilaginosa Applications in ).

6. The application of the compound antibacterial agent according to any one of claims 1 to 4 in open tissue culture of plants.

7. The application according to claim 6, characterized in that, The aforementioned compound antibacterial agent is used to inhibit Agrobacterium rhizogenes (Agrobacterium tumefaciens) Agrobacterium rhizogenes ) and / or red yeast ( Rhodotorula mucilaginosa ).

8. The application according to claim 6, characterized in that, The plant in question is Eucalyptus macrocarpa (Giant Eucalyptus) Eucalyptus grandis Eucalyptus urophylla (), Eucalyptus urophylla ) and the giant tail antler euphoria ( Eucalyptus grandis × urophylla At least one of the following.

9. The application according to claim 6, characterized in that, The final concentration of the active ingredient in the plant tissue culture medium is 200 to 1400 mg / L.

10. The application according to claim 6, characterized in that, The final concentration of the active ingredient in the plant tissue culture medium is 600 to 1400 mg / L.

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