A bacteriostatic composition, method, medium and culture system for reducing contamination of open plant tissue culture

Abstract

The application belongs to the technical field of plant tissue culture and particularly relates to a bacteriostatic composition, a method, a culture medium and a culture system for reducing open plant tissue culture pollution. The composition is composed of potassium sorbate, 1,2-benzisothiazolin-3-ketone and ketoconazole, the concentration of the potassium sorbate is 0.05-0.10 g / L, the concentration of the 1,2-benzisothiazolin-3-ketone is 0.025-0.05 g / L, and the concentration of the ketoconazole is 0.002-0.006 g / L. The bacteriostatic composition provided by the application can effectively reduce open plant tissue culture pollution.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, and specifically relates to an antibacterial composition, method, culture medium and culture system for reducing contamination in open plant tissue culture. Background Technology

[0002] Plant tissue culture is a technique that utilizes the cell division and differentiation characteristics of plants to cultivate and propagate plant tissues and organs under sterile conditions. Plant tissue culture plays a crucial role in plant detoxification, propagation, germplasm resource preservation, and variety creation. However, traditional plant tissue culture techniques typically require sterile conditions, demanding a high level of operational expertise and leading to increased overall production costs, significantly raising the economic cost of plant tissue culture. With ongoing research, researchers have proposed a new tissue culture technique—open tissue culture. This method overcomes the limitations of traditional sterile conditions by adding antibacterial agents to the culture medium, allowing the culture process to take place in a naturally occurring microbial environment, thus simplifying the operational procedures. However, a key bottleneck remains: the type and dosage of the selected antibacterial agent can inhibit the growth, development, and morphogenesis of explants, resulting in low propagation efficiency. This technical obstacle severely restricts its industrial application. Summary of the Invention

[0003] In view of the problems existing in the prior art, the present invention provides an antibacterial composition for reducing contamination in open plant tissue culture. This composition is used for plant tissue culture under sterile environmental conditions and can reduce culture medium contamination.

[0004] The specific technical solution provided by this invention is as follows: In a first aspect, the present invention provides an antibacterial composition for reducing contamination in open plant tissue cultures, comprising potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole, wherein: The concentration of potassium sorbate is 0.05 ~ 0.10 g / L; The concentration of 1,2-benzisothiazolin-3-one was 0.025 ~ 0.05 g / L; The concentration of ketoconazole is 0.002 ~ 0.006 g / L.

[0005] Preferably, in the composition, the concentrations of potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole are selected from any one of the following groups (a) to (c): (a) 0.05 g / L potassium sorbate, 0.025 g / L 1,2-benzisothiazolin-3-one, and 0.006 g / L ketoconazole; (b) 0.05 g / L potassium sorbate, 0.05 g / L 1,2-benzisothiazolin-3-one, and 0.002 g / L ketoconazole; (c) 0.10 g / L potassium sorbate, 0.025 g / L 1,2-benzisothiazolin-3-one, and 0.002 g / L ketoconazole.

[0006] Preferably, the concentrations of potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole in the composition are: potassium sorbate 0.05 g / L, 1,2-benzisothiazolin-3-one 0.05 g / L, and ketoconazole 0.002 g / L. This mixed antibacterial agent exhibits the best antibacterial effect, with a bacterial infection rate and contamination rate of 0.00% in purple sweet potato stem segments containing axillary buds, and the growth status is also good.

[0007] Preferably, the mass concentration ratio of 1,2-benzisothiazolin-3-one to ketoconazole is 5~25:1.

[0008] In a second aspect, the present invention provides a culture medium for open plant tissue culture, comprising a basal culture medium and the aforementioned antibacterial composition, wherein the concentrations of potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole, based on the total volume of the culture medium, are as follows: The concentration of potassium sorbate is 0.05 ~ 0.10 g / L; The concentration of 1,2-benzisothiazolin-3-one was 0.025 ~ 0.05 g / L; The concentration of ketoconazole is 0.002 ~ 0.006 g / L.

[0009] Preferably, the basal culture medium is MS medium, and the MS medium contains 25-30 g / L sucrose and 6.0-7.0 g / L agar, with a pH of 5.8.

[0010] More preferably, the MS medium contains 30 g / L sucrose and 6.5 g / L agar.

[0011] In a third aspect, the present invention provides a method for reducing contamination in open plant tissue culture, wherein plant explants are inoculated into the culture medium and cultured under sterile or non-strictly sterile environmental conditions.

[0012] In a fourth aspect, the present invention provides a plant tissue culture kit comprising the aforementioned antibacterial composition or the aforementioned culture medium.

[0013] In a fifth aspect, the present invention provides a plant tissue culture system comprising the aforementioned culture medium and a container for culturing plants in an open environment.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention combines three antibacterial agents with different mechanisms of action: potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole. In particular, the combination of 0.05 g / L potassium sorbate + 0.05 g / L BIT + 0.002 g / L ketoconazole achieved excellent antibacterial effects in open culture of purple sweet potato stem segments with axillary buds, with both the contamination rate and the pollution rate being 0.00%, reaching a cleanliness level close to that of traditional strictly aseptic culture.

[0015] This invention demonstrates through experiments that 1,2-benzisothiazolin-3-one, as the core broad-spectrum antibacterial agent, while effective at inhibiting bacteria when used alone at low concentrations (e.g., 0.1~0.5 g / L), also exhibits visible inhibition of alfalfa seed germination and growth. However, in the ternary compound system of this invention, the dosage of 1,2-benzisothiazolin-3-one can be significantly reduced to an extremely low level of 0.025~0.05 g / L. At this concentration, the antibacterial effect of 1,2-benzisothiazolin-3-one alone is limited, but in synergy with potassium sorbate and ketoconazole, it achieves an extreme antibacterial effect with 0% contamination rate in sweet potato stem segment culture, while maintaining good explant growth. This successfully solves the core technical bottleneck of severe inhibition of plant growth and development by antibacterial agents in traditional open tissue culture. This phenomenon indicates that the addition of potassium sorbate and ketoconazole produces a significant synergistic antibacterial effect with 1,2-benzisothiazolin-3-one. This allows 1,2-benzisothiazolin-3-one to exert antibacterial efficacy exceeding that of its own high-dose application at a "safe dose" that is far lower than its effective antibacterial effect without producing phytotoxicity, fundamentally resolving the traditional contradiction that highly effective antibacterial agents are inevitably accompanied by high phytotoxicity. Attached Figure Description

[0016] Figure 1 The growth characteristics of alfalfa seeds in MS medium without antibacterial agents and antibiotics for 14 days are shown.

[0017] Figure 2 The study investigated the 14-day growth of alfalfa seeds in MS medium containing different types and concentrations of acidic preservatives, with three parallel experiments per group.

[0018] Figure 3The growth characteristics of alfalfa seeds containing different types and concentrations of ester preservatives in MS medium were studied for 14 days, with 3 parallel experiments per group.

[0019] Figure 4 The growth characteristics of alfalfa seeds containing different concentrations of BIT preservative in MS medium for 14 days were compared, with 3 parallel experiments per group.

[0020] Figure 5 The growth characteristics of alfalfa seeds containing different types and concentrations of biopreservatives in MS medium were studied over 14 days, with three parallel experiments per group.

[0021] Figure 6 The growth characteristics of alfalfa seeds containing different types and concentrations of antibiotics in MS medium were observed after 14 days, with 3 parallel experiments per group.

[0022] Figure 7 The growth characteristics of purple sweet potato stem segments with axillary buds after 20 days in MS medium without antibacterial agents and antibiotics.

[0023] Figure 8 The growth characteristics of purple sweet potato stem segments with axillary buds were measured 20 days in MS medium containing four different concentrations of antibacterial agents, with three parallel experiments per group.

[0024] Figure 9 The growth characteristics of purple sweet potato stem segments with axillary buds in MS medium containing three different concentrations of antibacterial agents were studied over 20 days (single-factor experiment), with three parallel experiments in each group.

[0025] Figure 10 The growth characteristics of purple sweet potato stem segments with axillary buds in MS medium containing three different concentrations of antibacterial agents were studied over 20 days (orthogonal design experiment), with three parallel experiments in each group. Detailed Implementation

[0026] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0027] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0028] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that mutually excludes other embodiments. The present invention provides the following embodiments.

[0029] The culture medium used in the embodiments of the present invention is MS as the basic medium. All media contain 25-30 g / L sucrose and 6.0-7.0 g / L agar, and the pH is adjusted to 5.8. The media do not need to be autoclaved.

[0030] Example 1 Screening of preservatives and antibiotics 1. Plant materials and pretreatment The experiment used Tango (variety abbreviation TG) alfalfa seeds and Xu Zishu No. 8 (variety abbreviation XZ8) stem segments as materials. The TG seeds were provided by Klaus (Beijing) Ecological Technology Co., Ltd., and the XZ8 stem segments were provided by the invention team through constant temperature cultivation in their laboratory. The TG seeds and XZ8 stem segments were rinsed under running water for 30 min. In a clean bench, the explants were soaked in 75% alcohol for 30 s, then disinfected with 0.1% mercuric chloride solution for 8 min, followed by rinsing with sterile water (ddH2O) for 4 min. This process was repeated three times.

[0031] 2. Sterilize culture flasks, inoculation tools, and culture dishes. Using 100 ml wide-mouth Erlenmeyer flasks as inoculation containers, first, a small amount of dish soap was poured into a basin and the flasks were rinsed with tap water. Next, the flasks were soaked in 80 ml of 8% sodium hypochlorite solution for 10 seconds (the disinfectant can be reused). Then, they were rinsed with distilled water and finally dried in an oven (80℃) for later use. The inoculation forceps, scissors, petri dishes (including filter paper), and sterile water (ddH2O) were sterilized in an autoclave at 121℃ for 40 minutes.

[0032] 3. Effects of acidic preservatives on disinfection and growth in plant tissue culture MS medium was used as the basal medium, and three acidic preservatives were added: sodium benzoate, potassium sorbate, and sodium dehydroacetate. Three different concentration gradients (1.0, 2.0, and 3.0 g / L) were set up for each acidic preservative.

[0033] The operation method is as follows: (1) Preparation of culture medium for each treatment Measure 250 mL of distilled water, weigh 1.750 g of agar, add them to a beaker, and wait until completely dissolved. Then add 7.500 g of sucrose and 1.185 g of MS powder (Haibo Biotechnology HB8469-5), and boil. Adjust the pH to 5.8 with 1 mol / L NaOH, and finally add the appropriate concentration of preservative. Boil again and mix well. Dispense into 40 mL bottles, 6 bottles per treatment, and mark them with a marker (preparation date, culture medium number).

[0034] (2) Vaccination First, place the inoculation tools and necessary materials, including MS medium, scissors, tweezers, alcohol lamp, blue cap bottle, beaker, 75% ethanol, petri dish (with filter paper), marker pen, etc., into the inoculation room; then turn on the ultraviolet sterilization lamp to sterilize the inoculation space and the ultra-clean workbench for 30 min and ventilate for 10 min. Place the TG seeds that were sterilized in the above 1 experiment on the ultra-clean workbench and inoculate them into the MS medium prepared in (1). There are 5 bottles for each treatment, and 6 seeds are inoculated in each bottle. The medium without acidic preservatives is used as the control (CK).

[0035] (3) Cultivation The inoculated culture flasks were labeled with explant name, inoculation date, and culture medium number, and then transferred to the culture room and culture racks for cultivation. The culture room was kept under a light source of 16 h / d, a light intensity of 1800 lx, and a temperature of 25±2℃ for 14 days. After cultivation, the contamination and growth status were recorded.

[0036] 3. Effects of ester-based preservatives on disinfection and growth in plant tissue culture MS medium was used as the basal medium, and three ester-type preservatives were added: methylparaben, ethylparaben, and 1,2-benzisothiazolin-3-one. Three different concentration gradients (0.5, 1.0, and 1.5 g / L) were set up for each ester-type preservative. The operating procedure was the same as above.

[0037] 4. Effects of biological preservatives on disinfection and growth in plant tissue culture MS medium was used as the basal medium, and three biopreservatives—natamycin, polylysine, and lactate glycosides—were added. Three different concentration gradients (0.15, 0.25, and 0.35 g / L) were established for each biopreservative. The procedure was the same as described above.

[0038] 5. Effects of antibiotics on disinfection and growth in plant tissue culture MS medium was used as the basal medium, and four antibiotics were added: penicillin, gentamicin, tetracycline, and ketoconazole. Three different concentration gradients were set up for each antibiotic: penicillin (0.100, 0.150, 0.200 g / L), gentamicin (0.100, 0.150, 0.200 g / L), tetracycline (0.010, 0.015, 0.020 g / L), and ketoconazole (0.010, 0.015, 0.020 g / L). The operating procedure was the same as above.

[0039] 6. Data Analysis 6.1 Indicator Measurement After 14 days of open-field light cultivation, the contamination rate and bacterial infection rate were statistically analyzed from each treatment. Photos were then taken and the growth status was described. Three shoots of uniform growth were randomly selected from each treatment. The radicle and plumule were separated at the hypocotyl level below the cotyledon. The lengths of the radicle and plumule were measured with a ruler under moist conditions, and then the fresh weight was determined using an electronic balance. Germination was defined as a seed with a radicle length ≥ 2 mm and two cotyledons.

[0040] 6.2 Data Processing Data processing was performed using Microsoft Excel 2019 and IBM SPSS 27.0 software. Specific calculation formulas:

[0041] Germination rate = Number of germinated seeds / Total number of seeds inoculated × 100% (1) Contamination rate = Total number of contaminated explants / Total number of inoculated explants × 100% (2) Infection rate = Total infected area / Total inoculated area × 100% (3) 7. Analysis of Experimental Results 7.1 Effects of acidic preservatives on disinfection and growth of alfalfa tissue culture Depend on Figure 1 , Figure 2As shown in Table 4, compared with the control, different types and concentrations of acidic preservatives can effectively reduce microbial contamination during alfalfa tissue culture, but they also have adverse effects on alfalfa growth. For sodium benzoate, at 1.0 g / L, the germination rate, contamination rate, and bacterial infection rate were 3.33%, 23.33%, and 23.96%, respectively. At concentrations of 2.0–3.0 g / L, no seeds germinated, and the contamination rate and bacterial infection rate decreased with increasing concentration, with no contamination at 3.0 g / L. Potassium sorbate showed the best disinfection effect at a concentration of 2.0 g / L, with the effect slightly decreasing with increasing concentration. In potassium sorbate, at 1.0 g / L, the germination rate, contamination rate, and bacterial infection rate were 66.67%, 13.33%, and 11.24%, respectively. At concentrations of 2.0–3.0 g / L, it significantly inhibited alfalfa growth; although some seeds germinated, some cotyledons did not fully unfold, and the contamination rate and bacterial infection rate were significantly lower than at 1.0 g / L. In sodium dehydroacetate, germination of alfalfa seeds was significantly inhibited with increasing concentration; at 1.0 g / L, 2.0 g / L, and 3.0 g / L, the germination rates were only 46.67%, 13.33%, and 0.00%, respectively; the contamination rate and bacterial infection rate also decreased with increasing concentration.

[0042] Comprehensive analysis showed that potassium sorbate and sodium dehydroacetate had less adverse effects on alfalfa seed germination than sodium benzoate, with the least effect observed at a concentration of 1.0 g / L. The disinfection effect was in the order of sodium dehydroacetate > potassium sorbate > sodium benzoate, while the inhibition of alfalfa seed germination was in the order of potassium sorbate < sodium dehydroacetate < sodium benzoate. Therefore, potassium sorbate was ultimately selected as one of the compounding agents for further experiments.

[0043] Table 4 Effects of acidic preservatives on alfalfa tissue culture 7.2 Effects of ester-based preservatives on disinfection and growth of alfalfa tissue culture The effects of three different concentrations of ester-based preservatives on the disinfection and growth of alfalfa seeds under sterile conditions are shown in the figure. Figure 3 See Table 5. Among them, methylparaben and 1,2-benzisothiazolin-3-one showed the best disinfection effects, with no contamination at any concentration. Ethyl benzoate showed a contamination rate of 32.31% at 1.0 g / L, 1.89% at 3.0 g / L, and no contamination at 2.0 g / L. Compared with the control, all ester-type preservatives showed varying degrees of inhibition on seed germination with increasing concentration. Figure 1 , Figure 3(See Table 5). Methylparaben and ethylparaben failed to germinate at any concentration. 1,2-Benzisothiazolin-3-one, at 0.5 g / L, showed a germination rate of 66.67%, but its fresh weight decreased by 13.46% compared to the control group; germination did not occur at concentrations of 1.0–1.5 g / L. Further reductions in concentration, down to a minimum of 0.1 g / L, resulted in no contamination during cultivation, and the buds grew well. Figure 4 Therefore, 1,2-benzisothiazolin-3-one was ultimately chosen as one of the complexing factors for further experiments.

[0044] Table 5. Effects of ester-type preservative treatment on alfalfa tissue culture. 7.3 Effects of biological preservatives on disinfection and growth of alfalfa tissue culture The effects of three different concentrations of biological preservatives on the disinfection and growth of alfalfa seeds under a microbial environment are shown in the figure. Figure 5 And Table 3. All three biological preservatives at different concentrations reduced the contamination rate and bacterial infection rate, and had little effect on alfalfa seed germination and growth. Figure 1 , Figure 5 (See Table 6). Among the various concentrations of natamycin, the germinating shoots showed poor growth, but the contamination rate was low, and root growth was significantly inhibited. Although polylysine and nisin showed good antibacterial effects at 0.25 g / L, with contamination rates of 10.00% and 6.67% and bacterial contamination rates of 8.44% and 6.44%, respectively, both the contamination and bacterial contamination rates increased at 0.35 g / L. Therefore, natamycin was ultimately selected as one of the compounding factors for further experiments.

[0045] Table 6 Effects of bio-based preservatives on alfalfa tissue culture 7.4 Effects of antibiotics on disinfection and growth of alfalfa tissue culture The effects of four different concentrations of antibiotics on the disinfection and growth of alfalfa tissue culture under sterile environmental conditions are shown in [reference needed]. Figure 6 See Table 7. The results showed that penicillin had a poor antibacterial effect, with no significant difference in contamination rate and bacterial infection rate compared to the control group at different concentrations. In gentamicin, at a concentration of 0.20 g / L, the contamination rate and bacterial infection rate were significantly lower than the control, at 6.67% and 1.89%, respectively. In tetracycline, at concentrations ranging from 0.015 to 0.020 g / L, the contamination rate and bacterial infection rate were significantly lower than the control, but at 0.10 g / L, the antibacterial effect was not significantly different from the control. In ketoconazole, the contamination rate and bacterial infection rate were significantly lower than the control at all concentrations, but there was no significant difference in antibacterial effect among different concentrations.

[0046] Besides gentamicin, the other three antibiotics at different concentrations had no significant effect on the germination and growth of alfalfa seeds compared to the control. The order of inhibition from strongest to weakest was: gentamicin > penicillin > tetracycline > ketoconazole. Therefore, ketoconazole was ultimately selected as one of the compounding agents for further experiments.

[0047] Table 7 Effects of antibiotics on alfalfa tissue culture Based on the above experimental results, the four antibacterial agents selected as acidic preservative "potassium sorbate", ester preservative "1,2-benzisothiazolin-3-one", biological preservative "natamycin" and antibiotic "ketoconazole" were finally used as compound regulatory factors to explore the optimal antibacterial ratio for plant (purple sweet potato with sprouting progress) tissue culture.

[0048] Example 2 Effects of different combinations of antibacterial agents on disinfection and growth of purple sweet potato tissue culture 1. Method Refer to L9(3) 4 An orthogonal design was used to study the effects of natamycin, potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole on the sterilization and growth of purple sweet potato stem segments containing axillary buds (Table 8). Based on the experimental results in Table 8, a single-factor design was used to study the effects of potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole on the sterilization and growth of purple sweet potato stem segments containing axillary buds (Table 9). Finally, based on the results in Tables 1 and 2, an L9(3) orthogonal design was used to study the effects of potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole on the sterilization and growth of purple sweet potato stem segments containing axillary buds (Table 9). 4 An orthogonal design was used to study the effects of potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole on the sterilization and growth of purple sweet potato stem segments containing axillary buds (Table 10). MS medium was used as the basal medium in all experiments. All media contained 30 g / L sucrose and 6.5 g / L agar, and the pH was adjusted to 5.8. XZ8 stem segments were used as explants, with 5 bottles per treatment and 3 explants per bottle. After 20 days of culture in a constant temperature and light chamber, the contamination rate and bacterial infection rate were measured, and photographs and descriptions of growth status were taken.

[0049] 2. Data Analysis 2.1 Indicator Measurement After 14 days of open-field light cultivation, the contamination rate and bacterial infection rate were statistically analyzed from each treatment. Photos were then taken and the growth status was described. Three shoots of uniform growth were randomly selected from each treatment. The radicle and plumule were separated at the hypocotyl level below the cotyledon. The lengths of the radicle and plumule were measured with a ruler under moist conditions, and then the fresh weight was determined using an electronic balance. Germination was defined as a seed with a radicle length ≥ 2 mm and two cotyledons.

[0050] 2.2 Data Processing Data processing was performed using Microsoft Excel 2019 and IBM SPSS 27.0 software. Specific calculation formulas:

[0051] Germination rate = Number of germinated seeds / Total number of seeds inoculated × 100% (1) Contamination rate = Total number of contaminated explants / Total number of inoculated explants × 100% (2) Infection rate = Total infected area / Total inoculated area × 100% (3) 3. Results 3.1 Effects of Four Antibacterial Agents on Disinfection and Growth of Purple Sweet Potato Tissue Culture Table 8 shows that in the culture medium without added antibacterial agents (CK group), the bacterial infection rate and contamination rate of purple sweet potato stem segments containing axillary buds were both 100.00%, and the growth was poor, with some even dying. Figure 7 In culture media with added antibacterial agents, the contamination rate of purple sweet potato stem segments containing axillary buds was extremely low. R The values ​​were in the order of 1,2-benzisothiazolin-3-one > potassium sorbate = ketoconazole = natamycin; the optimal levels were natamycin (0.200 g / L), potassium sorbate (0.250 g / L), 1,2-benzisothiazolin-3-one (0.050 g / L), and ketoconazole (0.150 g / L) (Table 8). Furthermore, the highest infection rate (50.00%) was observed when both natamycin and 1,2-benzisothiazolin-3-one were at 0.000 g / L; the infection rate was 20.00% when natamycin was at 0.100 g / L and 1,2-benzisothiazolin-3-one was at 0.000 g / L; and the infection rate was 0.00% when natamycin was at 0.000 g / L and 1,2-benzisothiazolin-3-one was between 0.050 and 0.100 g / L. These results indicate that 1,2-benzisothiazolin-3-one has the best antibacterial effect on purple sweet potato stem segments with axillary buds and is the most important antibacterial agent. This invention also found that natamycin at a concentration of 0.200 g / L had the greatest inhibitory effect on the growth of purple sweet potato tissue culture seedlings. Figure 8 Meanwhile, although natamycin has a certain antibacterial effect on purple sweet potato stem segments containing axillary buds, it is not an essential factor. For example, combinations E2 and E3 without natamycin were uncontaminated, and the purple sweet potato stem segments containing axillary buds in combination E3 grew well. Figure 8 Therefore, natamycin is considered a bacteriostatic agent that can be eliminated.

[0052] Table 84 Effects of Antibacterial Agents on Purple Sweet Potato Tissue Culture Note: The more "+" signs there are, the better the growth status.

[0053] 3.2 Effects of three antibacterial agents on disinfection and growth of purple sweet potato tissue culture Based on the above experimental results, purple sweet potato stem segments with axillary buds were inoculated onto medium containing different concentrations of potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole, and cultured under light conditions for 14 days. The results were then statistically observed. Table 9 shows that adding different concentrations of antibacterial agents to MS medium had varying effects on the antibacterial effect and growth of purple sweet potato stem segments with axillary buds. From F1 to F3, it can be seen that as the concentration of potassium sorbate increased, there was no significant difference in the antibacterial effect; however, with increasing potassium sorbate concentration, the growth inhibition on purple sweet potato stem segments with axillary buds increased, especially resulting in smaller leaves. Figure 9 Furthermore, from F4 to F6, it can be seen that with the increase of 1,2-benzisothiazolin-3-one concentration, both the contamination rate and the infection rate decreased significantly. However, with the increase of concentration, the growth inhibition of purple sweet potato stem segments containing axillary buds also increased. Figure 9 Finally, from F7 to F9, it was observed that as the concentration of ketoconazole increased, there was no significant difference in the antibacterial effect, but the growth of purple sweet potato stem segments containing axillary buds was significantly inhibited. Figure 9 From F1 to F9, 1,2-benzisothiazolin-3-one showed the best antibacterial effect. Without this component, the contamination rate and infection rate were the highest, at 60.00% and 66.67%, respectively. However, at a concentration of 0.100 g / L, it significantly inhibited the growth of purple sweet potato stem segments containing axillary buds. Figure 9 Therefore, the optimal sterilization concentration for obtaining 1,2-benzisothiazolin-3-one is 0.050 g / L.

[0054] Table 93 Effects of Antibacterial Agents on Purple Sweet Potato Tissue Culture Note: The more "+" signs there are, the better the growth status.

[0055] Furthermore, the above experiments revealed that high concentrations of 1,2-benzisothiazolin-3-one and ketoconazole had no significant inhibitory effect on plant growth. To screen for the optimal formulation with the best antibacterial effect and the least growth inhibition, this invention further reduced the concentration, using L9(3) 4 An orthogonal design was used to study the effects of potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole on the inhibition of bacteria and growth of purple sweet potato stem segments containing axillary buds. Table 10 shows the range of bacterial contamination rates in purple sweet potato stem segments containing axillary buds. RThe values ​​were in the order of 1,2-benzisothiazolin-3-one > potassium sorbate = ketoconazole; the optimal levels of antibacterial effect were potassium sorbate (0.10 g / L), 1,2-benzisothiazolin-3-one (0.025 g / L), and ketoconazole (0.004 g / L). Analysis of variance (Table 11) showed that 1,2-benzisothiazolin-3-one had a highly significant effect on the contamination rate of purple sweet potato stem segments containing axillary buds. P ≤0.01), potassium sorbate and ketoconazole had no significant effect ( P >0.05). By Figure 10 Observations of growth showed that groups G5, G6, and G8 exhibited the best growth, with fully extended, green leaves and good antibacterial effects, with infection rates of 6.67%, 0.00%, and 4.44%, respectively. Among these, group G6, consisting of a mixture of 0.05 g / L potassium sorbate, 0.05 g / L 1,2-benzisothiazolin-3-one, and 0.002 g / L ketoconazole, showed the best antibacterial effect, with both infection and contamination rates of 0.00% in purple sweet potato stem segments containing axillary buds, and also exhibited good growth (Table 10 and...). Figure 10 G6 (China)

[0056] Table 103 Effects of Antibacterial Agents on Purple Sweet Potato Tissue Culture Note: The more "+" signs there are, the better the growth status.

[0057] Table 113 Analysis of Variance of Antibacterial Agents on Purple Sweet Potato Tissue Culture The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. An antibacterial composition for reducing contamination in open plant tissue cultures, characterized in that, Composed of potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole, wherein: The concentration of potassium sorbate is 0.05 ~ 0.10 g / L; The concentration of 1,2-benzisothiazolin-3-one was 0.025 ~ 0.05 g / L; The concentration of ketoconazole is 0.002 ~ 0.006 g / L.

2. The antibacterial composition according to claim 1, characterized in that, In the composition, the concentrations of potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole are selected from any one of the following groups (a) to (c): (a) Potassium sorbate 0.05 g / L, 1,2-benzisothiazolin-3-one 0.025 g / L, ketoconazole 0.006 g / L; (b) Potassium sorbate 0.05 g / L, 1,2-benzisothiazolin-3-one 0.05 g / L, ketoconazole 0.002 g / L; (c) Potassium sorbate 0.10 g / L, 1,2-benzisothiazolin-3-one 0.025 g / L, ketoconazole 0.002 g / L.

3. The antibacterial composition according to claim 1, characterized in that, In the composition, the concentrations of potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole are: potassium sorbate 0.05 g / L, 1,2-benzisothiazolin-3-one 0.05 g / L, and ketoconazole 0.002 g / L.

4. The antibacterial composition according to claim 1, characterized in that, The mass concentration ratio of 1,2-benzisothiazolin-3-one to ketoconazole is 5~25:

1.

5. A culture medium for open plant tissue culture, characterized in that, The medium comprises a basal culture medium and the antibacterial composition as described in any one of claims 1 to 4, wherein the concentrations of potassium sorbate, 1,2-benzisothiazolin-3-one, and ketoconazole, based on the total volume of the culture medium, are as follows: The concentration of potassium sorbate is 0.05 ~ 0.10 g / L; The concentration of 1,2-benzisothiazolin-3-one was 0.025 ~ 0.05 g / L; The concentration of ketoconazole is 0.002 ~ 0.006 g / L.

6. The culture medium according to claim 5, characterized in that, The basal culture medium is MS medium, which contains 25-30 g / L sucrose and 6.0-7.0 g / L agar, with a pH of 5.

8.

7. A method for reducing contamination in open-field plant tissue culture, characterized in that, Plant explants are inoculated into the culture medium as described in claim 5 or 6 and cultured under sterile or non-strictly sterile environmental conditions.

8. A plant tissue culture kit, characterized in that, It comprises the antibacterial composition as described in any one of claims 1 to 4 or the culture medium as described in any one of claims 5 to 6.

9. A plant tissue culture system, characterized in that, It includes the culture medium as described in claim 5 or 6, and a container for culturing plants in an open environment.

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