Method for open tissue culture of virus-free potato seedling

Abstract

The application discloses a kind of potato virus-free seedling open tissue culture method, belong to plant tissue culture and virus-free seedling rapid propagation technical field.The potato virus-free seedling open tissue culture method of the application includes the following steps: adding bacteriostatic agent in unsterilized culture medium, then in the environment treated by alcohol spraying and / or ultraviolet irradiation, potato virus-free seedling stem section inoculation is carried out, and sealing culture is carried out after inoculation is completed;Wherein, bacteriostatic agent is selected from any one of poly-manganese zinc, mancozeb, sodium hypochlorite, S106, yipei long and zhi pei ling.The bacteriostatic agent of the application can realize open tissue culture, overcome the strict aseptic environment required by traditional plant tissue culture technology, need specific instrument and equipment (ultraclean workbench, autoclave), production cost is high, operation procedure is complicated, and there is certain requirement to the technology of worker and so on.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture and rapid propagation of virus-free seedlings, and in particular to a method for open tissue culture of virus-free potato seedlings. Background Technology

[0002] potato( Solanum tuberosum Potatoes (L.), commonly known as "yam" or "potato," are an important annual herbaceous crop of the Solanaceae family. Originally from the mountainous regions of tropical America, they are now widely cultivated in temperate regions worldwide. Potatoes are rich in nutrients, characterized by high fiber, high vitamins, high protein, and low fat, low sugar, and low calories. Colored potatoes are also rich in anthocyanins, antioxidants that are extremely beneficial to the human body, earning them the nicknames "underground apples" and "second bread." Potatoes thrive in cool, dry climates and prefer loose, fertile sandy soil. They have a short growth cycle, high yields, and are drought-resistant, tolerant of poor soil, and highly adaptable.

[0003] The most common method of potato propagation is asexual reproduction using tubers. However, multi-generational planting of tubers can lead to the accumulation of various pathogens such as viruses, viroids, bacteria, and fungi, resulting in decreased potato yield and quality, severely hindering the healthy development of the potato industry. To overcome these problems and prevent disease-carrying seed potatoes, virus-free in vitro seed culture technology is currently widely used to produce virus-free seed potatoes, thereby increasing potato yield and improving quality. Traditional plant tissue culture technology requires a strictly sterile environment and specific equipment (clean benches, autoclaves), resulting in high production costs. Furthermore, the complex operating procedures demand a certain level of technical skill from the operators, significantly limiting the application and promotion of this technology. Therefore, there is an urgent need to find a plant tissue culture method that can simplify the operating procedures and reduce costs. Summary of the Invention

[0004] The purpose of this invention is to provide a method for open tissue culture of virus-free potato seedlings to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: a method for open tissue culture of virus-free potato seedlings, comprising the following steps: adding an antibacterial agent to an unsterilized culture medium, then inoculating potato virus-free seedling stem segments under an environment treated with alcohol spray and / or ultraviolet irradiation, and sealing the culture after inoculation.

[0006] Preferably, the potato variety is the 'Atlantic' potato.

[0007] Preferably, the antibacterial agent is selected from any one of manganese zinc (chemical pesticide), mancozeb (chemical pesticide), sodium hypochlorite (disinfectant), S106 (novel anti-pollution and antibacterial agent for plant tissue culture), Yipelong (novel anti-pollution and antibacterial agent for plant tissue culture), and Zhipeiling (novel anti-pollution and antibacterial agent for plant tissue culture).

[0008] Preferably, when the antibacterial agent is manganese zinc, its concentration in the culture medium is ≥0.25 g / L; When the antibacterial agent is mancozeb, its concentration in the culture medium is ≥0.20 g / L; When the antibacterial agent is sodium hypochlorite, its concentration in the culture medium is ≥0.01%; When the antibacterial agent is S106, its concentration in the culture medium is ≥0.20 ml / L; When the antibacterial agent is Eperazine, its concentration in the culture medium is ≥0.70 ml / L; When the antibacterial agent is Plantarazole, its concentration in the culture medium is ≥0.45%.

[0009] Preferably, when the antibacterial agent is manganese zinc, its concentration in the culture medium is 0.25 g / L; When the antibacterial agent is mancozeb, its concentration in the culture medium is 0.20~0.30 g / L; When the antibacterial agent is sodium hypochlorite, its concentration in the culture medium is 0.01~0.04%; When the antibacterial agent is S106, its concentration in the culture medium is 0.20~0.70 ml / L; When the antibacterial agent is Eperazine, its concentration in the culture medium is 0.70~0.80 ml / L; When the antibacterial agent is Plantarazole, its concentration in the culture medium is 0.45~0.65%.

[0010] Preferably, the culture medium is MS medium supplemented with agar and sucrose.

[0011] Preferably, the concentration of the agar added is 6-7 g / L; The concentration of added sucrose is 25~30g / L.

[0012] Preferably, the composition of the culture medium is: MS medium + 0.2 ml / L S106 + 6 g / L agar + 25 g / L sucrose.

[0013] The present invention discloses the following technical effects: The antibacterial agent of this invention can realize open tissue culture, overcoming the problems of traditional plant tissue culture technology, which requires a strict aseptic environment, specific instruments and equipment (clean bench, autoclave), high production cost, complicated operation procedures, and certain technical requirements for staff.

[0014] This invention realizes open tissue culture of 'Atlantic' potatoes, providing a direct technical reference for the industrialization of open tissue culture of 'Atlantic' potatoes. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The image shows the growth of virus-free seedlings under different concentrations of polymanganese zinc treatment in Example 2 of this invention. A represents the control (CK) treatment, B represents M1 (0.01 g / L polymanganese zinc) treatment, C represents M2 (0.05 g / L polymanganese zinc) treatment, D represents M3 (0.10 g / L polymanganese zinc) treatment, E represents M4 (0.15 g / L polymanganese zinc) treatment, F represents M5 (0.20 g / L polymanganese zinc) treatment, and G represents M6 (0.25 g / L polymanganese zinc) treatment. Figure 2 The effect of different concentrations of polymanganese zinc on the biomass of virus-free seedlings in Example 2 of the present invention, wherein A is the fresh weight of the aboveground part of the virus-free seedling, B is the fresh weight of the underground part of the virus-free seedling, C is the dry weight of the aboveground part of the virus-free seedling, and D is the dry weight of the underground part of the virus-free seedling. Figure 3 The image shows the growth of virus-free seedlings treated with different concentrations of mancozeb in Example 2 of this invention. A represents the control (CK) treatment, B represents D1 (0.05 g / L mancozeb) treatment, C represents D2 (0.10 g / L mancozeb) treatment, D represents D3 (0.15 g / L mancozeb) treatment, E represents D4 (0.20 g / L mancozeb) treatment, F represents D5 (0.25 g / L mancozeb) treatment, and G represents D6 (0.30 g / L mancozeb) treatment. Figure 4 The effect of different concentrations of mancozeb on the biomass of virus-free seedlings in Example 2 of the present invention is shown in the figure. In the figure, A is the fresh weight of the aboveground part of the virus-free seedling, B is the fresh weight of the underground part of the virus-free seedling, C is the dry weight of the aboveground part of the virus-free seedling, and D is the dry weight of the underground part of the virus-free seedling. Figure 5The image shows the growth of virus-free seedlings treated with different concentrations of sodium hypochlorite in Example 2 of this invention. A represents the control (CK) treatment, B represents C1 (0.005% sodium hypochlorite) treatment, C represents C2 (0.01% sodium hypochlorite) treatment, D represents C3 (0.02% sodium hypochlorite) treatment, E represents C4 (0.03% sodium hypochlorite) treatment, F represents C5 (0.04% sodium hypochlorite) treatment, and G represents C6 (0.05% sodium hypochlorite) treatment. Figure 6 The effect of different concentrations of sodium hypochlorite on the biomass of virus-free seedlings in Example 2 of the present invention is shown in the figure. In the figure, A is the fresh weight of the aboveground part of the virus-free seedling, B is the fresh weight of the underground part of the virus-free seedling, C is the dry weight of the aboveground part of the virus-free seedling, and D is the dry weight of the underground part of the virus-free seedling. Figure 7 The image shows the growth of virus-free seedlings treated with different concentrations of S106 in Example 2 of this invention. A represents the control (CK) treatment, B represents N1 (0.2 ml / L S106) treatment, C represents N2 (0.3 ml / L S106) treatment, D represents N3 (0.4 ml / L S106) treatment, E represents N4 (0.5 ml / L S106) treatment, F represents N5 (0.6 ml / L S106) treatment, and G represents N6 (0.7 ml / L S106) treatment. Figure 8 The virus-free seedlings cultured for 28 days in Example 2 of this invention are as follows: A is the CK treatment, B is the N1 (0.2 ml / L S106) treatment, C is the N2 (0.3 ml / L S106) treatment, D is the N3 (0.4 ml / L S106) treatment, E is the N4 (0.5 ml / L S106) treatment, F is the N5 (0.6 ml / L S106) treatment, and G is the N6 (0.7 ml / L S106) treatment. Figure 9 The effect of different concentrations of S106 on biomass in Example 2 of the present invention is shown in the figure. A is the fresh weight of the aboveground part of the virus-free seedling, B is the fresh weight of the underground part of the virus-free seedling, C is the dry weight of the aboveground part of the virus-free seedling, and D is the dry weight of the underground part of the virus-free seedling. Figure 10 The image shows the growth of virus-free seedlings treated with different concentrations of eperazine in Example 2 of this invention. A represents the control (CK) treatment, B represents Y1 (0.3 ml / L eperazine) treatment, C represents Y2 (0.4 ml / L eperazine) treatment, D represents Y3 (0.5 ml / L eperazine) treatment, E represents Y4 (0.6 ml / L eperazine) treatment, F represents Y5 (0.7 ml / L eperazine) treatment, and G represents Y6 (0.8 ml / L eperazine) treatment. Figure 11The effect of different concentrations of eperon on the biomass of virus-free seedlings in Example 2 of the present invention, wherein A is the fresh weight of the aboveground part of the virus-free seedling, B is the fresh weight of the underground part of the virus-free seedling, C is the dry weight of the aboveground part of the virus-free seedling, and D is the dry weight of the underground part of the virus-free seedling. Figure 12 The image shows the growth of virus-free seedlings under different concentrations of plant growth regulator in Example 2 of this invention. A represents the control (CK) treatment, B represents Z1 (0.15% plant growth regulator), C represents Z2 (0.25% plant growth regulator), D represents Z3 (0.35% plant growth regulator), E represents Z4 (0.45% plant growth regulator), F represents Z5 (0.55% plant growth regulator), and G represents Z6 (0.65% plant growth regulator). Figure 13 The effect of different concentrations of plant growth regulator on the biomass of virus-free seedlings in Example 2 of this invention, where A is the fresh weight of the aboveground part of the virus-free seedling, B is the fresh weight of the underground part of the virus-free seedling, C is the dry weight of the aboveground part of the virus-free seedling, and D is the dry weight of the underground part of the virus-free seedling. Figure 14 The image shows the growth of virus-free seedlings under different concentrations of sucrose treatment in Example 3 of this invention. In this image, A represents the control (CK) treatment, B represents the ZT1 (10 g / L sucrose) treatment, C represents the ZT2 (15 g / L sucrose) treatment, D represents the ZT3 (20 g / L sucrose) treatment, E represents the ZT4 (25 g / L sucrose) treatment, and F represents the ZT5 (30 g / L sucrose) treatment. Figure 15 The effect of different concentrations of sucrose on the biomass of virus-free seedlings in Example 3 of the present invention is shown in the figure. In the figure, A is the fresh weight of the aboveground part of the virus-free seedling, B is the fresh weight of the underground part of the virus-free seedling, C is the dry weight of the aboveground part of the virus-free seedling, and D is the dry weight of the underground part of the virus-free seedling. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0019] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0020] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0021] (I) The experimental materials used in the specific embodiments of the present invention are as follows: (1) The test material was the 'Atlantic' virus-free seedlings provided by Gansu Tianrun Potato Industry Co., Ltd., and the test site was the Potato Innovation Tissue Culture Center of Peili Vocational College, Shandan County, Zhangye City, Gansu Province.

[0022] (2) The test instruments and equipment include a clean bench, a 1 / 10,000 analytical balance, a 1 / 1000 electronic balance, a high-pressure steam sterilizer, an oven, a sealing machine, volumetric flasks, graduated cylinders, glass rods, beakers, test tube racks, filter paper, spatulas, funnels, rulers, bent scissors, vernier calipers, forceps, alcohol lamps, and sterile plastic tissue culture bottles.

[0023] (3) The test compounds included MS medium, sucrose, agar powder, 75% alcohol, 95% alcohol, manganese zinc (Sichuan Runer Technology Co., Ltd., 34% manganese zinc and 16% carbendazim, wettable powder), manganese zinc (Sichuan Runer Technology Co., Ltd., 70% manganese zinc, wettable powder), sodium hypochlorite (Xilong Scientific Co., Ltd.), S106 (Group AB, Jiangxi Wannian Innovation Tissue Culture Technology Research Institute), Yipeilong (Group AB, Shanghai Mingkun Biotechnology Co., Ltd.), and Zhipeiling (Shanghai Biotechnology Co., Ltd.).

[0024] (4) The culture environment is a culture room, the light source is LED fluorescent lamp, the culture temperature is maintained at 20~25°C, the light intensity is 2000 lx, and the daily light duration is 12 h.

[0025] (II) The index measurement methods used in the specific embodiments of the present invention are as follows: (1) Growth indicators and biomass After 28 days of cultivation, 5 virus-free seedlings were randomly selected from each treatment. The height from the base of the plant to the growing point was measured with a ruler to obtain the plant height. The diameter of the stem segment between the 3rd and 4th leaves at the top was measured with a vernier caliper to obtain the stem diameter. The length of all roots of a single plant was measured with a ruler and summed to obtain the total root length. The leaf area was determined by weighing. Four leaves were taken from each plant, punched with holes, and weighed (M1). The area of ​​the punched leaves (S1) was calculated. The total weight of all leaves (M2) was calculated. The total leaf area of ​​the plant (S2) was calculated as S1 × M2 / M1. The number of roots, leaves, and stem nodes were counted.

[0026] After washing the entire plant sample with tap water, rinse it repeatedly with pure water and wipe it dry. Cut the plant from the base of the stem node and divide it into above-ground and below-ground parts. Weigh the fresh weight of the upper and lower parts of the plant using a 1 / 10,000 analytical balance. Then pack them separately and dry them in an oven at 105°C for 30 minutes. After drying at 80°C until constant weight, weigh the dry weight of the upper and lower parts of the plant using a 1 / 10,000 analytical balance.

[0027] (2) Chlorophyll content Chlorophyll content (SPAD): The top third functional leaf was selected and measured using a portable chlorophyll meter. Five leaves were measured for each treatment.

[0028] (3) Other indicators Contamination rate (%) = (Contaminated stem segments with buds / Total number of inoculated stem segments with buds) × 100%; Whitening rate (%) = (Number of plants with whitened leaves / Number of inoculated plants) × 100%; Survival rate (%) = (Number of surviving stem segments with buds / Total number of inoculated stem segments with buds) × 100%; Germination rate (%) = (Number of stem segments that germinated new buds / Total number of inoculated stem segments with buds) × 100%.

[0029] (III) The data analysis methods used in the specific embodiments of the present invention are as follows: Data were processed using Microsoft Excel 2020, and ANOVA and Duncan's multiple comparisons were performed using DPS 20.05. P < 0.05, n = 5. Data were expressed as mean ± standard error. Principal component analysis and plotting were performed using Origin 2022.

[0030] (1) Entropy weight TOPSIS analysis First, establish the evaluation matrix for each process. X ij ( i= 1, 2, ... n ; j =1, 2, ... m ; n =7, m =17) where i Indicates processing, j The indicators are represented as follows: among all evaluation indicators, those with smaller values ​​are normalized using formula (1), while those with larger values ​​are normalized using formula (2). Then, the information entropy is calculated using formula (3). e j ); calculate the weights using formula (4) W j ); Multiply the processed index values ​​by their corresponding weights ( W j The weighted decision matrix is ​​obtained. The optimal solution is then derived from the weighted decision matrix. Z j + = max ( Z 1 , Z 2 , ... Z m and worst solutionZ j - = min ( Z 1 , Z 2 , ... Z m ); calculate the distance from each treatment to the positive ideal solution using formula (5). D i + ), calculate the distance from each treatment to the worst solution using formula (6) D i - Formula (7) is used to evaluate the degree of similarity between the object and the optimal solution. C i ), C i The higher the value, the better the evaluation object.

[0031] .

[0032] (2) Comprehensive evaluation and analysis of membership functions The weights of each principal component are obtained using formula (8). W k In the formula, P k Indicates the first k The contribution rate of each comprehensive indicator. The membership function value of each comprehensive indicator for each treatment is calculated using formula (9). μ(X) k ） In the formula X k For the first k A comprehensive indicator; X min For the first k The minimum value among the comprehensive indicators; X max Indicates the first k The maximum value among the comprehensive indicators. Calculate the comprehensive metric (D value) for each treatment using formula (10).

[0033] .

[0034] Example 1 Preparation of open culture medium: MS medium was prepared according to standard procedures, with 30 g / L sucrose added. Agar powder concentrations of 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, and 8 g / L were set. After dissolving, the medium was poured directly into clean tissue culture flasks without autoclaving. Traditional autoclaved MS medium (7 g / L agar, 30 g / L sucrose) was used as a control. A total of 8 treatments were performed (see Table 1), repeated 3 times, with 10 flasks per replicate. After standing for 1 hour, the solidification and firmness of the medium were observed and recorded. The results are shown in Table 2.

[0035] Table 1. Agar Concentration Settings for Open Tissue Culture Table 2. Effect of different agar concentrations on the solidification degree of the culture medium. Note: CK is the control group, and QZ1~QZ7 are the agar concentration treatment groups.

[0036] The amount of agar added is crucial as a solidifying agent for preparing solid culture media. If the concentration is too low, it will be difficult to effectively fix the plants, while if the concentration is too high, the culture medium will be too hard, affecting the inoculation operation and nutrient absorption.

[0037] As can be seen from Table 2, when the agar powder addition is 6 g / L, it can solidify quickly and has a moderate softness and hardness, with a smooth surface and no shrinkage or cracking. This ensures sufficient support and facilitates inoculation, making it the most suitable amount for open tissue culture of 'Atlantic' potatoes.

[0038] Example 2 Preparation of open tissue culture medium and open inoculation method: Before preparing the culture medium, the laboratory was sprayed with 75% alcohol to reduce dust and sterilize, and then irradiated with ultraviolet light for 30 min. Culture flasks, caps, and glassware were immersed in sodium hypochlorite solution, then removed and irradiated with ultraviolet light for 15 min before use. MS medium was prepared by adding 6 g / L agar, 30 g / L sucrose, and an antibacterial agent sequentially, stirring thoroughly, and then bringing the volume to a final depth. When the medium temperature dropped to 50–55°C, the pH was adjusted to 5.8. The medium was then dispensed into culture flasks while still warm, with a thickness of 1–1.5 cm. After dispensing, autoclaving was not performed; the medium was allowed to cool before use.

[0039] Before inoculation, the laboratory was sprayed with 75% alcohol to reduce dust and sterilize, and then irradiated with ultraviolet light for 30 minutes. During inoculation, the work surface and hands were wiped with alcohol swabs, and the inoculation instruments were wiped with alcohol and then sterilized by flaming with an alcohol lamp for later use. Virus-free potato seedlings were cut into stem segments with one bud, 1-1.5 cm in length, and quickly inoculated into a culture medium containing an antibacterial agent. During the inoculation process, the inoculation instruments were constantly immersed in 75% alcohol for sterilization; a laminar flow hood was not used. After inoculation, the tubes were sealed using a sealing machine.

[0040] The antibacterial agents used were manganese zinc, mancozeb, sodium hypochlorite, S106, Epclusa, or phytoalexin. Six concentration gradients were set for each antibacterial agent (see Table 3). Traditional tissue culture medium without antibacterial agents and aseptic inoculation methods served as the control (CK) (7 g / L agar, 30 g / L sucrose). A total of 37 treatments were set up, replicated three times, with 10 bottles per replicate. Ten stem segments of 'Atlantic' virus-free seedlings were inoculated into each bottle. The inoculated medium was placed in a culture room for cultivation, with a growth cycle of 28 days. The contamination rate, albinism rate, germination rate, survival rate, and growth indicators of the virus-free seedlings were recorded.

[0041] The traditional methods for preparing tissue culture media and aseptic inoculation are as follows: Prepare MS medium by adding 0.7% agar and 3% sucrose, stirring thoroughly, and bringing the volume to a final depth. When the medium temperature drops to 50-55℃, adjust the pH to 5.8. While still hot, dispense the medium into culture flasks, with a thickness of 1-1.5 cm. Then, sterilize in an autoclave at 121℃ and 0.1 MPa for 22 min. After sterilization, remove the flasks and place them in a sterile room to solidify before use.

[0042] Before inoculation, the laboratory was sprayed with 75% alcohol to reduce dust and sterilize, and then irradiated with ultraviolet light for 30 minutes. The laminar flow hood and ultraviolet light were turned on 30 minutes in advance. The ultraviolet light was turned off during inoculation. The workbench surface and inner walls were wiped with 75% alcohol, and the workbench surface and hands were wiped with alcohol swabs. Inoculation instruments were wiped with alcohol and then sterilized with an alcohol lamp for later use. Virus-free seedlings were cut into stem segments with one bud, 1–1.5 cm in length, and quickly inoculated into the culture medium. During the inoculation process, the inoculation instruments were constantly immersed in 75% alcohol for sterilization. The entire inoculation process must be carried out within the laminar flow hood. The inoculation was repeated three times, with 10 bottles per replicate, and 10 stem segments of 'Atlantic' virus-free seedlings inoculated into each bottle. After inoculation, the bottles were sealed using a sealing machine.

[0043] Table 3. Types and concentrations of antibacterial agents in MS culture medium The concentrations used for manganese zinc, mancozeb, S106, and eperazine are the concentrations of the formulation, while the concentrations used for sodium hypochlorite and phytochemicals are the concentrations of the active ingredients.

[0044] (I) Effects of different concentrations of polymanganese zinc on virus-free seedlings (1) Effects of different concentrations of polymanganese zinc on the contamination rate, whitening rate, survival rate and germination rate of virus-free seedlings (see Table 4).

[0045] Table 4. Effects of different concentrations of polymanganese zinc on the contamination rate, whitening rate, survival rate, and germination rate of virus-free seedlings. Note: CK is the control, and M1~M6 are the treatment groups with high manganese zinc concentrations, the same applies below.

[0046] As shown in Table 4, within the experimental concentration range, manganese zinc had no significant effect on the albino rate and survival rate of virus-free seedlings. The albino rate was 0% and the survival rate was 100% in all treatments, consistent with the control (CK). The fungal contamination rate was 0% in all treatments, but the bacterial contamination rate showed significant differences with concentration. The bacterial contamination rate was highest in treatments M1 and M2, reaching 93.33%, higher than the control (CK) (6.67%). The bacterial contamination rate was lowest in treatment M6, decreasing to 6.67%, consistent with the control (CK).

[0047] (2) Effects of different concentrations of polymanganese zinc on the growth and physiological indicators of virus-free seedlings (see Table 5 and 2010) Figure 1 ).

[0048] Table 5. Effects of different concentrations of polymanganese zinc on the growth and physiological indicators of virus-free seedlings. Note: Different letters represent P The difference was significant at the <0.05 level, and the same applies below.

[0049] From Table 5 and Figure 1 As can be seen, polymanganese zinc significantly inhibits the morphogenesis of virus-free seedlings. The inhibitory effect continues to increase with increasing concentration. At high concentrations, root development almost stops, and chlorophyll content is also significantly reduced, resulting in impaired photosynthetic capacity. Therefore, it is necessary to control the concentration of polymanganese zinc used.

[0050] (3) Effects of different concentrations of polymanganese zinc on the biomass of virus-free seedlings (see Figure 2 ).

[0051] from Figure 2 It can be seen that different concentrations of polymanganese zinc have a significant impact on the biomass accumulation of virus-free seedlings. P <0.05). Compared with the control (CK), the aboveground fresh weight, belowground fresh weight, aboveground dry weight, and belowground dry weight of each treatment were significantly reduced, and the inhibitory effect gradually increased with increasing concentration, further indicating that the concentration of polymanganese zinc used needs to be controlled.

[0052] (4) Entropy weight TOPSIS analysis (see Table 6).

[0053] Table 6 Information Entropy and Weights In the entropy-weighted TOPSIS, a lower information entropy value for a given indicator indicates greater dispersion and a larger impact (i.e., weight) on the TOPSIS evaluation results. If all indices are equal, the indicator has no effect on the overall evaluation. Table 6 shows that the top three weighted indicators are total root length, leaf area, and number of leaves, at 14.005%, 12.001%, and 10.437%, respectively. Fungal contamination rate, bleaching rate, and survival rate have the lowest weights, all at 0%.

[0054] The entropy weight TOPSIS method was used to analyze 17 indicators, including bacterial contamination rate, for 7 treatments. The results are shown in Table 7.

[0055] Table 7. Overall Score Results of TOPSIS Evaluation Method (II) Effects of different concentrations of mancozeb on virus-free seedlings (1) Effects of different concentrations of mancozeb on the contamination rate, whitening rate, survival rate and germination rate of virus-free seedlings (see Table 8).

[0056] Table 8. Effects of different concentrations of mancozeb on contamination rate, albinism rate, survival rate, and germination rate of virus-free seedlings. Note: CK is the control, and D1~D6 are the mancozeb concentration treatment groups, the same below.

[0057] Table 8 shows that within the experimental concentration range, mancozeb had no significant effect on the albino rate and survival rate of virus-free seedlings; the albino rate was 0% and the survival rate was 100% in all treatments, consistent with the control (CK). At the experimental concentrations, mancozeb effectively inhibited fungal contamination, with all treatments showing a fungal contamination rate of 0%. Treatment D1 had the highest bacterial contamination rate, reaching 100%. The inhibitory effect on bacterial contamination gradually increased with increasing concentration. The bacterial contamination rate in treatment D4 was consistent with the CK (6.67%), while the bacterial contamination rates in treatments D5 and D6 decreased to 0%, lower than the CK.

[0058] (2) Effects of different concentrations of mancozeb on the growth and physiological indicators of virus-free seedlings (see Table 9 and 1) Figure 3 ).

[0059] Table 9. Effects of different concentrations of mancozeb on the growth and physiological indicators of virus-free seedlings. From Table 9 and Figure 3As can be seen, mancozeb significantly inhibits the morphogenesis of virus-free seedlings. The inhibitory effect continues to increase with increasing concentration. At high concentrations, root development almost stops, and chlorophyll content is also significantly reduced, resulting in impaired photosynthetic capacity. Therefore, it is necessary to control the concentration of mancozeb used.

[0060] (3) Effects of different concentrations of mancozeb on the biomass of virus-free seedlings from Figure 4 The results show that different concentrations of mancozeb have a significant impact on the biomass accumulation of virus-free seedlings (P<0.05). Compared with the control (CK), mancozeb showed a trend of first promoting and then inhibiting the aboveground biomass, while exhibiting a continuous inhibitory effect on the underground biomass, further indicating the need to control the concentration of mancozeb used.

[0061] (4) Entropy weight TOPSIS analysis (see Table 10).

[0062] Table 10 Information Entropy and Weights As can be seen from Table 10, the top three weights are total root length, leaf area and aboveground fresh weight, which are 14.018%, 10.645% and 10.589% respectively. Fungal contamination rate, whitening rate and survival rate have the smallest weights, all of which are 0%.

[0063] The entropy weight TOPSIS method was used to analyze 17 indicators, including bacterial contamination rate, for 7 treatments. The results are shown in Table 11.

[0064] Table 11 Overall Score Results of TOPSIS Evaluation Method (III) Effects of different concentrations of sodium hypochlorite on virus-free seedlings (1) Effects of different concentrations of sodium hypochlorite on the contamination rate, whitening rate, survival rate and germination rate of virus-free seedlings (see Table 12).

[0065] Table 12 Effects of different concentrations of sodium hypochlorite on contamination rate, whitening rate, survival rate, and germination rate of virus-free seedlings. Note: CK is the control group, and C1~C6 are the sodium hypochlorite concentration treatment groups, the same below.

[0066] As can be seen from Table 12, sodium hypochlorite can effectively inhibit fungal contamination at the experimental concentration, bacterial contamination occurs at lower concentrations, and bacterial contamination can be completely inhibited at higher concentrations, but it will cause whitening of virus-free seedlings and have a significant inhibitory effect on the survival and germination of virus-free seedlings. Therefore, it is necessary to reasonably control the concentration of sodium hypochlorite used.

[0067] (2) Effects of different concentrations of sodium hypochlorite on the growth and physiological indicators of virus-free seedlings (see Table 13 and 14) Figure 5 ).

[0068] Table 13 Effects of different concentrations of sodium hypochlorite on the growth and physiological indicators of virus-free seedlings From Table 13 and Figure 5 As can be seen, with the increase of sodium hypochlorite concentration, the growth and physiological indicators of virus-free seedlings showed a trend of first increasing and then decreasing. At low concentrations, sodium hypochlorite had no significant inhibitory effect on the aboveground growth of virus-free seedlings, and some indicators were even significantly higher than the control (CK), but the root system showed significant inhibition. At high concentrations, both aboveground and underground parts were significantly inhibited. Therefore, the inhibitory effect of sodium hypochlorite is clearly concentration-dependent and organ-specific, with the root system being more sensitive than the aboveground parts.

[0069] (3) Effects of different concentrations of sodium hypochlorite on the biomass of virus-free seedlings (see Figure 6 ).

[0070] from Figure 6 As can be seen, different concentrations of sodium hypochlorite have a significant effect on the biomass accumulation of virus-free seedlings (P<0.05). Low concentrations of sodium hypochlorite can significantly promote the accumulation of aboveground biomass, but as the concentration increases, the promotion of aboveground biomass turns into inhibition; it has a continuous inhibitory effect on underground biomass, and the inhibitory effect on underground biomass is greater than that on aboveground biomass at high concentrations.

[0071] (4) Entropy weight TOPSIS analysis (see Table 14).

[0072] Table 14 Information Entropy and Weights As shown in Table 14, the top three weighted indicators are total root length, stem diameter, and fresh weight of underground parts, at 16.340%, 8.574%, and 8.484%, respectively. Fungal contamination rate has the lowest weight, at 0%. The entropy weight TOPSIS method was used to analyze 17 indicators, including bacterial contamination rate, for 7 treatments. The results are shown in Table 15.

[0073] Table 15 TOPSIS Evaluation Method Overall Score Results (iv) Effects of different concentrations of S106 on virus-free seedlings (1) Effects of different concentrations of S106 on the contamination rate, whitening rate, survival rate and germination rate of virus-free seedlings (see Table 16).

[0074] Table 16 Effects of different concentrations of S106 on contamination rate, albinism rate, survival rate, and germination rate of virus-free seedlings. Note: CK is the control group, and N1~N6 are the S106 concentration treatment groups, the same below.

[0075] As shown in Table 16, within the experimental concentration range, the fungal contamination rate and bleaching rate of each treatment were 0%, and the survival rate was 100%, consistent with the control (CK). This indicates that S106 can effectively inhibit fungal contamination without affecting the survival rate of virus-free seedlings or causing bleaching. With increasing treatment concentration, the inhibitory effect of S106 on bacterial contamination gradually increased, but the germination rate showed a decreasing trend.

[0076] (2) Effects of different concentrations of S106 on the growth and physiological indicators of virus-free seedlings (see Table 17 and 18) Figures 7-8 ).

[0077] Table 17 Effects of different concentrations of S106 on the growth and physiological indicators of virus-free seedlings From Table 17 and Figures 7-8 As can be seen, with the increase of S106 concentration, the growth and physiological indicators of virus-free seedlings generally showed a downward trend. At low concentrations, S106 had a certain promoting effect on the aboveground growth of virus-free seedlings, increasing the number of underground roots but shortening the root length; with the increase of concentration, the inhibitory effect rapidly increased, with the most significant decreases in leaf area and total root length.

[0078] (3) Effects of different concentrations of S106 on the biomass of virus-free seedlings (see Figure 9 ).

[0079] from Figure 9 It can be seen that different concentrations of S106 have a significant effect on the biomass accumulation of virus-free seedlings (P<0.05).

[0080] (4) Entropy weight TOPSIS analysis (see Table 18).

[0081] Table 18 Information Entropy and Weights As can be seen from Table 18, the top three weighted indicators are total root length, germination rate, and underground fresh weight, which are 13.684%, 13.096%, and 7.682%, respectively. The weights of fungal contamination rate, whitening rate, and survival rate are the smallest, all of which are 0%.

[0082] The entropy weight TOPSIS method was used to analyze 17 indicators, including bacterial contamination rate, for 7 treatments. The results are shown in Table 19.

[0083] Table 19 TOPSIS Evaluation Method Overall Score Results (V) Effects of different concentrations of eperazine on virus-free seedlings (1) Effects of different concentrations of epelonone on the contamination rate, albinism rate, survival rate and germination rate of virus-free seedlings (see Table 20) Table 20 Effects of different concentrations of eperin on contamination rate, albinism rate, survival rate, and germination rate of virus-free seedlings. Note: CK is the control group, and Y1~Y6 are the treatment groups for epelinone concentration, the same below.

[0084] As shown in Table 20, within the experimental concentration range, the fungal contamination rate and whitening rate were both 0% for each treatment, and the survival rate was 100%. This indicates that Yipelong can effectively inhibit fungal contamination without affecting the survival rate of virus-free seedlings or causing whitening. With increasing concentration, the inhibitory effect on bacterial contamination gradually increased, but the germination rate showed a decreasing trend.

[0085] (2) Effects of different concentrations of eperazine on the growth and physiological indicators of virus-free seedlings (see Table 21 and 2018) Figure 10 ).

[0086] Table 21 Effects of different concentrations of eperon on growth and physiological indicators of virus-free seedlings From Table 21 and Figure 10 It can be seen that at low concentrations, Yipelong has a certain promoting effect on the aboveground growth of virus-free seedlings, but it has a significant inhibitory effect on the root system. As the concentration increases, the inhibitory effect rapidly increases and extends to the aboveground parts, showing a clear "low-promoting and high-inhibiting" dose effect. Moreover, the inhibitory effect on the root system is always more significant, and at high concentrations, root development is almost completely stopped.

[0087] (3) Effects of different concentrations of eperazine on the biomass of virus-free seedlings (see Figure 11 ).

[0088] from Figure 11 As can be seen, different concentrations of epembrane have a significant effect on the biomass accumulation of virus-free seedlings (P<0.05). With increasing treatment concentration, the aboveground fresh weight and aboveground dry weight showed a trend of first increasing and then decreasing, both reaching their peak values ​​in treatment Y2.

[0089] (4) Entropy weight TOPSIS analysis (see Table 22).

[0090] Table 22 Information Entropy and Weights As can be seen from Table 22, the total root length, germination rate and bacterial contamination rate have the highest weights among all indicators, at 16.143%, 10.281% and 9.534% respectively, while the fungal contamination rate, whitening rate and survival rate have the lowest weights, all at 0%.

[0091] The entropy weight TOPSIS method was used to analyze 17 indicators, including bacterial contamination rate, for 7 treatments. The results are shown in Table 23.

[0092] Table 23 TOPSIS Evaluation Method Overall Score Results (vi) Effects of different concentrations of plant growth regulator on virus-free seedlings (1) Effects of different concentrations of plant growth regulator on the contamination rate, whitening rate, survival rate and germination rate of virus-free seedlings (see Table 24).

[0093] Table 24 Effects of different concentrations of plant growth regulator on contamination rate, albinism rate, survival rate, and germination rate of virus-free seedlings. Note: CK is the control group, and Z1~Z6 are the treatment groups for different concentrations of plant growth regulator, the same applies below.

[0094] As shown in Table 24, within the experimental concentration range, the fungal contamination rate and bleaching rate were both 0% for each treatment, and the survival rate was 100%. This indicates that the plant growth regulator can effectively inhibit fungal contamination without affecting the survival rate of virus-free seedlings or causing bleaching. With increasing treatment concentration, the inhibitory effect of the plant growth regulator on bacterial contamination gradually increased, but this led to a decrease in germination rate.

[0095] (2) Effects of different concentrations of plant growth regulator on the growth and physiological indicators of virus-free seedlings (see Table 25 and 2010) Figure 12 ).

[0096] Table 25 Effects of different concentrations of plant growth regulator on the growth and physiological indicators of virus-free seedlings As can be seen from Figure 12, with the increase of the concentration of plant growth regulator, the growth and physiological indicators of virus-free seedlings all showed a downward trend.

[0097] (3) Effects of different concentrations of plant growth regulator on the biomass of virus-free seedlings (see Figure 13 ).

[0098] from Figure 13 It can be seen that different concentrations of plant growth regulator have a significant effect on the biomass accumulation of virus-free seedlings (P<0.05). Compared with the control (CK), the aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight of each treatment were significantly reduced, and the inhibitory effect gradually increased with increasing concentration.

[0099] (4) Entropy weight TOPSIS analysis (see Table 26).

[0100] Table 26 Information Entropy and Weights As can be seen from Table 26, the top three weights among the indicators are underground fresh weight, total root length, and aboveground fresh weight, which are 17.718%, 13.262%, and 10.640%, respectively. Fungal contamination rate, whitening rate, and survival rate have the smallest weights, all of which are 0%.

[0101] The entropy weight TOPSIS method was used to analyze 17 indicators, including bacterial contamination rate, for 7 treatments. The results are shown in Table 27.

[0102] Table 27 TOPSIS Evaluation Method Overall Score Results (vii) Comprehensive evaluation of the optimal concentration of six antibacterial agents Comprehensive index values, membership function values, comprehensive evaluation D-values, and rankings of antibacterial agents at different concentrations The eigenvectors were obtained by factor loading, and the comprehensive evaluation D value was calculated by formulas (8), (9), and (10). The values ​​were sorted according to the size of the D value, and the results are shown in Table 28.

[0103] Table 28. Comprehensive index values, membership function values, comprehensive evaluation D-values, and rankings. Note: In the table, CI represents the comprehensive index value, CI1, CI2, and CI3 represent the comprehensive index values ​​of the corresponding principal components; μ represents the membership function value, μ1, μ2, and μ3 represent the membership function values ​​of the corresponding principal components. As can be seen from Table 28, the N1 treatment had the highest overall evaluation D value of 0.768, followed by CK at 0.760. This indicates that under suitable open tissue culture conditions, the addition of 0.2 ml / L of S106 antibacterial agent can enable virus-free seedlings to achieve or even surpass the growth status under traditional plant tissue culture conditions.

[0104] Example 3 Screening of sucrose concentration in open tissue culture: MS medium was prepared by adding 6 g / L agar, 0.2 ml / L S106, and different concentrations of sucrose (10 g / L, 15 g / L, 20 g / L, 25 g / L, and 30 g / L). A control (CK) was prepared using traditional tissue culture medium without antibacterial agents and aseptic inoculation (7 g / L agar, 30 g / L sucrose). Six treatments were set up (see Table 29), with three replicates of 10 bottles per replicate. Ten stem segments of 'Atlantic' virus-free seedlings were inoculated into each bottle. The inoculated medium was placed in a culture room and cultured for 28 days as one growth cycle. The contamination rate, albinism rate, germination rate, survival rate, and growth indicators of the virus-free seedlings were recorded.

[0105] Table 29 Sucrose Concentration Settings for Open Tissue Culture (1) Effects of different concentrations of sucrose on the contamination rate, albinism rate, survival rate and germination rate of virus-free seedlings (see Table 30).

[0106] Table 30 Effects of different concentrations of sucrose on the contamination rate, albinism rate, survival rate, and germination rate of virus-free seedlings. Note: CK is the control, ZT1~ZT6 are the sucrose concentration treatment groups, and the same applies below.

[0107] Table 30 shows that within the experimental concentration range, the whitening rate was 0% for all treatments, and the survival and germination rates were 100%, indicating that changes in sucrose addition did not affect the survival and germination rates of virus-free seedlings, nor did they cause whitening. However, the contamination of virus-free seedlings varied under different sucrose concentrations. The contamination rate increased with increasing sucrose concentration. The bacterial and fungal contamination rates of treatments ZT1-ZT3 were both 0%, the same as the control (CK), indicating that the sucrose environment in this concentration range did not provide suitable conditions for microbial reproduction, achieving aseptic growth under open tissue culture. With increasing sucrose concentration, the bacterial contamination rate of treatment ZT4 increased to 6.67%, and the fungal contamination rate increased to 3.33%, both higher than the CK. Under treatment ZT5, the contamination level further worsened, with the bacterial contamination rate rising to 13.33% and the fungal contamination rate remaining at 0%. This indicates that sucrose concentration has a direct impact on the contamination rate of open tissue culture, and higher concentrations increase the risk of contamination.

[0108] (2) Effects of different concentrations of sucrose on the growth and physiological indicators of virus-free seedlings (see Table 31 and 32) Figure 14 ).

[0109] Table 31 Effects of different sucrose concentrations on the growth and physiological indicators of virus-free seedlings From Table 31 and Figure 14 As can be seen, with the increase of sucrose concentration, most indicators of virus-free seedlings showed a trend of first increasing and then decreasing. This may be related to the fact that sucrose, as a carbon source, provides energy and skeletal substances for virus-free seedlings. At the same time, previous analysis showed that the added 0.2 ml / L S106 antibacterial agent also had a certain growth-promoting effect on the growth of virus-free seedlings. The synergistic effect of the two was most prominent in the ZT4 treatment, and the overall performance in promoting aboveground growth and root differentiation was better.

[0110] (3) Effects of different sucrose concentrations on the biomass of virus-free seedlings (see...) Figure 15 ).

[0111] from Figure 15As can be seen, different concentrations of sucrose have a significant effect on the biomass accumulation of virus-free seedlings (P<0.05). With increasing sucrose concentration, the aboveground fresh weight and aboveground dry weight showed a trend of first increasing and then decreasing, with the ZT4 treatment reaching the highest value, significantly increasing by 45.55% and 31.68% compared with the CK, respectively.

[0112] (4) Entropy weight TOPSIS analysis (see Table 32).

[0113] Table 32 Information Entropy and Weights As can be seen from Table 32, the top three weighted indicators are total root length, leaf area and chlorophyll content, which are 20.5998%, 9.651% and 8.398% respectively. The weights of bleaching rate, survival rate and germination rate are the smallest, all of which are 0%.

[0114] The entropy weight TOPSIS method was used to analyze 17 indicators, including bacterial contamination rate, for 7 treatments. The results are shown in Table 33.

[0115] Table 33 TOPSIS Evaluation Method Overall Score Results The results above show that different antibacterial agents have different inhibitory effects on crops. For the same antibacterial agent, low concentrations have less impact on plant growth, but the antibacterial effect cannot meet the ideal requirements. High concentrations of antibacterial agents have good antibacterial effects, but the high concentration can have side effects on plant growth. Therefore, the optimal concentration range of antibacterial agents should be selected by considering both the impact on plant growth and the antibacterial effect.

[0116] The optimal treatments for the six antimicrobial agents were M1 (0.01 g / L mancozeb), D1 (0.05 g / L mancozeb), C2 (0.01% sodium hypochlorite), N1 (0.2 ml / L S106), Y2 (0.4 ml / L Eppendorf), and Z1 (0.15% phytoalexin), representing the lowest or second-lowest concentrations of each agent. The results indicate that for open tissue culture of potatoes, higher concentrations of antimicrobial agents are not necessarily better. While lower concentrations may not have the optimal antimicrobial effect, they exhibit the weakest inhibitory effect on plant growth and offer better overall performance. Furthermore, 0.2 ml / L S106 showed the best overall performance in terms of both antimicrobial effect and virus-free seedling growth, and can be recommended as the optimal antimicrobial agent and concentration for 'Atlantic' potatoes. 0.4 ml / L Eppendorf also demonstrates good application potential and can be considered as an alternative antimicrobial treatment.

[0117] Agar concentration is a key factor affecting the physical properties of open tissue culture medium. Under open tissue culture conditions without autoclaving, a 6 g / L agar concentration is the optimal concentration for 'Atlantic' potato open culture medium. At this concentration, the medium has good solidification and moderate hardness, which can support the growth of virus-free seedlings and meet the operational requirements of open inoculation, laying the foundation for subsequent open tissue culture experiments.

[0118] Sucrose can provide a carbon source for the culture medium, but it is also a major cause of contamination in the medium. Through single-factor screening and entropy weight TOPSIS analysis, it was found that 25 g / L is the optimal sucrose concentration in the open culture medium of 'Atlantic' potato. At this concentration, the contamination control effect is good, and the virus-free seedlings grow vigorously with all growth indicators reaching the optimal level.

[0119] The optimal culture medium for open tissue culture of 'Atlantic' potato in this invention is: MS medium + 0.2 ml / L S106 + 6 g / L agar + 25 g / L sucrose.

[0120] This invention applies a multi-dimensional comprehensive evaluation system to the screening of antimicrobial agents in open tissue culture, thereby improving the scientific rigor of the evaluation. First, the optimal concentration of each antimicrobial agent is determined using the entropy-weighted TOPSIS method. Then, principal component analysis and membership function comprehensive evaluation are combined to rank the optimal concentrations of the six antimicrobial agents. This overcomes the limitations of single-index evaluation, making the screening results more scientific, objective, and reliable, and providing a new reference for the systematic evaluation of antimicrobial agents in open tissue culture.

[0121] This invention can control contamination within an acceptable range and achieve virus-free seedling growth results close to those of traditional tissue culture without using autoclaving and clean bench inoculation, thereby reducing equipment dependence and production costs.

[0122] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for open tissue culture of virus-free potato seedlings, characterized in that, Includes the following steps: Add antibacterial agents to unsterilized culture media, and then inoculate potato virus-free seedling stem segments under an environment treated with alcohol spray and / or ultraviolet irradiation. After inoculation, seal the culture medium.

2. The method according to claim 1, characterized in that, The antibacterial agent is selected from any one of manganese zinc, mancozeb, sodium hypochlorite, S106, Epclusa, and Phytoprotectant.

3. The method according to claim 2, characterized in that, When the antibacterial agent is manganese zinc, its concentration in the culture medium is ≥0.25 g / L; When the antibacterial agent is mancozeb, its concentration in the culture medium is ≥0.20 g / L; When the antibacterial agent is sodium hypochlorite, its concentration in the culture medium is ≥0.01%; When the antibacterial agent is S106, its concentration in the culture medium is ≥0.20 ml / L; When the antibacterial agent is Eperazine, its concentration in the culture medium is ≥0.70 ml / L; When the antibacterial agent is Plantarazole, its concentration in the culture medium is ≥0.45%.

4. The method according to claim 3, characterized in that, When the antibacterial agent is manganese zinc, its concentration in the culture medium is 0.25 g / L; When the antibacterial agent is mancozeb, its concentration in the culture medium is 0.20~0.30 g / L; When the antibacterial agent is sodium hypochlorite, its concentration in the culture medium is 0.01~0.04%; When the antibacterial agent is S106, its concentration in the culture medium is 0.20~0.70 ml / L; When the antibacterial agent is Eperazine, its concentration in the culture medium is 0.70~0.80 ml / L; When the antibacterial agent is Plantarazole, its concentration in the culture medium is 0.45~0.65%.

5. The method according to claim 1, characterized in that, The culture medium is MS medium supplemented with agar and sucrose.

6. The method according to claim 5, characterized in that, The concentration of agar added is 6~7 g / L; And / or, the concentration of added sucrose is 25~30g / L.

7. The method according to claim 6, characterized in that, The composition of the culture medium is: MS medium + 0.2 ml / L S106 + 6 g / L agar + 25 g / L sucrose.

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