Method for eliminating bacterial pollution in German iris tissue culture

Abstract

The invention relates to the technical field of plant tissue culture, and discloses a method for eliminating bacterial pollution of German iris tissue culture seedlings, which comprises the following steps: 1) surface sterilization: pretreating polluted tissue culture seedlings to remove epiphytic bacteria on the surfaces; 2) gradient degerming: inoculating the seedlings to a degerming culture medium I containing 0.1% of Kathon, culturing for 14-21 days, then transferring to a culture medium II containing 0.05% of Kathon, and culturing for 14-21 days, so as to thoroughly remove polluted bacteria; and 3) rejuvenation culture: inoculating the sterile seedlings to a rejuvenation culture medium, and culturing for 21-28 days to obtain healthy tissue culture plants.According to the technology, efficient degerming is realized through Kathon concentration gradient decrease (0.1%-0.05%), and detoxified seedlings are provided for German iris large-scale chemical plant production.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for eliminating bacterial contamination in German iris tissue culture. Background Art

[0002] German Iris ( Iris germanica As a perennial ornamental plant, L. is widely used in landscape gardening for its vibrant colors and unique flower shapes. However, traditional division propagation suffers from low reproduction coefficients (typically divisions are performed every three years, with each segment bearing only one or two buds), virus accumulation leading to varietal degeneration, and unstable germination rates, making it difficult to meet the demands of industrial production. (The optimal time for division propagation is after dormancy in autumn or before bud break in spring; transplanting individual plants can affect flowering rates.) While tissue culture allows for efficient propagation and virus-free seedlings, subculture often faces the challenge of bacterial contamination. The risk of contamination increases after three to five generations, leading to seedling rejection and increased costs. Long-term germplasm storage also carries higher contamination rates. Existing contamination control technologies have limitations: commonly used disinfectants (such as 0.1% HgCl₂ treatment) are highly toxic, easily causing browning of explants and environmental pollution; antibiotics (such as Timentin) can easily lead to drug resistance and have low bacterial removal rates. Therefore, the development of efficient, low-toxicity contamination solutions is urgently needed.

[0003] The present invention provides a method for eliminating bacterial contamination in German iris tissue culture. By combining surface sterilization with gradient cascade sterilization, the bacterial contamination rate is significantly reduced to below 5%. At the same time, the rejuvenation and germination rate of tissue culture seedlings is ≥95%, providing a healthy seed source for germplasm resource preservation and factory seedling cultivation. The specific steps are as follows: 1) Surface sterilization: Pre-treat the contaminated tissue culture seedlings to remove surface bacteria and set aside; 2) Gradient sterilization: First stage sterilization (high concentration rapid inhibition): The tissue culture seedlings obtained in step 1) are inoculated into sterilization medium I and cultured for 14-21 days to obtain sterilized tissue culture seedlings, wherein sterilization medium I is the basic formula and 0.01%-0.2% kason is added; Second stage sterilization (low concentration continuous bacterial control): The sterilized tissue culture seedlings are inoculated into sterilization medium II and cultured for another 14-21 days to obtain sterile tissue culture seedlings, wherein sterilization medium II is the basic formula and 0.005%-0.1% kason is added; 3) Rejuvenation culture: Inoculate the sterile tissue culture seedlings into the rejuvenation medium and perform rejuvenation culture for 21-28 days to obtain healthy and sterile German iris tissue culture seedlings.

[0004] Furthermore, in step 1), the pretreatment is specifically as follows: removing part of the leaves of the contaminated tissue culture seedlings, retaining a stem segment of about 1 cm, placing it in sterile water containing 0.1% carbazon, shaking at 110 rpm for 30 minutes, and then washing it with sterile water three times.

[0005] Further, in step 2), the basic formula of the sterilization medium I and the sterilization medium II is: MS medium + 6-BA 0.5 mg / L + NAA 0.05 mg / L + MES buffer (2-(N-morpholino)ethanesulfonic acid) 2 g / L + sucrose 30 g / L + agar powder 6 g / L, and the pH value is 5.8.

[0006] Further, in step 3), the rejuvenation medium is: MS medium + 6-BA 0.5 mg / L + NAA 0.05 mg / L + sucrose 30 g / L + agar powder 6 g / L, and the pH value is 5.8.

[0007] Further, the culture conditions in step 2) and step 3) are both: culture temperature 23 - 25°C, light intensity 2000 lux, and light cycle 12 h / d.

[0008] Further, in step 2), the sterilization medium I contains 0.1% Kathon, and the sterilization medium II contains 0.05% Kathon.

[0009] Further, the Kathon in step 2) is a mixture of 2-methyl-4-isothiazolin-3-one (MI) and 2-methyl-5-chloro-4-isothiazolin-3-one (CMI) and an inorganic salt stabilizer (CAS No. 2682-20-4).

[0010] Beneficial effects: The present invention significantly reduces the bacterial contamination rate of tissue-cultured seedlings of Iris germanica through a two-step gradient sterilization culture system.

[0011] 1. High-efficiency sterilization mechanism: Gradient Kathon concentration design: First-stage sterilization: Using a high concentration of 0.1% Kathon, culturing in the sterilization medium I for 14 - 21 days to quickly remove bacterial contamination; Second-stage continuous bacteria control: Using a low concentration of 0.05% Kathon, continuing to culture in the sterilization medium II for 14 - 21 days to further eliminate residual bacteria. Through the gradient transition from high concentration (0.1%) to low concentration (0.05%), it not only ensures thorough sterilization (the contamination rate is reduced to below 5%), but also reduces the toxic damage of the bacteriostatic agent Kathon to plant tissues.

[0012] 2. Applicability to large-scale production: Ease of operation: The whole culture conditions are unified (23 - 25°C, 2000 lux light, 12 h light cycle), reducing the operation complexity; Cost-effectiveness: Kathon is an industrial common reagent with a low price.

[0013] Other advantages, objectives, and features of the present invention will be elaborated in detail in the subsequent specification, and will be obvious to those skilled in the art based on the following embodiments, or can be inspired from the practice of the present invention. Detailed Embodiments

[0014] To make the technical solution clear, the present invention will be described in detail with reference to the attached tables of embodiments. It should be noted that the described embodiments are only illustrative and not an exhaustive list; accordingly, all derivative embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection.

[0015] The present invention provides a method for eliminating bacterial contamination in tissue culture of Iris germanica, comprising the following steps: 1. Surface sterilization: Place the tissue culture seedlings of Iris germanica contaminated with bacteria in a sterile operating table, remove the outer leaves and contaminated parts, and retain a stem segment about 1 cm in length. Immerse the treated stem segment in a sterile aqueous solution containing 0.1% (v / v) Kathon (active ingredients: a mixture of 2-methyl-4-isothiazolin-3-one and 2-methyl-5-chloro-4-isothiazolin-3-one, CAS No. 2682-20-4), place it on a constant temperature shaker and shake it at a speed of 110 rpm for 30 min. Take out the stem segment and rinse it 3 times with sterile water to remove the epiphytic bacteria on the surface.

[0016] 2. Gradient sterilization (1) First-stage sterilization: Inoculate the pre-treated tissue culture seedlings into sterilization medium I, the composition of which is: MS basal medium (Murashige & Skoog, 1962); plant growth regulator: 6-BA 0.5 mg / L + NAA 0.05 mg / L; buffer: MES (2-(N-morpholino)ethanesulfonic acid) 2 g / L; carbon source and solidifying agent: sucrose 30 g / L + agar powder 6 g / L; bacteriostatic agent: Kathon 0.1% (v / v); pH value 5.8 (adjusted with 0.1 M NaOH or HCl). Culture conditions: temperature 23–25°C, light intensity 2000 lux, light cycle 12 h / d, continuously culture for 14–21 d to obtain primary sterilized tissue culture seedlings.

[0017] (2) Second-stage sterilization: Transfer the primary sterilized tissue culture seedlings to sterilization medium II, the composition of which is the same as that of sterilization medium I (containing 2 g / L of MES); the concentration of Kathon is reduced to 0.05%. The culture conditions are the same as the previous steps, and continue to culture for 14 - 21 d to obtain sterile tissue culture seedlings. 3. Rejuvenation culture: Inoculate the aseptic tissue culture seedlings into the rejuvenation medium, which consists of: MS basal medium + 6-BA 0.5 mg / L + NAA 0.05 mg / L + sucrose 30 g / L + agar powder 6 g / L; pH value 5.8 (without adding MES buffer). The culture conditions are the same as before, and culture for 21 - 28 days to obtain healthy and aseptic tissue culture seedlings of Iris germanica. Example 1

[0018] Verification of the antibacterial effect of the gradient kasong sterilization medium and the control experiment 1. Surface sterilization Take the tissue culture seedlings of Iris germanica contaminated by bacteria (cultivar: 'Purple Snow Fluttering'), remove the outer contaminated leaves in the ultra-clean workbench, and retain a 1 cm stem segment; immerse it in a sterile aqueous solution containing 0.1% kasong; shake at 110 rpm for 30 min, and rinse with sterile water 3 times.

[0019] 2. Gradient sterilization (1) First-stage sterilization: Inoculate the pretreated tissue culture seedlings into 6 groups of sterilization medium I (basic formula: MS + 6-BA 0.5 mg / L + NAA 0.05 mg / L + MES buffer 2 g / L + sucrose 30 g / L + agar 6 g / L, pH 5.8), and add respectively: Number Antibacterial agent Concentration S0 Sterile water (control) - S1 Plant tissue culture antibacterial agent (PPM, commercially available) 0.1% (v / v) S2 Kathon 0.1% (v / v) S3 Phytomycin (commercially available, specific components unknown) 0.1% (v / v) S4 Timentin (antibiotic) 200 μg / L Culture conditions: 23 - 25°C, 2000 lux, 12 h / d light, culture for 21 days.

[0020] (2) Second-stage sterilization: Transfer the primary sterilized seedlings numbered S0 - S4 to sterilization medium II (the basic formula is the same as sterilization medium I, but the kasong concentration is 0.05%), and continue to culture for 21 days.

[0021] 3. Rejuvenation culture Transfer the aseptic tissue culture seedlings to the rejuvenation medium (MS + 6-BA 0.5 mg / L + NAA 0.05 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8), culture for 28 days, and count the contamination rate and germination rate.

[0022]

[0023] The results show that: The gradient kasong (S2) adopts a decreasing concentration gradient design. After stage II, the contamination rate drops to 2.1%, and the rejuvenation germination rate reaches 96.7%, which is significantly higher than other groups, reflecting its advantages. The contamination rate of single-step kasong is 10.67%, and it drops to 2.1% after the gradient, indicating that the gradient strategy is the key to sterilization.

[0024] Example 2 Optimization of the concentration of Kathon in the second stage of gradient sterilization and toxicity verification 1. Surface sterilization Take the tissue-cultured seedlings of Iris germanica contaminated by bacteria (cultivar: 'Purple Snow Flying'), in a laminar flow hood, remove the outer contaminated leaves, and retain a 1-cm stem segment; immerse it in a sterile aqueous solution containing 0.1% (v / v) Kathon; shake it at 110 rpm for 30 min, and rinse it 3 times with sterile water.

[0025] 2. Gradient sterilization First-stage sterilization Inoculate the pretreated tissue-cultured seedlings into sterilization medium I, composition: MS + 6-BA 0.5 mg / L + NAA 0.05 mg / L + MES buffer 2 g / L + sucrose 30 g / L + agar 6 g / L + 0.1% Kathon; pH 5.8, culture conditions: 23 - 25°C, 2000 lux, 12 h / d light, culture for 21 days.

[0026] Second-stage sterilization (concentration gradient experiment) Transfer the seedlings sterilized in the first stage to 5 groups of sterilization medium II (basic formula: MS + 6-BA 0.5 mg / L + NAA 0.05 mg / L + MES buffer 2 g / L + sucrose 30 g / L + agar 6 g / L, pH 5.8), and add respectively:

[0027] Culture conditions: the same as the first stage (23 - 25°C, 2000 lux, 12 h / d).

[0028] 3. Rejuvenation culture Transfer the seedlings in groups B0 - B4 to a unified rejuvenation medium (MS + 6-BA 0.5 mg / L + NAA 0.05 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8), culture for 28 days, and count the germination rate:

[0029] The results showed that: when using the gradient strategy, the 0.05% Kathon treatment group in the second stage showed the best comprehensive performance: the pollution rate was low (2.1%), significantly lower than that of the group without Kathon addition (B0: 16.67%); the rejuvenation germination rate reached 96.7%, and the seedlings were robust without significant physiological damage; compared with the 0.1% Kathon treatment group throughout the process (B3), although there was no significant difference in the pollution rate, 60% of the tissue-cultured seedlings in the B3 group showed browning, and the rejuvenation germination rate dropped sharply to 41.2%. Therefore, adopting the gradient strategy of 0.1% (stage I) first and then 0.05% (stage II) can effectively balance the antibacterial efficiency and safety.

[0030] Note: The embodiments of the present invention are only examples of the technical solutions and are not restrictive descriptions. Those of ordinary skill in the art can make equivalent replacements without departing from the essence of the technical solutions, and such modifications are all covered within the protection scope.

Claims

1. A method for eliminating bacterial contamination in tissue culture of Iris germanica, characterized in that, It includes the following steps: 1) Surface disinfection: Pretreat the contaminated tissue culture seedlings to remove the epiphytic bacteria on the surface and reserve them for use; 2) Gradient disinfection: The first-stage disinfection (rapid inhibition of bacteria at high concentration): Inoculate the tissue culture seedlings obtained in step 1 into the disinfection medium I and culture for 14 - 21 days to obtain disinfected tissue culture seedlings. The disinfection medium I is a basal medium, and additionally add 0.01% - 0.2% Kathon preservative; The second-stage disinfection (continuous control of bacteria at low concentration): Inoculate the disinfected tissue culture seedlings into the disinfection medium II and continue to culture for 14 - 21 days to obtain sterile tissue culture seedlings. The disinfection medium II is a basal medium, and additionally add 0.005% - 0.1% Kathon preservative; 3) Rejuvenation culture: Inoculate the sterile tissue culture seedlings into the rejuvenation medium and culture for 21 to 28 days to obtain healthy sterile tissue culture seedlings of Iris germanica.

2. The method according to claim 1, wherein: In step 1), the pretreatment is specifically: Remove some leaves of the contaminated tissue culture seedlings, retain about 1 cm of the stem segment, place it in sterile water containing 0.1% Kathon, shake at a speed of 110 rpm for 30 minutes, and then wash 3 times with sterile water.

3. The method according to claim 1, characterized in that: In step 2), the basic formula of the disinfection medium I and the disinfection medium II is: MS medium + 6 - BA 0.5 mg / L + NAA 0.05 mg / L + MES buffer (2-(N - morpholino)ethanesulfonic acid) 2 g / L + sucrose 30 g / L + agar powder 6 g / L, and the pH value is 5.

8.

4. The method according to claim 1, characterized in that: In step 4, the rejuvenation medium is: MS medium + 6 - BA 0.5 mg / L + NAA 0.05 mg / L + sucrose 30 g / L + agar powder 6 g / L, and the pH value is 5.

8.

5. The method according to claim 1, characterized in that: The culture conditions in steps 2) and 3) are: The culture temperature is 23 - 25°C, the light intensity is 2000 lux, and the light cycle is 12 h / d.

6. The method according to claim 1, characterized in that: In step 2), the disinfection medium I contains 0.1% Kathon, and the disinfection medium II contains 0.05% Kathon.

7. The method according to claim 1, wherein: The Kathon in step 2) is a mixture of 2 - methyl - 4 - isothiazolin - 3 - one (MI) and 2 - methyl - 5 - chloro - 4 - isothiazolin - 3 - one (CMI) and an inorganic salt stabilizer (CAS No. 2682 - 20 - 4).

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