Method for removing associated bacteria in tissue culture subculture multiplication culture of clerodendrum trichotomum and application
By treating associated bacteria with sterile gentamicin sulfate solution in tissue culture of Changshan hainanensis, the problem of red bacterial spot contamination was solved, achieving efficient removal and normal growth of tissue culture seedlings, while reducing resource waste and costs.
Patent Information
- Application Number
- CN202511404958.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2025-12-12
AI Technical Summary
In the practice of tissue culture in Changshan hainanensis, contamination by red associated bacteria led to the destruction of tissue culture seedlings and the extension of the propagation cycle, resulting in resource waste and increased costs.
Associated bacteria were treated with a sterile gentamicin sulfate solution of 200–1100 mg/L. The treatment process combined pretreatment and aseptic treatment steps, including explant disinfection, callus induction, redifferentiation and proliferation culture, and red bacterial plaques were treated with sterile gentamicin sulfate solution to ensure 100% removal rate.
It effectively removes red fungal spots, avoids damage to tissue culture seedlings, shortens the propagation cycle, reduces costs, improves the success rate of tissue culture seedlings, and has a proliferation coefficient of up to 16, resulting in robust growth of tissue culture seedlings.
Smart Images
Figure CN121100801A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of plant tissue culture technology, in particular to a method for removing associated bacteria in tissue culture subculture and proliferation of Clerodendrum trichotomum and application thereof. BACKGROUND
[0002] Clerodendrum trichotomum has high ornamental and medicinal value, and the demand for landscaping seedlings is increasing. Tissue culture technology is a key means to achieve rapid propagation. In the practice of tissue culture of Clerodendrum trichotomum, the problem of associated bacteria contamination is prominent when using stem segments with buds as explants for subculture and proliferation. Red associated bacteria rapidly multiply at this stage, forming red bacterial plaques that continuously expand and spread to the surface of the culture medium and the base of the buds. This bacterial contamination directly damages the tissue culture seedlings, leading to wilting and even death, and seriously hinders the expansion process of tissue culture.
[0003] According to the culture specifications of sterile seedlings, once bacterial contamination occurs, the contaminated tissue culture seedlings need to be discarded and sterilized, and then new explants need to be selected and a sterile system needs to be established. The traditional treatment method not only hinders the normal tissue culture process, leading to a prolonged expansion period, but also causes a large amount of loss of valuable explant materials and culture medium consumables, increases labor and time costs, and causes resource waste, which is a key bottleneck restricting the industrialization and high-efficiency production of tissue culture. Therefore, a targeted technical solution is urgently needed. SUMMARY
[0004] The present application aims to solve the above-mentioned problems in the prior art and provides a method for removing associated bacteria in tissue culture subculture and proliferation of Clerodendrum trichotomum and application thereof.
[0005] To solve the above technical problems, the technical solution adopted by the present application is as follows:
[0006] A method for removing associated bacteria in tissue culture subculture and proliferation of Clerodendrum trichotomum, taking stem segments with buds of Clerodendrum trichotomum plants as explants for pretreatment and sterilization; sequentially performing callus induction culture, callus redifferentiation culture, and proliferation culture; during the proliferation culture, using a sterile gentamicin sulfate solution with a concentration of 200-1100 mg / L to treat the associated bacteria, which are red spore bacteria.
[0007] Preferably, the concentration of the sterile gentamicin sulfate solution is 800 mg / L.
[0008] Preferably, when the proliferation culture process is contaminated by accompanying bacteria, the infected cluster shoots are transferred to new proliferation culture medium, 12-15 mL sterile gentamicin sulfate solution is added to the surface of 60 mL medium in each bottle, the culture bottle is shaken gently to make the sterile gentamicin sulfate solution evenly cover the surface of the medium and the gap at the base of the cluster shoots, and the treated cluster shoots are transferred to new proliferation culture medium for another 7 days of culture.
[0009] Preferably, the pretreatment method is as follows: the explants are washed with running water, soaked in a laundry detergent solution for 15 minutes, and the surface is gently brushed with a soft brush to ensure complete cleaning; then the explants are washed with running water for 4 hours to remove residual detergent.
[0010] Preferably, the sterilization method is as follows: the explants are soaked in 75% alcohol for 30 seconds, washed with sterile water for 2-3 times, then disinfected with 0.5% sodium hypochlorite solution for 8-10 minutes, and finally washed with sterile water for 4-5 times and dried with sterile filter paper.
[0011] Preferably, the induction culture is performed for 14 days to form callus, the callus is cultured for 7 days for redifferentiation to form adventitious shoots, and the adventitious shoots are cultured for 14 days for proliferation to form cluster shoots.
[0012] Preferably, the formula of the callus induction culture medium is: MS + 6-BA 0.75 mg·L -1 + NAA 0.25 mg·L -1 + sucrose 25.0 g·L -1 + agar 8 g·L -1 ; pH 5.8-6.2;
[0013] The callus induction culture conditions are: temperature 25±2℃, humidity 75%, light intensity 1800 lux, light treatment time 8h / d, and dark treatment time 16h / d.
[0014] Further preferably, the formula of the callus redifferentiation culture medium is: MS + 6-BA 0.4 mg·L -1 + IBA 0.5 mg·L -1 + sucrose 25.0 g·L -1 + agar 7.5 g·L -1 ; pH 5.8-6.2;
[0015] The callus redifferentiation culture conditions are: temperature 25±2℃, humidity 75%, light intensity 1800 lux, and light and dark treatment time each 12h / d.
[0016] Further preferably, the formula of the proliferation culture medium is: MS + 6-BA 0.2 mg·L -1 + IBA 0.25 mg·L-1 + sucrose 25.0 g·L -1 + agar 7.5 g·L -1 ; pH 5.8~6.2;
[0017] The proliferation culture conditions are: temperature 25±2℃, humidity 75%, light intensity 1800 lux, and light and dark treatment time each being 12h / d.
[0018] The application also provides application of the above method in seedling raising of Peristrophe roxburghiana.
[0019] The application has the following beneficial effects:
[0020] 1. The application effectively solves the industry problem of "bacterial contamination once occurring, tissue culture seedlings being completely discarded, and failure of sterile system establishment" in traditional tissue culture of Peristrophe roxburghiana. The problem of a large increase in propagation cycle, a large loss of explant materials, and an increase in labor and time cost caused by "contamination leading to abandonment and rework" in the traditional method is avoided, comprehensive cost is saved, and the success rate of tissue culture seedling raising is improved.
[0021] 2. The application can effectively remove red bacterial stains in 7 days without affecting the normal growth of tissue culture seedlings and reducing the proliferation efficiency, and the subsequent transfer does not relapse, the removal rate of associated bacteria reaches 100%, the effect is significant and stable; the sterile seedlings of Peristrophe roxburghiana can grow normally, the plants are healthy, no yellowing and vitrification phenomenon occurs, the height of the cluster buds reaches 4-5cm, and the proliferation coefficient reaches 16 (2 4 ), which realizes the simultaneous performance of "complete removal of associated bacterial contamination" and "normal growth and proliferation of tissue culture seedlings".
[0022] 3. The sterile gentamicin sulfate used for removing associated bacteria in the application has low cost, small dosage, is green and safe, does not affect the normal propagation of tissue culture seedlings, and reduces the cost of technical application. DETAILED DESCRIPTION
[0023] Figure 1 To be an effective dedifferentiation of the callus in example 1;
[0024] Figure 2 To be rediation of the callus to produce adventitious buds in example 1;
[0025] Figure 3 To be red associated bacterial contamination (plant yellowing, vitrification, wilting and death) in the proliferation culture process in example 1;
[0026] Figure 4 To be the normal growth state of the cluster buds after removal of the red associated bacteria in example 1;
[0027] Figure 5Axillary shoots without associated bacteria for rooting culture in Example 1;
[0028] Figure 6 Complete plants obtained after rooting culture in Example 1;
[0029] Figure 7 Red associated bacteria removal effect in Examples 2-4;
[0030] Figure 8 Red associated bacteria removal effect in Comparative Examples 1-3. DETAILED DESCRIPTION
[0031] The application will be further described below in conjunction with the accompanying drawings and specific examples.
[0032] Example 1
[0033] The method for removing associated bacteria in the subculture and proliferation culture of Clidemia hainanensis by tissue culture comprises the following steps:
[0034] 1. Selection and disinfection of explants
[0035] Selection of explants: the stem segments with axillary buds or terminal buds of Clidemia hainanensis plants are taken as the explants.
[0036] Pre-treatment: the explants are washed with running water, soaked in a laundry detergent solution for 15 minutes, and gently brushed with a soft brush to ensure complete cleaning. Then, the explants are washed with running water for 4 hours to remove the residual detergent.
[0037] Sterile treatment: the explants are transferred to a clean bench, soaked in 75% alcohol for 30 seconds, and then washed with sterile water for 2-3 times. Then, the explants are disinfected with a 0.5% sodium hypochlorite solution for 8-10 minutes, and then washed with sterile water for 4-5 times. Finally, the explants are placed on sterile filter paper and dried.
[0038] 2. Callus induction (dedifferentiation)
[0039] Under sterile conditions, the explants subjected to sterile treatment are transferred to callus induction medium for culture.
[0040] The optimal medium for callus induction is MS+6-BA 0.75 mg·L -1 +NAA 0.25 mg·L -1 + sucrose 25.0 g·L -1 + agar 8 g·L -1 ; pH 5.8;
[0041] Culturing conditions: temperature 25±2℃, humidity 75%, light intensity 1800 lux, light treatment duration 8h / d, and dark treatment duration 16h / d.
[0042] The callus was green, full and compact (Fig. 2) and the effective callus induction rate was 100%. Figure 1
[0043] 3. Redifferentiation of callus
[0044] The callus was transferred to the redifferentiation medium for culture.
[0045] Medium formula: MS + 6-BA 0.4 mg·L -1 + IBA 0.5 mg·L -1 + sucrose 25.0 g·L -1 + agar 7.5 g·L -1 , pH 6.2;
[0046] Culture conditions: temperature 25 ± 2℃, humidity 75%, light intensity 1800 lux, light and dark treatment time 12 h / d.
[0047] After 7 days of culture, the callus redifferentiated into adventitious buds and the redifferentiation rate was 100%. The buds germinated and grew to 0.5 cm, the leaves unfolded 2-3 pieces, and formed adventitious buds (Fig. 3) with good growth potential, laying a foundation for subsequent proliferation. Figure 2
[0048] 4. Proliferation culture (induction of clustered buds)
[0049] (1) The adventitious buds were cut from the callus for proliferation culture.
[0050] Proliferation medium formula: MS + 6-BA 0.2 mg·L -1 + IBA 0.25 mg·L -1 + sucrose 25.0 g·L -1 + agar 7.5 g·L -1 , pH 6.2;
[0051] Culture conditions: temperature 25 ± 2℃, humidity 75%, light intensity 1800 lux, light and dark treatment time 12 h / d.
[0052] (2) When the associated bacteria infected, the treatment method was as follows: after 3 days of proliferation culture, the surface of the culture medium was observed to be pus-like, mucous red bacterial spots (area 5 × 5 cm 2 ), the plants appeared yellowing and wilting, and even wilted to death (Fig. 4). Figure 3 The presence of red basal bacteria indicates contamination. In this case, the infected bud clusters were transferred to a new proliferation medium. A sterile gentamicin sulfate solution (800 mg / L) was added dropwise to the surface of the medium (under sterile conditions, gentamicin injection stock solution was taken with a sterile pipette, injected into a sterile container, and diluted with sterile water to a final volume, then shaken well to prepare an 800 mg / L sterile gentamicin sulfate solution) at a concentration of 12 mL per 60 mL of medium. The culture flask was gently shaken to ensure the sterile gentamicin sulfate solution evenly covered the surface of the medium and the gaps at the base of the bud clusters. The culture was continued under the original conditions for 7 days. The red bacterial patches disappeared, and the medium returned to its original state (see attached). Figure 4 The treated shoot clusters were transferred to a new proliferation medium for further culture. No red associated bacteria were observed within 7 days, indicating a 100% clearance rate. Tissue culture seedlings treated with gentamicin sulfate grew vigorously, without yellowing or vitrification. The height of the tissue culture shoot clusters was 4–5 cm, and the proliferation coefficient was as high as 16(2). 4 ).
[0053] (3) Figure 5 As shown, adventitious buds taller than 4cm were selected and transferred to conventional rooting medium for rooting culture. After 14 days of culture, the plant height reached 5cm-6cm, with 3-5 roots forming a root system 2-3cm in length and 0.2-0.3mm in diameter, achieving a 100% rooting rate and forming a complete plant (see attached image). Figure 6 This fully demonstrates that the treatment of red associated bacteria in this embodiment does not affect the normal growth of clustered buds at all.
[0054] Example 2
[0055] The method for establishing the rapid propagation system of *Changshania hainanensis* tissue culture in this embodiment is the same as that in Example 1. The difference is that when red associated bacteria infection occurs during the propagation culture, this embodiment uses a sterile gentamicin sulfate solution with a concentration of 200 mg / L for treatment.
[0056] The red fungal plaque area was observed to have decreased by 70%. After treatment, the clustered shoots were transferred to a new culture medium, and no red fungal plaque recurrence was observed within 3 days. (See attached image) Figure 7 As shown, the leaves of the tissue culture seedlings treated with sterile gentamicin sulfate were dark green, without yellowing or vitrification. The height of the tissue culture bud clusters was 2.5 cm, and the proliferation coefficient was 2. In this example, the treatment with red symbiotic bacteria affected the normal growth of the bud clusters.
[0057] Example 3
[0058] The method for establishing the rapid propagation system of *Changshania hainanensis* tissue culture in this embodiment is the same as that in Example 1. The difference is that when red associated bacteria infection occurs during the propagation culture, this embodiment uses a sterile gentamicin sulfate solution with a concentration of 500 mg / L for treatment.
[0059] The red bacterial plaque area was observed to shrink by 80%, and the treated cluster buds were transferred to new culture medium for continued culture, and the red bacterial plaque reappeared within 3 days. As shown in the accompanying Figure 7 The leaves of the tissue culture seedlings treated with sterile gentamicin sulfate were dark green, and the plants did not have yellowing or vitrification, the cluster buds had a height of 3.5 cm, and the proliferation coefficient was 4. In this example, the treatment of the red companion bacteria affected the normal growth of the cluster buds.
[0060] Example 4
[0061] In this example, the method for establishing the tissue culture and rapid propagation proliferation system of Radix Bupleuri was the same as in Example 1, except that when red companion bacteria infection occurred during the proliferation culture, sterile gentamicin sulfate solution with a concentration of 1100 mg / L was used for treatment.
[0062] The red bacterial plaque was observed to disappear, and the treated cluster buds were transferred to new culture medium for continued culture, and no red bacterial plaque reappeared within 3 days. As shown in the accompanying Figure 7 The leaves of the tissue culture seedlings treated with sterile gentamicin sulfate were yellow, and the edges of the leaves were observed to curl and wilt, the cluster buds had a height of 4 cm, and the proliferation coefficient was 8. In this example, the treatment of the red companion bacteria affected the normal growth of the cluster buds.
[0063] Comparative Example 1
[0064] In this comparative example, the method for establishing the tissue culture and rapid propagation high proliferation system of Radix Bupleuri was the same as in Example 1, except that when companion bacteria contamination occurred during step 4, the proliferation culture (cluster bud induction), instead of using the “(2) treatment method when companion bacteria infection occurs” in Example 1, the following three methods were used for treatment.
[0065] Method 1, the bacterial cluster buds were transferred to new sterile proliferation culture medium for culture for 14 days.
[0066] Method 2, the bacterial cluster buds were cut into small pieces and re-sterilized, specifically soaked in 75% alcohol for 30 seconds, washed with sterile water for 2-3 times, sterilized with 0.5% sodium hypochlorite solution for 8-10 minutes, washed with sterile water for 4-5 times, and then dried with sterile filter paper; after treatment, they were transferred to new sterile proliferation culture medium for culture for 14 days.
[0067] Method 3, the bacteria-carrying cluster shoots are transferred to new propagation medium, and 12 mL of sterile streptomycin sulfate solution with a concentration of 800 mg / L is added dropwise to the surface of the medium (under sterile conditions, the streptomycin sulfate injection stock solution is taken with a sterile pipette, injected into a sterile container, diluted with sterile water to a concentration of 800 mg / L of sterile streptomycin sulfate solution), and the culture bottle is gently shaken to evenly cover the surface of the culture medium and the gap at the base of the cluster shoots with the sterile streptomycin sulfate solution. Continue to culture under the original culture conditions for 7 days, and then transfer to new propagation medium for another 7 days of culture.
[0068] The culture results after the treatments of methods 1-3 are shown in Table 1 and the attached Figure 8
[0069] Table 1 Culture results after treatment by methods 1-3
[0070]
[0071] The specification and drawings of the present application are considered to be illustrative rather than restrictive, and based on the disclosed technical content, those skilled in the art can make some substitutions and modifications to some technical features without creative labor, which are all within the protection scope of the present application.
Claims
1. A method for eliminating associated bacteria in the tissue culture and subculture propagation of *Changshania hainanensis*, characterized in that, Stem segments with buds from *Changshania hainanensis* plants were used as explants for pretreatment and aseptic treatment; then, callus induction culture, callus redifferentiation culture, and proliferation culture were carried out sequentially; during the proliferation culture, a sterile gentamicin sulfate solution with a concentration of 200-1100 mg / L was used to treat the associated bacteria, which were red spore-forming bacteria.
2. The method according to claim 1, characterized in that, The concentration of the sterile gentamicin sulfate solution is 800 mg / L.
3. The method according to claim 1, characterized in that, When contamination by associated bacteria occurs during the proliferation culture process, the infected bud clusters are transferred to a new proliferation culture medium. 12-15 mL of sterile gentamicin sulfate solution is added to the surface of each 60 mL culture medium bottle. The culture bottle is gently shaken to ensure that the sterile gentamicin sulfate solution evenly covers the surface of the culture medium and the gaps at the base of the bud clusters. The culture is continued for 7 days. Then, the treated bud clusters are transferred to a new proliferation culture medium and cultured for another 7 days.
4. The method according to claim 1, characterized in that, The pretreatment method is as follows: rinse the explant with running water, soak it in a laundry detergent solution for 15 minutes, and gently scrub the surface with a soft brush to ensure thorough cleaning; then rinse with running water for 4 hours to remove residual detergent.
5. The method according to claim 1, characterized in that, The aseptic treatment method is as follows: soak in 75% alcohol for 30 seconds, then rinse with sterile water 2-3 times, then disinfect with 0.5% sodium hypochlorite solution for 8-10 minutes, finally rinse with sterile water 4-5 times, and then blot dry with sterile filter paper.
6. The method according to claim 1, characterized in that, Induction culture for 14 days to form callus tissue; callus tissue redifferentiation culture for 7 days to form adventitious buds; finally, proliferation culture for 14 days to form clustered buds.
7. The method according to claim 1, characterized in that, The formula for callus induction medium is: MS + 6-BA 0.75 mg·L⁻¹ -1 +NAA 0.25 mg·L -1 +25.0g·L of sucrose -1 +8g / L agar -1 pH 5.8–6.2; The conditions for callus induction culture were: temperature 25±2℃, humidity 75%, light intensity 1800 lux, light treatment time 8h / d, and dark treatment time 16h / d.
8. The method according to claim 1, characterized in that, The formula for callus redifferentiation medium is: MS + 6-BA 0.4 mg·L⁻¹ -1 +IBA 0.5 mg·L -1 +25.0g·L of sucrose -1 +7.5g / L agar -1 pH 5.8–6.2; The conditions for callus redifferentiation culture were: temperature 25±2℃, humidity 75%, light intensity 1800 lux, and light and dark treatment durations of 12 h / d each.
9. The method according to claim 1, characterized in that, The culture medium formula is: MS + 6-BA 0.2 mg·L⁻¹ -1 +IBA 0.25 mg·L -1 +25.0g·L of sucrose -1 +7.5g / L agar -1 pH 5.8–6.2; The propagation culture conditions were: temperature 25±2℃, humidity 75%, light intensity 1800 lux, and light and dark treatment durations of 12 h / d each.
10. The application of the method according to any one of claims 1-9 in tissue culture seedlings of *Changshania hainanensis*.