Method for establishing banana explant sterile system

Abstract

The invention belongs to the technical field of agricultural biology, and discloses a method for establishing a banana explant sterile system, which comprises the following steps: taking available banana suction buds, wiping and disinfecting the surfaces with 75% alcohol, stripping banana stem tips, inoculating the banana stem tips into a screening culture medium, and carrying out dark culture; when macroscopic pyosis appears on the banana stem tips in the screening culture medium, the banana stem tips are inserted into a sterilization culture medium with bactericide liquid drops added on the surface for dark culture until the banana stem tips start to have differentiation characteristics and no pyosis appears, and the sterile explants are obtained. According to the method disclosed by the invention, the explants with pyosis are subjected to cover type sterilization and induction simultaneously after preliminary screening, so that the explants can be effectively sterilized while the differentiation of the explants is not influenced, the sterile bodies are obtained, the death probability of the explants caused by bacterial pollution is reduced to a greater extent, the success rate of subsequent conventional tissue culture is improved, and the method is suitable for large-scale popularization and application. The cultivation efficiency is improved, cost is saved, operation is easy, and popularization is facilitated.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and particularly relates to a method for establishing a banana explant aseptic system. Background Art

[0002] Plant tissue culture technology has broad market prospects in seedling production, virus removal, and germplasm improvement. By screening excellent strains and obtaining explants, tissue culture technology is used to generate complete plants. It has the characteristics of low raw material demand, no restrictions on time and location, high reproduction coefficient, and the ability to maintain the excellent traits of the mother plant. It can efficiently breed healthy seedlings of excellent strains on a large scale and is widely used in plant seedling breeding.

[0003] In the plant tissue culture system, the establishment of a sterile system is one of the key factors in plant tissue culture. Among them, ensuring the sterility of explants is the basis and prerequisite for successful tissue culture. Wild plant resources are an important source of materials for the cultivation of new crop varieties. Since wild resources, especially tropical wild plant resources, have been in the wild and hot natural environment for a long time, there are many types of pests and diseases and large numbers. It is difficult to ensure that each explant can be completely disinfected and sterile using traditional disinfection technology, resulting in the failure of subsequent tissue culture of incompletely disinfected explants, affecting the survival rate of introduced and collected resources.

[0004] Bananas originated in southern Asia and are native to Southeast Asia, with the center probably being the Malay Peninsula and the islands of Indonesia. Bananas on the islands in the distribution area have evolved into a variety of types, with more specific performance in terms of morphology, resistance, and nutritional components. They are an important target for the introduction of banana resources and have a more important significance for the cultivation of banana varieties and the development of the industry in my country.

[0005] Most bananas are triploid, with a small number of diploid and tetraploid. Some diploids have seeds, while the rest do not. Therefore, banana resource collection mainly involves digging and sucking buds. Banana resources introduced from abroad or collected from places far from the laboratory in China have become moldy and rotten after long-term transportation. The material collection (banana stem tip) and disinfection steps are cumbersome, and incomplete disinfection and repeated infection are prone to occur during the operation, resulting in the problem of bacterial infection in the banana stem tip (explant), which not only affects the subsequent tissue culture process, but also consumes more banana materials, resulting in increased costs, which is not conducive to the development and utilization of excellent varieties and industrial development. Summary of the invention

[0006] Therefore, the object of the present invention is to provide a method for establishing a sterile system for banana explants, which adopts a mode of first screening and then covering sterilization for sterilization treatment, so as to ensure that banana stem tips are cultured in vitro to obtain sterile bodies, so as to improve the success rate of cultivation, improve the utilization rate of introduced or collected materials, improve cultivation efficiency, and save cultivation costs.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for establishing a banana explant sterile system comprises the following steps:

[0009] (1) First treatment: Take available banana suckers, disinfect the surface with a disinfectant, dry them on a clean bench, peel off a 0.5-1.0 mm stem tip as an explant with the help of a dissecting microscope, and inoculate it into a screening medium for screening culture, wherein the screening medium is an induction differentiation medium for banana stem tips;

[0010] The banana stem tips were infected with bacteria and sterile screened using an induced differentiation medium. The culture medium not only facilitated the expansion and differentiation of the banana stem tips, providing convenience for subsequent operations, but also facilitated the rapid reproduction of bacteria. Bacterial pus appeared on the infected explants, and the infected explants could be screened out as soon as possible.

[0011] (2) Second treatment: when visible bacterial pus appears on the stem tip in the screening culture medium, droplets of bactericide are added to the surface of the bactericidal culture medium, the stem tip with bacterial pus is inserted into the bactericide droplets so that the stem tip is covered with the bactericide, and bactericidal culture is performed; if the bacterial pus does not disappear after the bactericidal culture until the stem tip shows obvious differentiation, the banana stem tip is moved to a new bactericidal culture medium and the bactericidal culture process is repeated; the bactericidal culture medium is an induction differentiation culture medium for banana stem tips;

[0012] (3) The sterile (the pus disappears) and swollen banana stem tips are inoculated into a new differentiation induction medium for normal tissue culture.

[0013] In an optional embodiment, the disinfectant is 75% alcohol.

[0014] In an optional embodiment, the differentiation induction medium is MS+6-BA 2.0-3.0 mg / L+NAA 0.1-0.5 mg / L+sucrose 25-30 g / L+carrageenan 6-8 g / L, with a pH of 5.8.

[0015] In an optional embodiment, the bactericide is PPM, and the dosage is 10 to 20 μl.

[0016] In an optional embodiment, the screening culture or sterilization culture conditions are: temperature of 25-30° C., humidity of 30-50%, and dark culture.

[0017] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0018] (1) The present invention performs covering sterilization and induction on banana stem tips with bacterial pus after preliminary screening, and can effectively sterilize the explants without affecting the differentiation of the explants, thereby obtaining sterile bodies, and reducing the probability of explant contamination to a greater extent, laying a foundation for the establishment of a subsequent tissue culture sterile system, improving the success rate of tissue culture of introduced or collected resources, and reducing the cost required for repeated introduction or collection; after directly dripping a bactericide on a solid culture medium, the stem tips contaminated with bacteria are transferred, which is simple to operate, has good effects, and is conducive to promotion.

[0019] (2) The aseptic system obtained by the method of the present invention can be used for normal induction differentiation, proliferation, seedling strengthening and rooting culture to obtain healthy seedlings of banana resources. This technology can effectively solve the problem that a large number of resources in the introduction and collection of banana sucker resources are in poor condition and cannot be kept alive, and plays an important technical support role in the introduction of excellent banana germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The following are comparison diagrams of the materials used in the embodiments of the present invention before and after treatment. A: Cutting of sucker buds, 5 cm on the left and 2 cm on the right; B: Surface state of the material after 15 days of treatment at room temperature and high temperature; C: State of the material after 15 days of treatment at room temperature and high temperature after the epidermis is cut off.

[0021] Figure 2 This is the first treatment effect of the present invention. A: Microscopic photo of the peeled stem tip; B: The stem tip is cultured in a φ3.5 cm disposable plastic culture dish.

[0022] Figure 3 This is the second treatment picture of the present invention. A: stem tip with bacterial pus; B: stem tip that has been freed from bacteria and has begun to grow and differentiate.

[0023] Figure 4 Adventitious buds are differentiated after the stem apex is sterilized. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are further described in detail below in conjunction with the examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0025] Experimental materials: Simulate the processing method of imported resources from abroad, cut the newly dug banana suckers into pieces, such as Figure 1As shown in A in the figure (5 cm on the left and 2 cm on the right). According to the transportation time and temperature of the imported resources, the sucker bud decay process was simulated: the sucker bud cut pieces were wrapped with absorbent paper, sealed in plastic bags, and placed in a dark place at room temperature (25°C) and high temperature (32°C) for 15 days. After removing and opening the absorbent paper, the sucker bud cut pieces were observed. Figure 1 As shown in B (the upper row is treated with high temperature, the lower row is treated with normal temperature), most of the materials have turned brown and soft rot. After the treated materials were cut off from the skin, the materials that were all black and soft rot were dead and could not be used; the materials that were partially soft rotten but still retained white at the stem tip and the materials that had not changed color were used as the materials for the following experiments, such as Figure 1 As shown in C.

[0026] Example 1

[0027] A method for establishing a sterile system for banana sucker bud stem tips comprises the following steps:

[0028] (1) First treatment: Take available suckers, wipe the surface with 75% alcohol for disinfection, dry it on a clean bench, and use a dissecting microscope to carefully peel off the stem tip of about 1 mm (such as Figure 2 The culture medium was inoculated into a disposable sterile plastic culture dish (preferably 3.5 cm in diameter) loaded with screening medium (MS + 6-BA 2.0 mg / L + NAA 0.5 mg / L + sucrose 25 g / L + carrageenan 6 g / L, pH 5.8). One stem tip was inoculated per dish, with the base of the stem tip facing downward (as shown in A). Figure 2 The culture was carried out in the dark at 25°C and 45% humidity. The infection of the stem tip was observed at any time.

[0029] (2) Second treatment: Transfer the sterile stem tip to the differentiation induction medium for routine culture. Screen the stem tip with bacterial pus (infected with bacteria) and set aside. Add 10 μl of PPM droplets on the surface of the sterilization medium (the composition is the same as the screening medium), and carefully insert the stem tip with bacterial pus into the PPM droplet (so that the PPM covers the stem tip). Figure 3 As shown in A, the material was cultured in the dark at a temperature of 25°C and a humidity of 45%. When no pus appeared in the material and it had begun to swell and differentiate, it indicated that a sterile body had been obtained and a sterile system had been established.

[0030] (3) Repeat treatment: If the second treatment cannot completely remove bacteria and visible pus still exists on the stem tip, move the stem tip to a new sterilization culture medium and repeat the sterilization culture process until the pus disappears.

[0031] (4) Subsequent culture: After the sterile system is established, culture in the dark for 45 days. When the stem tip is obviously enlarged and differentiated (such as Figure 3The stem tip was cut into two pieces and inoculated into the induction differentiation medium (with the same composition as the screening medium) without the fungicide PPM. The temperature was 25°C, the humidity was 45%, and the light intensity was 25 μmol·m -2 ·s -1 , cultured under a light intensity of 10 h / d, adventitious buds can be differentiated, such as Figure 4 shown.

[0032] Example 2

[0033] A method for establishing a sterile system for banana sucker bud stem tips comprises the following steps:

[0034] (1) First treatment: Take available suckers, wipe the surface with 75% alcohol for disinfection, dry on a clean bench, carefully peel off a 1 mm long shoot tip with the help of a dissecting microscope, and inoculate it on a disposable sterile plastic culture dish (preferably with a diameter of 3.5 cm) loaded with screening medium (MS+6-BA3.0 mg / L+NAA0.2 mg / L+sucrose 28 g / L+carrageenan 8 g / L, pH 5.8), inoculate one shoot tip per dish, with the base of the shoot tip facing downward, and incubate in the dark at a temperature of 27°C and a humidity of 40%. Observe the infection of the shoot tip at any time.

[0035] (2) Second treatment: Transfer the sterile stem tips to the induction differentiation medium for routine culture. Screen the stem tips with bacterial pus (already infected) and set aside. Add 15 μl of PPM droplets on the surface of the sterilization medium (the ingredients are the same as the screening medium), carefully insert the stem tip with bacterial pus into the PPM droplet (so that the PPM covers the stem tip), and culture in the dark at a temperature of 27°C and a humidity of 40%. When there is no bacterial pus in the material and it has begun to swell and differentiate, it indicates that a sterile body has been obtained and a sterile system has been established.

[0036] (3) Repeat treatment: If the second treatment cannot completely remove bacteria and visible pus still exists on the stem tip, move the stem tip to a new sterilization culture medium and repeat the sterilization culture process until the pus disappears.

[0037] (4) Subsequent culture: After the aseptic system was established, the cells were cultured in the dark for 45 days. When the stem tip was obviously swollen and differentiated, the stem tip was cut into two pieces and inoculated into the induction differentiation medium (with the same composition as the screening medium) without the fungicide PPM. The culture medium was kept at 27°C, 40% humidity, and 25 μmol·m -2 ·s -1 , cultured under a light intensity of 10h / d, adventitious buds can be differentiated.

[0038] Example 3

[0039] A method for establishing a sterile system for banana sucker bud stem tips comprises the following steps:

[0040] (1) First treatment: Take available suckers, wipe the surface with 75% alcohol for disinfection, dry on a clean bench, carefully peel off a 1mm stem tip with the help of a dissecting microscope, and inoculate it on a disposable sterile plastic culture dish (preferably with a diameter of 3.5 cm) loaded with screening medium (MS+6-BA2.6 mg / L+NAA0.5 mg / L+sucrose 30 g / L+carrageenan 7 g / L, pH 5.8). Inoculate one stem tip per dish, with the base of the stem tip facing downward, and incubate in the dark at a temperature of 30°C and a humidity of 50%. Observe the infection of the stem tip at any time.

[0041] (2) Second treatment: Transfer the sterile stem tips to the induction differentiation medium for routine culture. Screen the stem tips with bacterial pus (already infected) and set aside. Add 20 μl of PPM droplets on the surface of the sterilization medium (with the same ingredients as the screening medium), carefully insert the stem tip with bacterial pus into the PPM droplet (so that the PPM covers the stem tip), and culture in the dark at a temperature of 30°C and a humidity of 50%. When there is no bacterial pus in the material and it has begun to swell and differentiate, it indicates that a sterile body has been obtained and a sterile system has been established.

[0042] (3) Repeat treatment: If the second treatment cannot completely remove bacteria and visible pus still exists on the stem tip, move the stem tip to a new sterilization culture medium and repeat the sterilization culture process until the pus disappears.

[0043] (4) Subsequent culture: After the aseptic system was established, the cells were cultured in the dark for 45 days. When the stem tip was obviously swollen and differentiated, the stem tip was cut into two pieces and inoculated into the induction differentiation medium (with the same composition as the screening medium) without the fungicide PPM. The culture medium was kept at 30°C, 50% humidity, and 25 μmol·m -2 ·s -1 , cultured under a light intensity of 10h / d, adventitious buds can be differentiated.

[0044] Comparative Example 1

[0045] A banana tissue culture method, compared with Example 1, the steps of subsequent tissue culture are the same, the difference is that no second treatment is performed in the early stage, the sucker buds are wiped with alcohol, the stem tips of about 1 mm are peeled off, and are directly inoculated into an induction differentiation medium for induction differentiation culture.

[0046] Experimental effect identification

[0047] (1) Experimental materials: Prepare 64 tubers with rotten suckers that are still white at the tip of the rhizome.

[0048] (2) All the stem tips were randomly divided into 4 groups, namely, Group 1, Group 2, Group 3, and Group 4, with 16 stem tips in each group, wherein Group 1, Group 2, Group 3, and Group 4 correspond to Example 1, Example 2, Example 3, Example 4, and Comparative Example 1, respectively. The number of stem tip contaminations was counted after the first treatment. The results are shown in Table 1.

[0049] Table 1 Bacterial contamination of banana explants

[0050] project Number of stem tip contamination after the first treatment (pcs) Group 1 (Example 1) 8 Group 2 (Example 2) 8 Group 3 (Example 3) 6 Group 4 (Comparative Example 1) 7

[0051] (3) Grouping: The contaminated stem tips of Group 1, Group 2, and Group 3 were treated for the second time, and all the stem tips of Group 4 were not treated. 16 culture dishes were prepared, each of which was added with differentiation induction medium. The 16 culture dishes were divided into 4 groups, each with 4 culture dishes, and the culture dishes in each group were numbered 1, 2, 3, and 4 respectively.

[0052] (4) Inoculation: Four groups of culture dishes were inoculated with the stem tips of the first group (Example 1), the second group (Example 2), the third group (Example 3) and the comparative example 1 that had not been treated for the second time, respectively, at a temperature of 25° C., a humidity of 45%, and a light intensity of 25 μmol·m -2 ·s -1 , cultured under a light intensity of 10 h / d, with 4 stem tips inoculated in each culture dish.

[0053] (5) Observation: During the culture process, observe the condition of the shoot tips in each culture dish and record the number of shoot tips infected with bacteria.

[0054] (6) Statistics: The recorded data are statistically analyzed and the results are shown in Table 2.

[0055] Table 2 Bacterial infection of stem apex

[0056]

[0057] *Note: The number of infected shoot tips refers to the number of shoot tips infected with bacteria that have been treated for the first time but not for the second time. The number of previously infected shoot tips refers to the number of shoot tips infected with bacteria that have been treated for the first time and then for the second time.

[0058] From the above results, it can be seen that the average contamination rate of the banana suckers before sterilization treatment is about 50%, which is related to the rot of the suckers after the simulated long-distance transportation. When the stem tips after sterilization treatment in the embodiment were inoculated into the differentiation induction medium for culture, all stem tips were no longer infected; when the untreated stem tips of comparative example 1 were inoculated into the differentiation induction medium for culture, the stem tips were infected with bacteria. As the culture time was prolonged, the probability of contamination increased. When the material was cultured for 45 days, the infection rate of the material without sterilization treatment was about 70% on average.

[0059] In summary, in the process of banana sucker shoot tip tissue culture, the method of the present invention is used in time for aseptic treatment of the shoot tip contaminated with bacteria, which can effectively remove bacteria, significantly improve the probability of successful cultivation, reduce the probability of death of introduced or collected banana resources during the cultivation process, save the cost of repeated introduction or collection, and is easy to operate and conducive to promotion. It can effectively solve the problem that a large number of resources in the introduction and collection of banana resources are in poor state and cannot be preserved after introduction (collection), and plays an important technical support role in the introduction of excellent banana germplasm resources.

[0060] Although the present invention has been described using the above preferred embodiments, it is not intended to limit the scope of protection of the present invention. Any person skilled in the art who makes various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention still fall within the scope of protection of the present invention.

Claims

1. A method for establishing a banana explant sterile system, characterized in that: The following steps are involved: (1) First treatment: Take available banana suckers, disinfect the surface with a disinfectant, dry them on a clean bench, peel off a 0.5-1.0 mm stem tip as an explant with the help of a dissecting microscope, and inoculate it into a screening medium for screening culture, wherein the screening medium is an induction differentiation medium for banana stem tips; (2) Second treatment: when visible bacterial pus appears on the stem tip in the screening culture medium, droplets of bactericide are added to the surface of the bactericidal culture medium, the stem tip with bacterial pus is inserted into the bactericide droplets so that the stem tip is covered with the bactericide, and bactericidal culture is performed; if the bacterial pus does not disappear after the bactericidal culture until the stem tip shows obvious differentiation, the banana stem tip is moved to a new bactericidal culture medium and the bactericidal culture process is repeated; the bactericidal culture medium is an induction differentiation culture medium for banana stem tips; (3) Inoculate the sterile, swollen banana stem tips into a new differentiation induction medium for normal tissue culture.

2. The method according to claim 1, wherein the banana explant aseptic system is set up, The disinfectant is 75% alcohol.

3. The method according to claim 1, wherein the banana explant aseptic system is set up, The induction differentiation medium is MS+6-BA 2.0-3.0 mg / L+NAA 0.1-0.5 mg / L+sucrose 25-30 g / L+carrageenan 6-8 g / L, with a pH of 5.

8.

4. The method according to claim 1, wherein the banana explant aseptic system is set up, The bactericide is PPM, and the dosage is 10-20 μl.

5. The method according to claim 1, wherein the banana explant aseptic system is set up, The conditions of the screening culture or sterilization culture are: temperature of 25-30° C., humidity of 30-50%, and dark culture.

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