CN113854155A - High-throughput breeding method of vanilla virus-free seedlings - Google Patents
High-throughput breeding method of vanilla virus-free seedlings Download PDFInfo
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Abstract
The invention provides a culture medium combination of vanilla detoxified seedlings, which is characterized by comprising a sterile seed germination culture medium, a cluster bud induction culture medium and a proliferation and rooting culture medium; the sterile seed germination medium comprises: 0.3-1.0 mg/L6-BA, 0.1-1.0 mg/L NAA and 0.1-0.5 mg/L GA3(ii) a The cluster bud induction medium comprises: 1.0-2.5 mg/L6-BA and 0.1-0.5 mg/LNAA; the proliferation and rooting culture medium comprises: 0.1-0.5 mg/L NAA and 0.05-0.1 mg/L GA3. The invention ensures that the vanillia tissue is free from virus infection by a specific disinfection means, provides sterile seeds as explant materials and a specific induction culture medium for regeneration of the vanillia virus-free seedlings, obtains cluster buds, improves the seedling breeding efficiency, obtains a large number of virus-free tissue culture seedlings with consistent states, and has important significance for high-throughput breeding of the vanillia virus-free seedlings in the future.
Description
Technical Field
The invention relates to the technical field of tissue culture, seedling detoxification and high-throughput seedling breeding, in particular to a high-throughput breeding method of vanilla detoxified seedlings.
Background
Vanilla (Vanilla planifolia Andrews), also known as Vanilla, Orchidaceae, with perennial vine climbing plants. Vanilla is known as the king of natural food spice, is widely used in food, medicine and cosmetic industries, and has wide application and high added value. With the pursuit of people for high-quality fragrant life, the demand of vanilla pods is gradually increased, the planting area and the yield of vanilla are limited worldwide, and the product is short of the international market. The plant tissue culture has very important significance for the fine breed rapid propagation, the virus-free seedling culture, the transgenic plant cultivation, the germplasm preservation and the like of the vanilla.
At present, the vanilla seedling breeding mainly adopts asexual breeding, wherein the asexual breeding is to select stem and vine of a healthy stock plant with excellent growth vigor to breed seedlings, the genetic background of the seedlings bred by the method is single, the excellent properties of the stock plant can be kept, and the consumed time is short. The sexual breeding method is adopted firstly, virus-free detoxified seeds can be obtained, and a new variety can be cultivated in a cross breeding mode, but the seeds rarely sprout under natural conditions. By adopting the tissue culture technology, the sterile germination of the detoxified seeds in a room can be ensured, the interference of environmental factors is avoided, the detoxified seedlings are obtained, the plant regeneration is carried out through cluster bud induction, the limitation on parent plant materials can be eliminated, the breeding efficiency of the vanilla detoxified seedlings is greatly improved, the purpose of breeding the seedlings in a high-flux manner is achieved, and the yield and the economic benefit of the vanilla seedlings are improved. Therefore, the tissue culture technology has important practical value for the detoxification of the vanilla seedlings and the seedling breeding.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method for breeding vanilla virus-free seedlings at high throughput, which can make nontoxic vanilla seeds germinate into cluster buds, and carry out proliferation and differentiation, and regenerate roots into complete plants, thereby realizing vanilla seedling virus-free, improving seedling breeding efficiency, and avoiding the problem of endophyte pollution in primary induction.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a culture medium combination of vanilla detoxified seedlings, which comprises a sterile seed germination culture medium, a cluster bud induction culture medium and a proliferation and rooting culture medium;
the sterile seed germination medium comprises: 0.3-1.0 mg/L6-BA, 0.1-1.0 mg/L NAA and 0.1-0.5 mg/L GA3；
The cluster bud induction medium comprises: 1.0-2.5 mg/L6-BA and 0.1-0.5 mg/L NAA;
the proliferation and rooting culture medium comprises: 0.1-0.5 mg/L NAA and 0.05-0.1 mg/L GA3。
Preferably, the sterile seed germination culture medium contains 0.5 mg/L6-BA, 0.1mg/L NAA and 0.1mg/L GA3The MS modified medium of (1).
Preferably, the cluster bud induction medium contains 1.0 mg/L6-BA, 0.1mg/L NAA and 0.1mg/L GA3The MS modified medium of (1).
Preferably, the proliferation and rooting culture medium contains 0.1mg/L NAA and 0.05mg/L GA3VM medium of (1).
The invention provides a high-throughput breeding method of vanilla virus-free seedlings, which is characterized by comprising the following steps:
A) sterilizing vanilla pods to obtain sterilized pods;
B) taking out seeds from the sterilized bean pods, inoculating the seeds to the sterile seed germination culture medium of any one of claims 1-4, and performing sterile germination on the seeds to obtain detoxified bud segments;
C) inoculating the detoxified bud segments onto the cluster bud induction medium of any one of claims 1 to 4 to induce cluster buds to obtain cluster buds;
D) after the multiple shoots are divided into plants, the plants are inoculated to the propagation and rooting culture medium of any one of claims 1 to 4 for propagation and rooting, and then complete plants are obtained.
Preferably, the sterilization method in the step a) is specifically as follows: sequentially soaking the surfaces of vanilla pods in a carbendazim solution, soaking in a chlorothalonil solution and cleaning with sterile water; and then transferring the bean pods onto a sterile workbench, disinfecting the surfaces of the bean pods by using 75% alcohol, washing the bean pods with sterile water, soaking the bean pods with mercuric chloride, and washing the bean pods with the sterile water to obtain the sterilized bean pods.
Preferably, the mass concentration of the carbendazim solution in the step A) is 0.05 wt%; soaking the carbendazim solution for 1-2 h; the mass concentration of the chlorothalonil solution is 0.1 wt%; the soaking time of the chlorothalonil solution is 0.5-1 h; the number of times of cleaning with sterile water is 4-5; the concentration of the mercuric chloride is 0.1 wt%.
Preferably, the time of sterile germination is 20-30 days; the induction time of the cluster buds is 35-45 days, and the proliferation and rooting time is 20-35 days.
Preferably, the temperature for inducing the cluster buds is 26-30 ℃; the cluster bud induction needs to be carried out under the condition of illumination, the illumination intensity is 1000-1200 lux, and the illumination period is 8 h/d.
Preferably, the temperature of the proliferation rooting is 26-30 ℃; the proliferation and rooting are carried out under the condition of illumination, the illumination intensity is 800-1000 lux, and the illumination period is 12 h/d.
Compared with the prior art, the invention provides a high-throughput breeding method of vanilla virus-free seedlings, which comprises the following steps: A) sterilizing vanilla pods to obtain sterilized pods; B) taking out seeds from the sterilized bean pods, inoculating the seeds to the sterile seed germination culture medium of any one of claims 1-4, and performing sterile germination on the seeds to obtain detoxified bud segments; C) inoculating the detoxified bud segments onto the cluster bud induction medium of any one of claims 1-4 to induce cluster buds to obtain cluster buds; D) after the multiple shoots are divided into plants, the plants are inoculated to the propagation and rooting culture medium of any one of claims 1 to 4 for propagation and rooting, and then complete plants are obtained.
The invention takes different explant detoxification methods and culture media as comparison methods, and the method is cultured under the same environmental conditions as the method, and the results show that only by combining 0.05% carbendazim solution, 0.1% chlorothalonil, 75% alcohol and 0.1% mercury bichloride as a disinfection and detoxification method, a detoxification stem segment can be obtained, and finally a detoxification cluster bud is obtained, only by using 75% alcohol as the disinfection and detoxification method, the seed infectious microbe can not germinate in the culture, and the detoxification stem segment or the detoxification cluster bud can not be obtained, so the method is obviously superior to each comparison method in the aspects of vanilla seed detoxification germination and cluster detoxification bud regeneration.
The method selects detoxified vanilla seeds as explants, and the sterile seed germination culture medium formula is as follows: adding 6-benzylamino adenine (6-BA), naphthylacetic acid (NAA) and Gibberellin (GA) into MS modified culture medium without organic elements3) (ii) a The formula of the cluster bud induction culture medium comprises: MS is taken as a basic culture medium, and 6-benzylamino adenine (6-BA) and naphthylacetic acid (NAA) are added, so that sterile seeds can have the differentiation rate of more than 10 virus-free cluster buds through culture, and the method has important significance for high-throughput breeding and genetic engineering research of the vanilla virus-free seedlings in the future.
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FIG. 1 is a high throughput breeding technique for the vanilla detoxified seedlings of the present invention.
Detailed Description
The invention provides a high-throughput breeding method of vanilla virus-free seedlings, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a culture medium combination of vanilla detoxified seedlings, which comprises a sterile seed germination culture medium, a cluster bud induction culture medium and a proliferation and rooting culture medium;
the sterile seed germination medium comprises: 0.3-1.0 mg/L6-BA, 0.1-1.0 mg/L NAA and 0.1-0.5 mg/L GA3；
The cluster bud induction medium comprises: 1.0-2.5 mg/L6-BA and 0.1-0.5 mg/L NAA.
The proliferation and rooting culture medium comprises: 0.1-0.5 mg/L NAA and 0.05-0.1 mg/L GA3。
The culture medium combination of vanilla detoxified seedling provided by the invention comprises a sterile seed germination culture medium; the sterile seed germination medium comprises: 0.3-1.0 mg/L6-BA, 0.1-1.0 mg/L NAA and 0.1-0.5 mg/L GA3(ii) a Preferably, the sterile seed germination culture medium contains 0.5 mg/L6-BA, 0.1mg/L NAA and 0.1mg/L GA3The MS modified medium of (1).
The culture medium combination of vanilla detoxified seedling provided by the invention comprises a cluster bud induction culture medium; the cluster bud induction medium comprises: 1.0-2.5 mg/L6-BA and 0.1-0.5 mg/L NAA; preferably, the cluster bud induction medium contains 1.0 mg/L6-BA, 0.1mg/L NAA and 0.1mg/L GA3The MS modified medium of (1).
The culture medium composition of vanilla detoxicated seedling includes proliferation rooting culture medium; the proliferation and rooting culture medium comprises: 0.1-0.5 mg/L NAA and 0.05-0.1 mg/L GA3(ii) a Preferably, the proliferation and rooting culture medium contains 0.1mg/L NAA and 0.05mg/L GA3VM medium of (1).
The invention provides a high-throughput breeding method of vanilla virus-free seedlings, which is characterized by comprising the following steps:
A) sterilizing vanilla pods to obtain sterilized pods;
B) taking out seeds from the sterilized bean pods, inoculating the seeds to the sterile seed germination culture medium of any one of claims 1-4, and performing sterile germination on the seeds to obtain detoxified bud segments;
C) inoculating the detoxified bud segments to the cluster bud induction medium of any one of claims 1 to 4 to induce cluster buds to obtain cluster buds;
D) after the multiple shoots are divided into plants, the plants are inoculated to the proliferation and rooting culture medium of any one of claims 1 to 4 for proliferation and rooting, and then complete plants are obtained.
The invention provides a high-throughput breeding method of vanilla virus-free seedlings.
The disinfection method of the invention specifically comprises the following steps: sequentially soaking the surfaces of vanilla pods in a carbendazim solution, soaking in a chlorothalonil solution and cleaning with sterile water; and then transferring the bean pods onto a sterile workbench, disinfecting the surfaces of the bean pods by using 75% alcohol, washing the bean pods with sterile water, soaking the bean pods with mercuric chloride, and washing the bean pods with the sterile water to obtain the sterilized bean pods.
Sequentially soaking the surfaces of vanilla pods in a carbendazim solution; the mass concentration of the carbendazim solution is 0.05 wt%; the preferred soaking time of the carbendazim solution is 1-2 h; more preferably 2 h; the number of times of cleaning with sterile water is 4-5;
soaking in chlorothalonil solution, and washing with sterile water; the mass concentration of the chlorothalonil solution is 0.1 wt%; the number of times of cleaning with sterile water is 4-5; the soaking time of the chlorothalonil solution is preferably 0.5-1 h; more preferably 0.5 h.
Then transferring the pod to a sterile workbench, disinfecting the surface of the pod with 75% alcohol, and washing with sterile water; the number of times of washing with sterile water is 3; soaking with mercuric chloride, and washing with sterile water to obtain sterilized bean pod.
The concentration of the mercuric chloride is 0.1 wt%; the soaking time is 0.5 h. Washing with sterile water for 4-5 times;
taking out the seeds of the sterilized bean pods, and inoculating the seeds to the sterile seed germination culture medium in any one of the technical schemes for sterile germination of the seeds to obtain the virus-free bud segments. The sterile seed germination medium has been clearly defined above, and is not described in detail herein.
The germination condition of the sterile seeds is 26-30 ℃; and (5) dark culture. The sterile germination time is 20-30 days;
inoculating the detoxified bud segment to the cluster bud induction culture medium of any one of the technical schemes for cluster bud induction to obtain cluster buds. The invention has been clearly defined for the said cluster bud induction medium, and is not described herein again.
The temperature for inducing the cluster buds is 26-30 ℃; dark illumination; the induction time of the cluster buds is 35-45 days.
Preferably, the cluster bud induction needs to be carried out under the condition of illumination, the illumination intensity is 1000-1200 lux, and the illumination period is 8 h/d.
After the cluster buds are divided into plants, the plant buds are inoculated to the propagation and rooting culture medium of any one of the technical schemes for propagation and rooting to obtain a complete plant.
The temperature of the proliferation and rooting is 26-30 ℃; the proliferation and rooting are carried out under the condition of illumination, the illumination intensity is 800-1000 lux, and the illumination period is 12 h/d. The time for proliferation and rooting is 20-35 d.
The invention provides a high-throughput breeding method of vanilla virus-free seedlings, which comprises the following steps: A) sterilizing vanilla pods to obtain sterilized pods; B) taking out seeds from the sterilized bean pods, inoculating the seeds to the sterile seed germination culture medium of any one of claims 1-4, and performing sterile germination on the seeds to obtain detoxified bud segments; C) inoculating the detoxified bud segments onto the cluster bud induction medium of any one of claims 1-4 to induce cluster buds to obtain cluster buds; D) after the multiple shoots are divided into plants, the plants are inoculated to the propagation and rooting culture medium of any one of claims 1 to 4 for propagation and rooting, and then complete plants are obtained.
The invention takes different explant detoxification methods and culture media as comparison methods, and the method is cultured under the same environmental conditions as the method, and the results show that only by combining 0.05% carbendazim solution, 0.1% chlorothalonil, 75% alcohol and 0.1% mercury bichloride as a disinfection and detoxification method, a detoxification stem segment can be obtained, and finally a detoxification cluster bud is obtained, only by using 75% alcohol as the disinfection and detoxification method, the seed infectious microbe can not germinate in the culture, and the detoxification stem segment or the detoxification cluster bud can not be obtained, so the method is obviously superior to each comparison method in the aspects of vanilla seed detoxification germination and cluster detoxification bud regeneration.
The method selects detoxified vanilla seeds as explants, and the sterile seed germination culture medium formula is as follows: adding 6-benzylamino adenine (6-BA), naphthylacetic acid (NAA) and Gibberellin (GA) into MS modified culture medium without organic elements3) (ii) a The formula of the cluster bud induction culture medium comprises: MS is taken as a basic culture medium, and 6-benzylamino adenine (6-BA) and naphthylacetic acid (NAA) are added, so that sterile seeds can have the differentiation rate of more than 10 virus-free cluster buds through culture, and the method has important significance for high-throughput breeding and genetic engineering research of the vanilla virus-free seedlings in the future.
The invention ensures that the vanillia tissue is free from virus infection by a specific disinfection means, provides sterile seeds as explant materials and a specific induction culture medium for regeneration of the vanillia virus-free seedlings, obtains cluster buds, improves the seedling breeding efficiency, obtains a large number of virus-free tissue culture seedlings with consistent states, and has important significance for high-throughput breeding of the vanillia virus-free seedlings in the future.
In order to further illustrate the present invention, the following will describe the method for breeding vanilla seedlings in high throughput in detail with reference to the examples.
The invention relates to the technical field of tissue culture and high-throughput seedling breeding of vanilla and discloses a rapid propagation technical method of virus-free seedlings by taking vanilla sterile seeds as explants. The method of the invention adopts the technology that the surface of the mature and healthy vanilla pod is disinfected by 0.05 percent of carbendazim solution, 0.1 percent of chlorothalonil, 75 percent of alcohol and 0.1 percent of mercury bichloride, and the black vanilla detoxified seeds in the pod are taken out as explants; inoculating the detoxified explant to a seed germination culture medium for germination for about 60d, and then inoculating the detoxified explant to a cluster bud proliferation induction culture medium for cluster bud proliferation induction for about 30d to obtain cluster buds so as to achieve the purpose of high-throughput breeding of detoxified seedlings. The method finally realizes that the sterile seed is used for sprouting the detoxified stem segment as the explant to carry out the propagation and differentiation of the cluster buds, and the sterile seed can have more than 10 detoxified cluster buds, thereby greatly improving the breeding efficiency of the seedlings. The in vitro regeneration method of the vanilla virus-free seedlings provides technical support for the industrialized high-throughput breeding and genetic engineering breeding research of the vanilla virus-free seedlings in the future.
Example 1: the method of the invention
Soaking the surface of mature and healthy vanilla pod in 0.05% carbendazim solution for 2h, soaking in 0.1% chlorothalonil for 0.5h, and washing with sterile water for 4-5 times; transferring to a sterile workbench, fully wiping the surfaces of the pods with 75% of hotel to sterilize, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride for 0.5h, washing with sterile water for 4-5 times, draining off water, and standing for inoculation.
Taking out the seeds of the sterilized bean pods, inoculating the seeds to an aseptic seed germination culture medium for aseptic germination of the seeds to obtain detoxified stem sections; the sterile seed germination culture medium contains 0.5 mg/L6-BA, 0.1mg/L NAA and 0.1mg/L GA3The MS modified culture medium without organic elements.
Inducing the induced detoxified stem segment to perform cluster bud proliferation induction, inoculating the stem segment to a cluster bud proliferation induction culture medium, and culturing at the temperature of 28 +/-2 ℃ under 2000lux illumination to perform cluster bud proliferation, wherein the culture medium comprises MS +1.5 mg/L6-benzylamino adenine (6-BA), 0.3mg/L naphthylacetic acid (NAA) and 0.1mg/L Gibberellin (GA)3)。
Dividing the induced cluster buds into single buds, inoculating to a proliferation and rooting culture medium, culturing at 28 + -2 deg.C under 1000lux illumination, and culturing to obtain proliferation and rooting culture medium with VM +0.1mg/L NAA and 0.05mg/L GA3。
The culture effect of each period in the tissue culture rapid propagation technology of the embodiment is shown in figure 1.
Realizes the detoxification culture of the sterile seeds and can have the differentiation rate of more than 10 detoxification cluster buds.
Comparative example 1:
the method for sterilizing and detoxifying by combining 75% alcohol and 0.1% mercury bichloride adopts the culture method in the embodiment 1, and comprises the following steps:
putting mature and healthy vanilla pods on an aseptic workbench, fully wiping the surfaces of the pods with 75% of a hotel to sterilize, washing the pods with aseptic water for 3 times, then soaking the pods with 0.1% of mercuric chloride for 0.5h, washing the pods with the aseptic water for 4-5 times, draining water, and waiting for inoculation after draining the water.
Taking out the seeds of the sterilized bean pods, inoculating the seeds to an aseptic seed germination culture medium for aseptic germination of the seeds to obtain detoxified stem sections; the sterile seed germination culture medium contains 0.5 mg/L6-BA, 0.1mg/L NAA and 0.1mg/L GA3The MS modified culture medium without organic elements. Some of the seeds are contaminated and cannot germinate.
Inducing the induced detoxified stem segment to perform cluster bud proliferation induction, inoculating the stem segment to a cluster bud proliferation induction culture medium, and culturing at the temperature of 28 +/-2 ℃ under 2000lux illumination to perform cluster bud proliferation, wherein the culture medium comprises MS +1.5 mg/L6-benzylamino adenine (6-BA), 0.3mg/L naphthylacetic acid (NAA) and 0.1mg/L Gibberellin (GA)3)。
Dividing the induced cluster buds into single buds, inoculating to a proliferation and rooting culture medium, culturing at 28 + -2 deg.C under 1000lux illumination, and culturing to obtain proliferation and rooting culture medium with VM +0.1mg/L NAA and 0.05mg/L GA3。
The sterile seeds have poor detoxification effect, and the sterile seeds can have the differentiation rate of more than 10 detoxification cluster buds after being cultured.
Comparative example 2:
the culture medium for inducing and proliferating the cluster buds in example 1 was changed to MS +1.0mg/L Kinetin (KT) +0.1mg/L naphthaleneacetic acid (NAA) +0.1mg/LGA3. The method comprises the following specific steps:
soaking the surface of mature and healthy vanilla pod in 0.05% carbendazim solution for 2h, soaking in 0.1% chlorothalonil for 0.5h, and washing with sterile water for 4-5 times; transferring to a sterile workbench, fully wiping the surfaces of the pods with 75% of hotel to sterilize, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride for 0.5h, washing with sterile water for 4-5 times, draining off water, and standing for inoculation.
Taking out the seeds of the sterilized bean pods, inoculating the seeds to an aseptic seed germination culture medium for aseptic germination of the seeds to obtain detoxified stem sections; the sterile seed germination culture medium contains 0.5 mg/L6-BA, 0.1mg/L NAA and 0.1mg/L GA3The MS modified culture medium without organic elements.
Inducing the induced detoxified stem segment into cluster bud, inoculating to cluster bud proliferation inducing culture medium, culturing at 28 + -2 deg.C under 2000lux illumination to proliferate and root, wherein the culture medium formula is MS +1.0mg/L Kinetin (KT) +0.1mg/L naphthylacetic acid (NAA).
Dividing the induced cluster buds into single buds, inoculating to a proliferation and rooting culture medium, culturing at 28 + -2 deg.C under 1000lux illumination, and culturing to obtain proliferation and rooting culture medium with VM +0.1mg/L NAA and 0.05mg/L GA3。
Realizes the detoxification culture of the sterile seeds and can induce the differentiation rate of about 5 detoxification cluster buds.
Comparative example 3:
the sterile seed germination medium in example 1 was changed to 0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D), 0.5mg/L Kinetin (KT), 0.1mg/L GA3The MS modified culture medium without organic elements. The method comprises the following specific steps:
soaking the surface of mature and healthy vanilla pod in 0.05% carbendazim solution for 2h, soaking in 0.1% chlorothalonil for 0.5h, and washing with sterile water for 4-5 times; transferring to a sterile workbench, fully wiping the surfaces of the pods with 75% of hotel to sterilize, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride for 0.5h, washing with sterile water for 4-5 times, draining off water, and standing for inoculation.
Taking out the seeds of the sterilized bean pods, inoculating the seeds to an aseptic seed germination culture medium for aseptic germination of the seeds to obtain detoxified stem sections; the sterile seed germination culture medium contains 0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D), 0.5mg/L Kinetin (KT) and 0.1mg/L GA3The MS modified culture medium without organic elements.
Inducing the induced detoxified stem segment into cluster bud, inoculating to cluster bud induction proliferation culture medium, culturing at 28 + -2 deg.C under 2000lux illumination to perform proliferation and rooting, wherein the culture medium comprises MS +1.5 mg/L6-benzylamino adenine (6-BA) and 0.3mg/L naphthylacetic acid (NAA).
Dividing the induced cluster buds into single buds, inoculating to a proliferation and rooting culture medium, culturing at 28 + -2 deg.C under 1000lux illumination, and culturing to obtain proliferation and rooting culture medium with VM +0.1mg/L NAA and 0.05mg/L GA3。
Some sterile seeds can not germinate, and callus can possess 3 cluster buds after induction.
Comparative example 4:
the culture medium for inducing and proliferating the cluster buds in example 1 is changed to MS +0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +0.5 mg/L6-BA +0.1mg/L GA3. The method comprises the following specific steps:
soaking the surface of mature and healthy vanilla pod in 0.05% carbendazim solution for 2h, soaking in 0.1% chlorothalonil for 0.5h, and washing with sterile water for 4-5 times; transferring to a sterile workbench, fully wiping the surfaces of the pods with 75% of hotel to sterilize, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride for 0.5h, washing with sterile water for 4-5 times, draining off water, and standing for inoculation.
Taking out the seeds of the sterilized bean pods, inoculating the seeds to an aseptic seed germination culture medium for aseptic germination of the seeds to obtain detoxified stem sections; the sterile seed germination culture medium contains 0.5 mg/L6-BA, 0.1mg/L NAA and 0.1mg/L GA3The MS modified culture medium without organic elements.
Inducing the induced detoxified stem segment into cluster bud, inoculating to cluster bud proliferation inducing culture medium, culturing at 28 + -2 deg.C under 2000lux illumination to proliferate and root, wherein the culture medium formula is MS +0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +0.5 mg/L6-BA.
Dividing the induced cluster buds into single buds, inoculating to a proliferation and rooting culture medium, culturing at 28 + -2 deg.C under 1000lux illumination, and culturing to obtain proliferation and rooting culture medium with VM +0.1mg/L NAA and 0.05mg/L GA3。
Realizes the detoxification culture of the sterile seeds and can induce the differentiation rate of about 3 detoxification cluster buds.
Comparative example 5:
soaking stem segment surface of health vanilla mother plant in 0.05% carbendazim solution for 2 hr, soaking in 0.1% chlorothalonil for 0.5 hr, and washing with sterile water for 4-5 times; transferring to a sterile workbench, fully wiping the surfaces of the pods with 75% of hotel to sterilize, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride for 0.5h, washing with sterile water for 4-5 times, draining off water, and standing for inoculation.
Taking out the seeds of the sterilized bean pods, inoculating the seeds to an aseptic seed germination culture medium for aseptic germination of the seeds to obtain detoxified stem sections; the sterile seed germination culture medium contains 0.5 mg/L6-BA, 0.1mg/L NAA and 0.1mg/L GA3The MS modified culture medium without organic elements.
And (3) after carrying out cluster bud proliferation induction on the induced detoxified stem segments, inoculating the stem segments to a cluster bud proliferation induction culture medium, and carrying out cluster bud proliferation by 2000lux illumination culture at the temperature of 28 +/-2 ℃, wherein the formula of the culture medium comprises MS +1.5 mg/L6-benzylamino adenine (6-BA) and 0.3mg/L naphthylacetic acid (NAA). After the stem section of the stock plant is inoculated and cultured, most of the stem section begins to brown and die.
Dividing the induced cluster buds into single buds, inoculating to a proliferation and rooting culture medium, culturing at 28 + -2 deg.C under 1000lux illumination, and culturing to obtain proliferation and rooting culture medium with VM +0.1mg/L NAA and 0.05mg/L GA3。
The stem section can not be detoxified, most of the stem section begins to brown and die after being cultured, and the stem section can induce the cluster buds with about 5 roots.
Test example 1: comparative test
The test was carried out according to the methods of example 1 and comparative examples 1 to 5, and the germination rate of sterile seeds and the proliferation induction rate of cluster buds were counted, and the results are shown in Table 1.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
TABLE 1 comparison of explant contamination rate and cluster bud induction rate for different culture methods
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A culture medium composition of vanilla detoxified seedling is characterized by comprising a sterile seed germination culture medium, a cluster bud induction culture medium and a proliferation and rooting culture medium;
the sterile seed germination medium comprises: 0.3-1.0 mg/L6-BA, 0.1-1.0 mg/L NAA and 0.1-0.5 mg/L GA3；
The cluster bud induction medium comprises: 1.0-2.5 mg/L6-BA and 0.1-0.5 mg/L NAA;
the proliferation and rooting culture medium comprises: 0.1-0.5 mg/L NAA and 0.05-0.1 mg/L GA3。
2. The medium according to claim 1, wherein the sterile seed germination medium is a medium containing 0.5 mg/L6-BA, 0.1mg/L NAA, 0.1mg/L GA3The MS modified medium of (1).
3. The medium according to claim 1, wherein the multiple shoot induction medium comprises 1.0 mg/L6-BA, 0.1mg/L NAA, 0.1mg/L GA3The MS modified medium of (1).
4. The culture medium of claim 1, wherein the rooting culture medium comprises 0.1mg/L NAA and 0.05mg/L NAAmg/L GA3VM medium of (1).
5. A high-throughput breeding method of vanilla virus-free seedlings is characterized by comprising the following steps:
A) sterilizing vanilla pods to obtain sterilized pods;
B) taking out seeds from the sterilized bean pods, inoculating the seeds to the sterile seed germination culture medium of any one of claims 1-4, and performing sterile germination on the seeds to obtain detoxified bud segments;
C) inoculating the detoxified bud segments onto the cluster bud induction medium of any one of claims 1 to 4 to induce cluster buds to obtain cluster buds;
D) after the multiple shoots are divided into plants, the plants are inoculated to the proliferation and rooting culture medium of any one of claims 1 to 4 for proliferation and rooting, and then complete plants are obtained.
6. Method according to claim 5, characterized in that step A) of sterilization is in particular: sequentially soaking the surfaces of vanilla pods in a carbendazim solution, soaking in a chlorothalonil solution and cleaning with sterile water; and then transferring the bean pods onto a sterile workbench, disinfecting the surfaces of the bean pods by using 75% alcohol, washing the bean pods with sterile water, soaking the bean pods with mercuric chloride, and washing the bean pods with the sterile water to obtain the sterilized bean pods.
7. The method as claimed in claim 6, wherein the carbendazim solution of step A) has a mass concentration of 0.05 wt%; soaking the carbendazim solution for 1-2 h; the mass concentration of the chlorothalonil solution is 0.1 wt%; the soaking time of the chlorothalonil solution is 0.5-1 h; the number of times of cleaning with sterile water is 4-5; the concentration of the mercuric chloride is 0.1 wt%.
8. The method according to claim 5, wherein the time for sterile germination is 20-30 days; the induction time of the cluster buds is 35-45 days; the time for proliferation and rooting is 20-35 d.
9. The method according to claim 1, wherein the temperature for inducing the cluster buds is 26-30 ℃; the cluster bud induction needs to be carried out under the condition of illumination, the illumination intensity is 1000-1200 lux, and the illumination period is 8 h/d.
10. The method according to claim 1, wherein the temperature of the proliferation rooting is 26-30 ℃; the proliferation and rooting are carried out under the condition of illumination, the illumination intensity is 800-1000 lux, and the illumination period is 12 h/d.
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| CN117561982A (en) * | 2024-01-19 | 2024-02-20 | 深圳市兰科植物保护研究中心 | Culture medium group for aseptic culture of beancurd blue Mao Eshan coral, application and culture method thereof |
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| CN117561982B (en) * | 2024-01-19 | 2024-03-26 | 深圳市兰科植物保护研究中心 | Culture medium group for aseptic culture of beancurd blue Mao Eshan coral, application and culture method thereof |
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