CN113854155B - High-throughput breeding method of vanilla virus-free seedlings - Google Patents

Abstract

The invention provides a culture medium combination of vanilla detoxified seedlings, which is characterized by comprising a sterile seed germination culture medium and a clump growthA 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

A kind of high-throughput breeding method of vanilla orchid detoxification seedlings

technical field

The invention relates to the technical field of tissue culture, seedling detoxification and seedling high-throughput breeding, in particular to a high-throughput breeding method for vanilla orchid virus-free seedlings.

Background technique

Vanilla planifolia Andrews, also known as vanilla, vanilla, vanilla, vanilla, orchid perennial tropical vine climbing plant. Vanilla orchid is known as the "king of natural food flavors" and is widely used in the food, pharmaceutical and cosmetic industries with a wide range of uses and high added value. With people's pursuit of high-quality fragrant life, the demand for vanilla orchid commercial pods has gradually increased, while the planting area and output of vanilla orchid are limited worldwide, and the product is in short supply in the international market. Plant tissue culture is of great significance for the rapid propagation of vanilla orchid, the cultivation of virus-free seedlings, the cultivation of transgenic plants, and the preservation of germplasm.

At present, the breeding of vanilla orchid seedlings mainly adopts vegetative breeding, and vegetative breeding is to select the stems and vines of healthy mother plants with excellent growth. However, with this method, large-scale high-throughput breeding of seedlings is often impossible due to the influence of the environmental climate and the state of the mother plant. In addition, the vanilla orchid seedlings have the phenomenon of seedling virus accumulation, resulting in a decrease in seedling yield and affecting economic benefits. The first use of sexual breeding methods can obtain virus-free and virus-free seeds, and new varieties can be cultivated through cross-breeding, but the seeds rarely germinate under natural conditions. Adopting tissue culture technology can ensure aseptic germination of detoxified seeds indoors, avoid the interference of environmental factors, obtain detoxified seedlings, and conduct plant regeneration through cluster bud induction, which can get rid of the limitation of mother plant materials, extremely It greatly improves the breeding efficiency of vanilla orchid detoxification seedlings, achieves the purpose of high-throughput breeding of seedlings, and improves the yield and economic benefits of vanilla orchid seedlings. Therefore, tissue culture technology has important practical value for vanilla orchid seedling detoxification and seedling breeding.

SUMMARY OF THE INVENTION

In view of this, the technical problem to be solved by the present invention is to provide a high-throughput breeding method for vanilla orchid detoxification seedlings, which can make non-toxic vanilla orchid seeds germinate into cluster buds, and carry out proliferation and differentiation to regenerate roots. The complete plant is realized, the vanilla orchid seedling is detoxified, the breeding efficiency of the seedling is improved, and the problem of endophyte contamination in the primary induction is avoided.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a medium combination for vanilla orchid detoxification seedlings, including a sterile seed germination medium, a cluster bud induction medium and a proliferation rooting medium;

The sterile seed germination medium comprises: 0.3-1.0 mg/L 6-BA, 0.1-1.0 mg/L NAA and 0.1-0.5 mg/L GA ₃ ;

The clump bud induction medium comprises: 1.0-2.5 mg/L 6-BA and 0.1-0.5 mg/L NAA;

The proliferation rooting medium includes: 0.1-0.5 mg/L NAA and 0.05-0.1 mg/L GA ₃ .

Preferably, the sterile seed germination medium is an MS modified medium containing 0.5 mg/L 6-BA, 0.1 mg/L NAA, and 0.1 mg/LGA ₃ .

Preferably, the clump bud induction medium is an MS modified medium containing 1.0 mg/L 6-BA, 0.1 mg/L NAA, and 0.1 mg/L GA ₃ .

Preferably, the proliferation rooting medium is VM medium containing 0.1 mg/L NAA and 0.05 mg/L GA ₃ .

The invention provides a high-throughput breeding method for vanilla orchid detoxification seedlings, characterized in that it comprises the following steps:

A) sterilizing vanilla orchid pods to obtain sterilized pods;

B) taking out the seeds from the sterilized pods, inoculating them on the sterile seed germination medium of any one of claims 1 to 4, and performing aseptic germination of the seeds to obtain detoxified bud segments;

C) inoculate the detoxified bud segment on the clump bud induction medium described in any one of claims 1 to 4 to induce the clump bud to obtain the clump bud;

D) After dividing the clump buds, inoculate them on the proliferation and rooting medium according to any one of claims 1 to 4 for proliferation and rooting to obtain a complete plant.

Preferably, the disinfection method described in step A) is specifically: the surface of vanilla bean pods is soaked in carbendazim solution, soaked in chlorothalonil solution, and cleaned with sterile water in sequence; The surface of the pods is disinfected, rinsed with sterile water, soaked with mercury chloride, and rinsed with sterile water to obtain sterilized pods.

Preferably, the mass concentration of the carbendazim solution in step A) is 0.05wt%; the soaking time of the carbendazim solution is 1-2h; the mass concentration of the chlorothalonil solution is 0.1wt%; the chlorothalonil solution is soaked The time is 0.5-1 h; the number of times of washing with sterile water is 4-5 times; the concentration of the mercury chloride is 0.1 wt %.

Preferably, the time for aseptic germination is 20d to 30d; the time for induction of the clump buds is 35d to 45d, and the time for proliferation and rooting is 20d to 35d.

Preferably, the temperature for inducing the clump buds is 26-30°C; the induction of the clump buds needs to be carried out under the condition of illumination, the illumination intensity is 1000-1200 lux, and the illumination period is 8h/d.

Preferably, the temperature of the proliferation and rooting is 26-30°C; the proliferation and rooting needs to be carried out under the condition of light, the light intensity is 800-1000 lux, and the light period is 12h/d.

Compared with the prior art, the present invention provides a high-throughput breeding method for vanilla orchid detoxification seedlings, comprising the following steps: A) sterilizing the vanilla orchid pods to obtain sterilized pods; B) sterilizing the vanilla orchid pods Take out seeds from the rear pods, inoculate on the sterile seed germination medium described in any one of claims 1 to 4, and carry out aseptic germination of seeds to obtain detoxified bud segments; C) must inoculate detoxified bud segments into claims Inducing clump buds on the clump bud induction medium according to any one of 1 to 4 to obtain clump buds; D) dividing the clump buds into divisions, and then inoculating the clump buds into the proliferation and rooting according to any one of claims 1 to 4 Proliferation and rooting were carried out on the medium to obtain complete plants.

In the present invention, different explant detoxification methods and culture medium are used as control methods, and the method is cultured under the same environmental conditions as the method of the present invention. The results show that only 0.05% carbendazim solution, 0.1% chlorothalonil, 75% alcohol The combination of 0.1% mercuric chloride and 0.1% mercuric chloride is the disinfection and detoxification method to obtain detoxified stem segments and finally detoxified cluster buds. Only 75% alcohol is used as the disinfection and detoxification method. During the culture, the germs cannot germinate, and detoxification is not obtained. Stem segments or detoxified clump buds, therefore, the method of the present invention is significantly better than the control methods in terms of the detoxification germination of vanilla orchid seeds and the regeneration of detoxified clump buds.

The method of the present invention selects detoxified vanilla orchid seeds as explants, and the formula of sterile seed germination medium: MS improved medium without organic elements, added with 6-benzylaminoadenine (6-BA), naphthalene acetic acid (NAA) ) and gibberellin (GA ₃ ); cluster bud induction medium formula: MS-based medium, supplemented with 6-benzylaminoadenine (6-BA) and naphthalene acetic acid (NAA), to achieve sterile seeds through culture , can have a differentiation rate of more than 10 detoxified cluster buds, which is of great significance to the high-throughput breeding and genetic engineering research of vanilla orchid detoxification seedlings in the future.

Description of drawings

Fig. 1 is the high-throughput breeding technology of vanilla orchid detoxification seedlings of the present invention.

Detailed ways

The invention provides a high-throughput breeding method for vanilla orchid detoxification seedlings, and those skilled in the art can learn from the content of this article and appropriately improve process parameters to achieve. It should be particularly pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the protection scope of the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.

The invention provides a medium combination for vanilla orchid detoxification seedlings, including a sterile seed germination medium, a cluster bud induction medium and a proliferation rooting medium;

The sterile seed germination medium comprises: 0.3-1.0 mg/L 6-BA, 0.1-1.0 mg/L NAA and 0.1-0.5 mg/L GA ₃ ;

The clump bud induction medium includes: 1.0-2.5 mg/L 6-BA and 0.1-0.5 mg/L NAA.

The proliferation rooting medium includes: 0.1-0.5 mg/L NAA and 0.05-0.1 mg/L GA ₃ .

The invention provides a medium combination for vanilla orchid detoxification seedlings, including a sterile seed germination medium; the sterile seed germination medium comprises: 0.3-1.0 mg/L 6-BA, 0.1-1.0 mg/L L NAA and 0.1-0.5 mg/LGA ₃ ; preferably, the sterile seed germination medium is MS modified medium containing 0.5 mg/L 6-BA, 0.1 mg/L NAA and 0.1 mg/L GA ₃ .

The invention provides a medium combination for vanilla orchid detoxification seedlings, including a clump bud induction medium; the clump bud induction medium comprises: 1.0-2.5mg/L 6-BA and 0.1-0.5mg/L NAA ; Preferably, the clump bud induction medium is an MS modified medium containing 1.0 mg/L 6-BA, 0.1 mg/L NAA, and 0.1 mg/L GA ₃ .

The invention provides a medium combination for vanilla orchid detoxification seedlings, including a proliferation and rooting medium; the proliferation and rooting medium comprises: 0.1-0.5 mg/L NAA and 0.05-0.1 mg/L GA ₃ ; preferably , the proliferation rooting medium is VM medium containing 0.1 mg/L NAA and 0.05 mg/L GA ₃ .

The invention provides a high-throughput breeding method for vanilla orchid detoxification seedlings, which is characterized in that comprising the following steps:

A) sterilizing vanilla orchid pods to obtain sterilized pods;

B) taking out the seeds from the sterilized pods, inoculating them on the sterile seed germination medium of any one of claims 1 to 4, and performing aseptic germination of the seeds to obtain detoxified bud segments;

C) inoculate the obtained detoxified bud segment on the clump bud induction medium described in any one of claims 1 to 4 to carry out clump bud induction to obtain a clump bud;

D) After dividing the clump buds, inoculate them on the proliferation and rooting medium according to any one of claims 1 to 4 for proliferation and rooting to obtain a complete plant.

The invention provides a high-throughput breeding method for vanilla orchid detoxification seedlings. First, the vanilla orchid pods are sterilized to obtain sterilized pods.

The disinfection method of the invention is specifically as follows: the surface of vanilla orchid pods is soaked in carbendazim solution, soaked in chlorothalonil solution, and washed with sterile water in sequence; then transferred to a sterile workbench, and the surface of the pods is disinfected with 75% alcohol , rinsed with sterile water, soaked with mercuric chloride, rinsed with sterile water to obtain sterilized pods.

The surface of the vanilla pods is soaked in a carbendazim solution in turn; the mass concentration of the carbendazim solution is 0.05wt%; the soaking time of the carbendazim solution is preferably 1-2h; more preferably 2h; the number of times of cleaning with sterile water 4 to 5 times;

The chlorothalonil solution is soaked and washed with sterile water; the mass concentration of the chlorothalonil solution is 0.1 wt %; the number of times of washing with sterile water is 4 to 5 times; the soaking time of the chlorothalonil solution is preferably 0.5 to 1 h; more Preferably it is 0.5h.

Then transfer to a sterile workbench, disinfect the surface of the pods with 75% alcohol, and rinse with sterile water; the number of times of rinsing with sterile water is 3; pod.

The concentration of the mercury chloride is 0.1wt%; the soaking time is 0.5h. After soaking, rinse with sterile water 4-5 times;

The seeds of the sterilized pods are taken out and inoculated onto the sterile seed germination medium described in any one of the above technical solutions for aseptic germination of the seeds to obtain detoxified bud segments. The present invention has clearly defined the above-mentioned sterile seed germination medium, which will not be repeated here.

The conditions for germination of the sterile seeds of the present invention are 26-30° C.; dark culture. The aseptic germination time is 20d～30d;

It is necessary to inoculate the detoxified bud segment on the clump bud induction medium described in any one of the above technical solutions to induce the clump bud to obtain the clump bud. The present invention has clearly defined the above-mentioned cluster bud induction medium, which is not repeated here.

The temperature for inducing the cluster buds of the present invention is 26-30° C.; the light is dark; the time for inducing the cluster buds is 35d-45d.

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 clump buds are divided into divisions, they are inoculated onto the proliferation and rooting medium described in any one of the above technical solutions for proliferation and rooting to obtain a complete plant.

The temperature of the proliferation and rooting of the present invention is 26-30°C; the proliferation and rooting need to be carried out under the condition of light, the light intensity is 800-1000 lux, and the light period is 12h/d. The time for the proliferation and rooting is 20d-35d.

The invention provides a high-throughput breeding method for vanilla orchid detoxification seedlings, comprising the following steps: A) sterilizing vanilla orchid pods to obtain sterilized pods; B) taking out seeds from the sterilized pods and inoculating them Carry out aseptic germination of seeds on the sterile seed germination medium described in any one of claims 1 to 4 to obtain detoxified bud segments; C) must inoculate the detoxified bud segments into any one of claims 1 to 4. Inducing the clump bud on the described clump bud induction medium to obtain the clump bud; D) after the clump bud is divided into divisions, inoculate on the proliferation rooting medium described in any one of claims 1 to 4 to carry out proliferation and rooting, Obtain complete plants.

In the present invention, different explant detoxification methods and culture medium are used as control methods, and the method is cultured under the same environmental conditions as the method of the present invention. The results show that only 0.05% carbendazim solution, 0.1% chlorothalonil, 75% alcohol The combination of 0.1% mercuric chloride and 0.1% mercuric chloride is the disinfection and detoxification method to obtain detoxified stem segments and finally detoxified cluster buds. Only 75% alcohol is used as the disinfection and detoxification method. During the culture, the germs cannot germinate, and detoxification is not obtained. Stem segments or detoxified clump buds, therefore, the method of the present invention is significantly better than the control methods in terms of the detoxification germination of vanilla orchid seeds and the regeneration of detoxified clump buds.

The method of the present invention selects detoxified vanilla orchid seeds as explants, and the formula of sterile seed germination medium: MS improved medium without organic elements, added with 6-benzylaminoadenine (6-BA), naphthalene acetic acid (NAA) ) and gibberellin (GA ₃ ); cluster bud induction medium formula: MS-based medium, supplemented with 6-benzylaminoadenine (6-BA) and naphthalene acetic acid (NAA), to achieve sterile seeds through culture , can have a differentiation rate of more than 10 detoxified cluster buds, which is of great significance to the high-throughput breeding and genetic engineering research of vanilla orchid detoxification seedlings in the future.

The invention ensures that the vanilla orchid tissue is free from virus infection by a specific disinfection method, provides sterile seeds as explant materials and a specific induction medium for the regeneration of the vanilla orchid detoxification seedling, and obtains clustered buds, thereby improving the seedling breeding efficiency and efficiency. Obtaining a large number of virus-free tissue culture seedlings in the same state is of great significance for the high-throughput breeding of vanilla orchid virus-free seedlings in the future.

In order to further illustrate the present invention, a high-throughput breeding method for a vanilla orchid detoxification seedling provided by the present invention is described in detail below with reference to the examples.

The invention relates to the technical field of vanilla orchid tissue culture and seedling high-throughput breeding, and discloses a detoxification seedling rapid propagation technology method using vanilla orchid sterile seeds as explants. According to the method of the present invention, the surface of the mature and healthy vanilla orchid pods is sterilized with 0.05% carbendazim solution, 0.1% chlorothalonil, 75% alcohol and 0.1% mercuric chloride, and the blackened vanilla orchid in the pods is taken out and removed. The poisonous seeds were used as explants; the detoxified explants were inoculated on the seed germination medium for germination for about 60 days, and then received on the cluster bud proliferation induction medium for about 30 days to induce the proliferation of the cluster buds to obtain the cluster buds. To achieve the purpose of high-throughput breeding of virus-free seedlings. The method finally realizes the proliferation and differentiation of cluster buds by using sterile seeds to germinate detoxified stem segments as explants, and can have more than 10 detoxified cluster buds, which greatly improves the breeding efficiency of seedlings. The in vitro regeneration method of the detoxified seedlings of vanilla orchid provides technical support for the future research on industrialized high-throughput breeding and genetic engineering breeding of the detoxified seedlings of vanilla orchid.

Example 1: The method of the present invention

Soak the surface of mature and healthy vanilla orchid pods in 0.05% carbendazim solution for 2h, then soak in 0.1% chlorothalonil for 0.5h, and wash with sterile water for 4-5 times; transfer to a sterile workbench, first use 75 % Hotel fully wipe the surface of the pods to disinfect, rinse with sterile water for 3 times, then soak in 0.1% mercuric chloride for 0.5h, rinse with sterile water for 4-5 times, drain the water, and wait for inoculation after draining the water.

The bean pods after sterilization and disinfection are taken out seeds, inoculated on sterile seed germination medium for aseptic germination of seeds to obtain detoxified stem segments; MS modified medium without organic elements at mg/L NAA, 0.1 mg/LGA ₃ .

The induced detoxified stem segments are inoculated into the clump bud induction and proliferation medium after inducing the proliferation of clump buds, and the clump bud proliferation is carried out under 2000lux light culture at 28±2°C. The medium formula is MS+1.5mg/L 6 - Benzylaminoadenine (6-BA), 0.3 mg/L naphthalene acetic acid (NAA), 0.1 mg/L gibberellin (GA ₃ ).

The induced clump buds were divided into single buds, then inoculated on the proliferation and rooting medium, and cultured under 1000 lux light at 28±2°C for proliferation and rooting. The medium formula was VM+0.1mg/L NAA, 0.05mg/ _LGA3 .

Figure 1 shows the culture effect of each period in the tissue culture rapid propagation technique of the present embodiment.

The detoxification culture of sterile seeds is realized, and the differentiation rate of more than 10 detoxified cluster buds can be obtained.

Comparative Example 1:

The combination of 75% alcohol and 0.1% mercuric chloride is used as a sterilization and detoxification method, and the culture method in Example 1 is adopted, as follows:

Put the mature and healthy vanilla orchid pods on the sterile workbench, first wipe the surface of the pods with 75% hotel for disinfection, rinse with sterile water 3 times, then soak with 0.1% mercuric chloride for 0.5h, rinse with sterile water 4-5 times, Drain the water and wait for inoculation after draining the water.

The bean pods after sterilization and disinfection are taken out seeds, inoculated on aseptic seed germination medium to carry out aseptic germination of seeds, to obtain detoxified stem segments; MS modified medium without organic elements at mg/L NAA, 0.1 mg/LGA ₃ . Some of the seeds were contaminated and could not germinate.

The induced detoxified stem segments are inoculated into the clump bud induction and proliferation medium after inducing the proliferation of clump buds, and the clump bud proliferation is carried out under 2000lux light culture at 28±2°C. The medium formula is MS+1.5mg/L 6 - Benzylaminoadenine (6-BA), 0.3 mg/L naphthalene acetic acid (NAA), 0.1 mg/L gibberellin (GA ₃ ).

The induced clump buds were divided into single buds, then inoculated on the proliferation and rooting medium, and cultured under 1000 lux light at 28±2°C for proliferation and rooting. The medium formula was VM+0.1mg/L NAA, 0.05mg/ _LGA3 .

The detoxification effect of sterile seeds is not good, and they can have a differentiation rate of more than 10 detoxified clump buds after culture.

Comparative Example 2:

The clump shoot-inducing proliferation medium in Example 1 was changed to MS+1.0 mg/L kinetin (KT)+0.1 mg/L naphthalene acetic acid (NAA)+0.1 mg/LGA ₃ . details as follows:

Soak the surface of mature and healthy vanilla orchid pods in 0.05% carbendazim solution for 2h, then soak in 0.1% chlorothalonil for 0.5h, and wash with sterile water for 4-5 times; transfer to a sterile workbench, first use 75 % Hotel fully wipe the surface of the pods to disinfect, rinse with sterile water for 3 times, then soak in 0.1% mercuric chloride for 0.5h, rinse with sterile water for 4-5 times, drain the water, and wait for inoculation after draining the water.

The bean pods after sterilization and disinfection are taken out seeds, inoculated on aseptic seed germination medium to carry out aseptic germination of seeds, to obtain detoxified stem segments; MS modified medium without organic elements at mg/L NAA, 0.1 mg/LGA ₃ .

The induced detoxified stem segments were induced to clump buds, inoculated on the clump bud induction and proliferation medium, and cultured under 28±2°C under 2000lux light for proliferation and rooting. The medium formula was MS+1.0mg/L kinetin (KT ) + 0.1 mg/L naphthalene acetic acid (NAA).

The induced clump buds were divided into single buds, then inoculated on the proliferation and rooting medium, and cultured under 1000 lux light at 28±2°C for proliferation and rooting. The medium formula was VM+0.1mg/L NAA, 0.05mg/ _LGA3 .

The detoxification culture of sterile seeds is realized, and the differentiation rate of about 5 detoxified cluster buds can be induced.

Comparative Example 3:

The sterile seed germination medium in Example 1 was changed to 0.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg/L kinetin (KT), 0.1 mg/L GA ₃ of MS modified medium without organic elements. details as follows:

Soak the surface of mature and healthy vanilla orchid pods in 0.05% carbendazim solution for 2h, then soak in 0.1% chlorothalonil for 0.5h, and wash with sterile water for 4-5 times; transfer to a sterile workbench, first use 75 % Hotel fully wipe the surface of the pods to disinfect, rinse with sterile water for 3 times, then soak in 0.1% mercuric chloride for 0.5h, rinse with sterile water for 4-5 times, drain the water, and wait for inoculation after draining the water.

The bean pods after the sterilization and disinfection are taken out of the seeds, inoculated on a sterile seed germination medium for aseptic germination of seeds, to obtain a detoxified stem segment; the sterile seed germination medium contains 0.1 mg/L 2,4-di Chlorphenoxyacetic acid (2,4-D), 0.5 mg/L kinetin (KT), 0.1 mg/L GA ₃ MS modified medium without organic elements.

The induced detoxified stem segments were induced to clump buds, inoculated into the clump bud induction proliferation medium, and cultured under 2000lux light at 28±2°C for proliferation and rooting. The medium formula was MS+1.5mg/L 6-benzylamino Adenine (6-BA), 0.3 mg/L naphthalene acetic acid (NAA).

The induced clump buds were divided into single buds, then inoculated on the proliferation and rooting medium, and cultured under 1000 lux light at 28±2°C for proliferation and rooting. The medium formula was VM+0.1mg/L NAA, 0.05mg/ _LGA3 .

A part of the sterile seeds could not germinate, but the callus became callus, and could have about 3 clump buds after induction.

Comparative Example 4:

Change the cluster bud induction proliferation medium in Example 1 to MS+0.1mg/L 2,4-dichlorophenoxyacetic acid (2,4-D)+0.5mg/L 6-BA+0.1mg/L GA ₃ . details as follows:

Soak the surface of mature and healthy vanilla orchid pods in 0.05% carbendazim solution for 2h, then soak in 0.1% chlorothalonil for 0.5h, and wash with sterile water for 4-5 times; transfer to a sterile workbench, first use 75 % Hotel fully wipe the surface of the pods to disinfect, rinse with sterile water for 3 times, then soak in 0.1% mercuric chloride for 0.5h, rinse with sterile water for 4-5 times, drain the water, and wait for inoculation after draining the water.

The bean pods after sterilization and disinfection are taken out seeds, inoculated on aseptic seed germination medium to carry out aseptic germination of seeds, to obtain detoxified stem segments; MS modified medium without organic elements at mg/L NAA, 0.1 mg/LGA ₃ .

The induced detoxified stem segments were induced to clump buds, inoculated into the clump bud induction proliferation medium, and cultured under 2000lux light at 28±2°C for proliferation and rooting. The medium formula was MS+0.1mg/L 2,4- Dichlorophenoxyacetic acid (2,4-D) + 0.5 mg/L 6-BA.

The induced clump buds were divided into single buds, then inoculated on the proliferation and rooting medium, and cultured under 1000 lux light at 28±2°C for proliferation and rooting. The medium formula was VM+0.1mg/L NAA, 0.05mg/ _LGA3 .

The detoxification culture of sterile seeds is realized, and the differentiation rate of about 3 detoxified clump buds can be induced.

Comparative Example 5:

After soaking the stem surface of the healthy vanilla orchid mother plant in 0.05% carbendazim solution for 2 hours, then soaking in 0.1% chlorothalonil for 0.5 hours, and washing with sterile water for 4-5 times; Fully wipe the surface of the pods with 75% hotel for disinfection, rinse with sterile water 3 times, then soak in 0.1% mercuric chloride for 0.5h, rinse with sterile water for 4-5 times, drain the water, and wait for inoculation after draining the water.

The bean pods after sterilization and disinfection are taken out seeds, inoculated on aseptic seed germination medium to carry out aseptic germination of seeds, to obtain detoxified stem segments; MS modified medium without organic elements at mg/L NAA, 0.1 mg/LGA ₃ .

The induced detoxified stem segments are inoculated into the clump bud induction and proliferation medium after inducing the proliferation of clump buds, and the clump bud proliferation is carried out under 2000lux light culture at 28±2°C. The medium formula is MS+1.5mg/L 6 - Benzylaminoadenine (6-BA), 0.3 mg/L naphthalene acetic acid (NAA). After the stem segment of the mother plant is inserted into the culture, most of them begin to brown and die.

The induced clump buds were divided into single buds, then inoculated on the proliferation and rooting medium, and cultured under 1000 lux light at 28±2°C for proliferation and rooting. The medium formula was VM+0.1mg/L NAA, 0.05mg/ _LGA3 .

Detoxification of stem segments cannot be achieved, and most of them begin to brown and die after culture, and can induce clustered buds with about 5 roots.

Test Example 1: Comparative Test

Experiments were carried out according to the methods of Example 1 and Comparative Examples 1-5, and the germination rate of sterile seeds and the proliferation induction rate of clump buds were counted. The results are shown in Table 1.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Table 1 Comparison of explant contamination rate and cluster bud induction rate in different culture methods

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should 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 culture medium contains: 0.3-1.0 mg/L6-BA, 0.1-1.0 mg/L NAA and 0.1-0.5 mg/L GA₃The MS modified culture medium without organic elements;

the cluster bud induction culture medium is an MS culture medium containing 1.0-2.5 mg/L of 6-BA and 0.1-0.5 mg/L of NAA;

the proliferation and rooting culture medium contains 0.1-0.5 mg/L NAA and 0.05-0.1 mg/L GA₃VM medium of (1).

2. The media combination of claim 1, wherein the sterile seed germination media is a medium comprising 0.5 mg/L6-BA, 0.1mg/L NAA, 0.1mg/L GA₃The MS modified culture medium without organic elements.

3. The media combination of claim 1, wherein the multiple shoot induction medium comprises 1.0 mg/L6-BA, 0.1mg/L NAA, 0.1mg/L GA₃The MS medium of (1).

4. The culture medium combination of claim 1, wherein the rooting medium comprises 0.1mg/L NAA and 0.05mg/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 to carry out 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 d; the induction time of the cluster buds is 35-45 d, and the proliferation and rooting time is 20-35 d.

9. The method according to claim 5, 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 5, wherein the temperature of the proliferation root 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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