Grape detoxification culture medium method

Abstract

The invention relates to the technical field of grape detoxification, in particular to a grape detoxification culture medium method, and aims to solve the problems of non-uniform detoxification effect and low detoxification success rate of grape explants due to the fact that the existing grape detoxification technology is traditional in detoxification mode of the grape explants and lacks a strictly standard detoxification process. According to the scheme, the method comprises the following steps of S1, primary preparation, S2, explant disinfection, S3, primary culture, S4, subculture, S5, heat treatment detoxification and S6, stem tip tissue differentiation, and aims at achieving the purposes that detoxification is conducted through stem tip tissue and heat treatment, a strict and normative detoxification process is set, the detoxification effect is unified, the detoxification success rate is increased, and the survival rate is increased. Meanwhile, the detoxified grape explants are subjected to a planting test, and test results are compared, so that the effect authenticity of the detoxification method is improved.

Description

Technical Field

[0001] The invention relates to the technical field of grape detoxification, in particular to a grape detoxification culture medium method. Background Art

[0002] Grapes are tall, twining vines with strong anti-cancer properties. Young stems are bald or slightly covered with cotton wool. The leaves are papery, oval or round. The inflorescence is large and long. The calyx is very small and yellow-green cup-shaped. The style is very short and conical. The berries are oval to oval-oblong. When ripe, they are purple-black or red with blue. The flowering period is June and the fruiting period is September-October. Grapes have the effects of replenishing qi and blood, relaxing tendons and collaterals, and promoting urination. It is mainly used to treat qi and blood deficiency, cough due to lung deficiency, palpitations and night sweats, thirst, rheumatic pain, gonorrhea, edema, and acne. Its efficacy is recorded in "Ben Jing" and "Bie Lu". Grapes are regarded as precious fruits and are praised as the first of the four major fruits in the world. They are not only nutritious, widely used, beautiful in color, fragrant, and delicious, but also a good fruit. They can be eaten fresh and made into wine. Moreover, the fruit, roots, and leaves can all be used as medicine. The whole body is a treasure. Grapes are also an important fruit tree in my country. my country's grape production and area are among the highest in the world. However, grape virus diseases are common in my country, which have an adverse effect on production and quality. Grape virus disease is an important disease that affects grape production and quality. Therefore, when building a vineyard, virus-free seedlings should be selected as much as possible.

[0003] However, the existing grape detoxification technology has the problem of traditional detoxification methods for grape explants and lack of strict and standardized detoxification process, resulting in inconsistent detoxification effects of grape explants and low detoxification success rate. Therefore, we propose a grape detoxification culture medium method to solve the above problems. Summary of the invention

[0004] The purpose of the present invention is to solve the problems that the existing grape detoxification technology has a traditional detoxification method for grape explants and lacks a strictly standardized detoxification process, resulting in inconsistent detoxification effects on grape explants and a low detoxification success rate, and a grape detoxification culture medium method is proposed to unify the detoxification effects of grape explants and improve the detoxification success rate.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A grape detoxification culture medium method comprises the following steps:

[0007] S1: Preliminary preparation: Preliminary preparation is carried out by professionals;

[0008] S2: Explant disinfection: Explant disinfection is performed by professionals;

[0009] S3: Primary culture: Professionals prepare rooting medium and use it to culture the explants.

[0010] S4: Subculture: After the primary culture is completed, professionals will carry out subculture;

[0011] S5: Heat treatment and detoxification: Professionals will perform heat treatment and detoxification on the regenerated buds;

[0012] S6: Shoot tip tissue differentiation: After heat treatment, the stem tip tissue differentiation is performed by professionals;

[0013] S7: Rooting culture of regenerated buds: Rooting culture of regenerated buds is carried out by professionals;

[0014] Preferably, in S1, preliminary preparation is performed by professionals, wherein during the preliminary preparation, professionals first select grape explants through the grape field, and collect the explants selected from the grape field, and the selected explants are buds that have just protruded and have not germinated on semi-lignified branches, and the distance between two adjacent spores is not less than 3 cm, and the selected grape explants are processed by professionals at the same time, wherein during the processing, the professionals remove excess leaves and tendrils from the collected explants, wrap them with gauze, and rinse them with running water for 4 hours, pinch off the heads and tails of the washed branches, retain the middle section, and cut them into 3 cm small sections with pruning shears, wherein 2 cm is below the bud and 1 cm is above the bud;

[0015] Preferably, in S2, the explants are disinfected by professionals, wherein when the explants are disinfected, the professionals transfer the cleaned stem segments to a sterile beaker or a triangular flask on a clean bench, and use 75% ethanol for preliminary disinfection for 30 seconds, and use sterile water to wash twice after the preliminary disinfection, and use 0.1% mercuric chloride for secondary disinfection for 8 minutes after the washing, and after the secondary disinfection, pour out the disinfectant and wash with sterile water 3-5 times, and each washing time is 5 minutes, and the number of explant stem segments in the explant disinfection process is not less than 50;

[0016] Preferably, in S3, a professional prepares a rooting medium, and primary culture is performed on the explants using the rooting medium, wherein the rooting medium is prepared using a formula of MS+1.0mg / L 6-BA+0.5mg / L KT+0.2mg / L IBA+30g / L sucrose+7g / L agar+0.1% PPM, the medium pH is 5.8, and IBA and PPM are added when the temperature drops below 60°C after sterilization;

[0017] Preferably, in S4, after the primary culture is completed, a professional performs subculture, wherein during the subculture, the sterile young shoots obtained after 2 weeks of primary culture are transferred to different subculture culture media, and the production of adventitious shoots is observed by a professional, and the total number of inoculated shoots is counted after 30 days of culture, and the wilting rate and the value-added rate are calculated by statistical data, and the preparation formula of the culture medium used in the subculture is MS+0.2mg / L6-BA+0.2mg / L IBA+7g / L agar+20g / L sucrose, and the pH of the culture medium is 5.8;

[0018] Preferably, in S5, professionals perform heat treatment to detoxify the regenerated buds, transfer the plants to a constant temperature incubator, and culture them for 7 days in an environment of 32°C for 16 hours of light and 30°C for 8 hours of darkness. After 7 days of culture, the temperature is raised to 38°C for 16 hours of light and 32°C for 8 hours of darkness for 20 days of heat treatment. After 20 days of culture, professionals observe and calculate the survival rate.

[0019] Preferably, in S6, after the heat treatment, the stem apex tissue differentiation is performed by professionals, wherein the stem apex tissue differentiation is performed by professionals from the detoxified stem segment under a stereo microscope to peel off 1 mm of the stem apex and transfer it to a differentiation medium, wherein the differentiation medium formula is MS+2.0 mg / L6-BA+0.2 mg / L NAA+0.5 mg / L KT+30 g / L sucrose, and the pH is 5.8, wherein when peeling the stem apex, the detoxified stem segment is first placed on sterile filter paper to absorb moisture, and the stem apex is cut off with a sterile surgical blade, and the cut surface is placed on the above-mentioned differentiation medium;

[0020] Preferably, in S7, professional personnel perform rooting culture of regenerated buds, wherein when performing rooting culture of regenerated buds, differentiated seedlings of >2 cm are selected and transferred to a newly prepared rooting medium, and professional personnel observe the rooting situation and count the rooting rate, and the culture time for rooting culture of regenerated buds is 30 days, and after the rooting of the regenerated buds is completed, professional personnel perform virus detection, and process according to the virus detection result, wherein if the virus detection result shows that there is no virus in the grape seedlings, it is judged that the virus removal is successful, and if the virus detection result shows that there is virus in the grape seedlings, it is judged that the virus removal fails, and if the result is successful, professional personnel plant the virus-free grape seedlings, and if the result is unsuccessful, professional personnel perform a virus removal step inspection, and re-detoxify the grape seedlings, and at the same time professional personnel calculate the virus removal success rate of the grape seedlings according to the virus detection result, and compare the calculated data with the existing data, wherein the preparation formula of the rooting medium is 1 / 2MS+0.2mg / L 6-BA+0.2mg / L IBA+20g / L sucrose, the pH of the culture medium is 5.8, and IBA is added when the temperature drops below 60°C after sterilization.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention improves the efficiency of detoxification by formulating a strictly standardized detoxification process.

[0023] The purpose of the present invention is to perform detoxification by utilizing stem tip tissue and heat treatment, and to set a strict and standardized detoxification process, thereby unifying the detoxification effect and improving the detoxification success rate. At the same time, a planting test is performed on the detoxified grape explants, and the test results are compared to improve the effect authenticity of the detoxification method. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention provides a flow chart of a grape detoxification culture medium method. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0026] Embodiment 1

[0027] Reference Figure 1 , a grape detoxification culture medium method, comprising the following steps:

[0028] S1: Preliminary preparation: Preliminary preparation is performed by professionals, wherein the professionals first select grape explants from the grape field, and collect the explants selected from the grape field, and the selected explants are buds that have just protruded and have not germinated on semi-lignified branches, and the distance between two adjacent buds is not less than 3 cm, and the selected grape explants are processed by professionals at the same time, wherein during the processing, the professionals remove excess leaves and tendrils from the collected explants, wrap them with gauze, and rinse them with running water for 4 hours, pinch off the heads and tails of the washed branches, retain the middle section, and cut them into 3 cm small sections with pruning shears, wherein 2 cm is below the bud and 1 cm is above the bud;

[0029] S2: Explant disinfection: Professionals are responsible for explant disinfection, wherein the professional transfers the cleaned stem segments to a sterile beaker or a triangular flask on a clean bench, and uses 75% ethanol for preliminary disinfection for 30 seconds, washes twice with sterile water after the preliminary disinfection, and uses 0.1% mercuric chloride for secondary disinfection for 8 minutes after the washing, and after the secondary disinfection, pours out the disinfectant and washes 4 times with sterile water, and each washing time is 5 minutes, and the number of explant stem segments in the explant disinfection process is not less than 50;

[0030] S3: Primary culture: Professionals prepare rooting medium and perform primary culture on explants using the rooting medium, wherein the rooting medium is prepared with a formula of MS+1.0mg / L 6-BA+0.5mg / L KT+0.2mg / L IBA+30g / L sucrose+7g / L agar+0.1% PPM, the medium pH is 5.8, and IBA and PPM are added when the temperature drops below 60°C after sterilization;

[0031] S4: Subculture: After the primary culture is completed, the professional personnel will carry out subculture, wherein the sterile young shoots obtained after 2 weeks of primary culture are transferred to different subculture culture media, and the professional personnel will observe the production of adventitious shoots, and the total number of inoculated shoots is counted after 30 days of culture, and the wilting rate and the value-added rate are calculated by statistical data, and the preparation formula of the culture medium used in the subculture is MS+0.2mg / L 6-BA+0.2mg / LIBA+7g / L agar+20g / L sucrose, and the pH of the culture medium is 5.8;

[0032] S5: Heat treatment and detoxification: Professionals will perform heat treatment and detoxification on the regenerated buds, transfer the plants to a constant temperature incubator, and culture them for 7 days at 32℃ for 16 hours of light and 30℃ for 8 hours of darkness. After 7 days of culture, the temperature will be raised to 38℃ for 16 hours of light and 32℃ for 8 hours of darkness for 20 days. After 20 days of culture, professional personnel will observe and calculate the survival rate.

[0033] S6: stem apex tissue differentiation: After heat treatment, stem apex tissue differentiation is performed by professionals. When performing stem apex tissue differentiation, professionals need to peel off 1mm stem apex from the detoxified stem segment under a stereo microscope and transfer it into differentiation medium. The differentiation medium formula is MS+2.0mg / L6-BA+0.2mg / L NAA+0.5mg / L KT+30g / L sucrose, and the pH is 5.8. When peeling off the stem apex, the detoxified stem segment is first placed on sterile filter paper to absorb water, and the stem apex is cut off with a sterile surgical blade, and the cut surface is placed on the above differentiation medium;

[0034] S7: Rooting culture of regenerated buds: Professionals carry out rooting culture of regenerated buds, wherein when carrying out rooting culture of regenerated buds, seedlings of >2 cm after differentiation are selected and transferred to newly prepared rooting medium, and professional personnel observe the rooting situation and calculate the rooting rate, and the culture time for rooting culture of regenerated buds is 30 days, and after the rooting of regenerated buds is completed, professional personnel carry out virus detection, and process according to the virus detection result, wherein if the virus detection result shows that there is no virus in the grape seedlings, it is judged that the virus removal is successful, and if the virus detection result shows that there is virus in the grape seedlings, it is judged that the virus removal fails, and if the result is judged that the virus removal is successful, professional personnel will plant the virus-free grape seedlings, and if the result is judged that the virus removal fails, professional personnel will check the virus removal step and re-detoxify the grape seedlings, and at the same time professional personnel will calculate the virus removal success rate of the grape seedlings according to the virus detection result, and compare the calculated data with the existing data, wherein the preparation formula of the rooting medium is 1 / 2MS+0.2mg / L 6-BA+0.2mg / L IBA+20g / L sucrose, the pH of the culture medium is 5.8, and IBA is added when the temperature drops below 60°C after sterilization.

[0035] Embodiment 2

[0036] Reference Figure 1 , a grape detoxification culture medium method, comprising the following steps:

[0037] S1: Preliminary preparation: Preliminary preparation is performed by professionals, wherein during the preliminary preparation, professionals first select grape explants from the grape field and collect the explants selected from the grape field, and the selected explants are buds that have just protruded and have not germinated on semi-lignified branches, and the distance between two adjacent spores is not less than 3 cm;

[0038] S2: Explant disinfection: Explant disinfection is performed by professionals, wherein the professionals transfer the cleaned stem segments to a sterile beaker or a triangular flask on a clean bench, and use 75% ethanol for preliminary disinfection for 30 seconds, and then use sterile water to wash twice after the preliminary disinfection. After the washing, use 0.1% mercuric chloride for secondary disinfection for 8 minutes. After the secondary disinfection, pour out the disinfectant and wash three times with sterile water, and each washing time is 5 minutes. The number of explant stem segments in the explant disinfection process is not less than 50;

[0039] S3: Primary culture: Professionals prepare rooting medium and perform primary culture on explants using the rooting medium, wherein the rooting medium is prepared with a formula of MS+1.0mg / L 6-BA+0.5mg / L KT+0.2mg / L IBA+30g / L sucrose+7g / L agar+0.1% PPM, the medium pH is 5.8, and IBA and PPM are added when the temperature drops below 60°C after sterilization;

[0040] S4: Subculture: After the primary culture is completed, the professional personnel will carry out subculture, wherein the sterile young shoots obtained after 2 weeks of primary culture are transferred to different subculture culture media, and the professional personnel will observe the production of adventitious shoots, and the total number of inoculated shoots is counted after 30 days of culture, and the wilting rate and the value-added rate are calculated by statistical data, and the preparation formula of the culture medium used in the subculture is MS+0.2mg / L 6-BA+0.2mg / LIBA+7g / L agar+20g / L sucrose, and the pH of the culture medium is 5.8;

[0041] S5: Heat treatment and detoxification: Professionals will perform heat treatment and detoxification on the regenerated buds, transfer the plants to a constant temperature incubator, and culture them for 7 days at 32℃ for 16 hours of light and 30℃ for 8 hours of darkness. After 7 days of culture, the temperature will be raised to 38℃ for 16 hours of light and 32℃ for 8 hours of darkness for 20 days. After 20 days of culture, professional personnel will observe and calculate the survival rate.

[0042] S6: stem apex tissue differentiation: After heat treatment, stem apex tissue differentiation is performed by professionals. When performing stem apex tissue differentiation, professionals need to peel off 1mm stem apex from the detoxified stem segment under a stereo microscope and transfer it into differentiation medium. The differentiation medium formula is MS+2.0mg / L6-BA+0.2mg / L NAA+0.5mg / L KT+30g / L sucrose, and the pH is 5.8. When peeling off the stem apex, the detoxified stem segment is first placed on sterile filter paper to absorb water, and the stem apex is cut off with a sterile surgical blade, and the cut surface is placed on the above differentiation medium;

[0043] S7: Rooting culture of regenerated buds: Professionals carry out rooting culture of regenerated buds, wherein when carrying out rooting culture of regenerated buds, seedlings of >2 cm after differentiation are selected and transferred to newly prepared rooting medium, and professional personnel observe the rooting situation and calculate the rooting rate, and the culture time for rooting culture of regenerated buds is 30 days, and after the rooting of regenerated buds is completed, professional personnel carry out virus detection, and process according to the virus detection result, wherein if the virus detection result shows that there is no virus in the grape seedlings, it is judged that the virus removal is successful, and if the virus detection result shows that there is virus in the grape seedlings, it is judged that the virus removal fails, and if the result is judged that the virus removal is successful, professional personnel will plant the virus-free grape seedlings, and if the result is judged that the virus removal fails, professional personnel will check the virus removal step and re-detoxify the grape seedlings, and at the same time professional personnel will calculate the virus removal success rate of the grape seedlings according to the virus detection result, and compare the calculated data with the existing data, wherein the preparation formula of the rooting medium is 1 / 2MS+0.2mg / L 6-BA+0.2mg / L IBA+20g / L sucrose, the pH of the culture medium is 5.8, and IBA is added when the temperature drops below 60°C after sterilization.

[0044] Embodiment 3

[0045] Reference Figure 1 , a grape detoxification culture medium method, comprising the following steps:

[0046] S1: Preliminary preparation: Preliminary preparation is performed by professionals, wherein the professionals first select grape explants from the grape field, and collect the explants selected from the grape field, and the selected explants are buds that have just protruded and have not germinated on semi-lignified branches, and the distance between two adjacent buds is not less than 3 cm, and the selected grape explants are processed by professionals at the same time, wherein during the processing, the professionals remove excess leaves and tendrils from the collected explants, wrap them with gauze, and rinse them with running water for 4 hours, pinch off the heads and tails of the washed branches, retain the middle section, and cut them into 3 cm small sections with pruning shears, wherein 2 cm is below the bud and 1 cm is above the bud;

[0047] S2: Explant disinfection: Professionals will disinfect the explants. When disinfecting the explants, professionals will transfer the cleaned stem segments to a sterile beaker or a triangular flask on a clean bench and initially disinfect them with 75% ethanol for 30 seconds.

[0048] S3: Primary culture: Professionals prepare rooting medium and perform primary culture on explants using the rooting medium, wherein the rooting medium is prepared with a formula of MS+1.0mg / L 6-BA+0.5mg / L KT+0.2mg / L IBA+30g / L sucrose+7g / L agar+0.1% PPM, the medium pH is 5.8, and IBA and PPM are added when the temperature drops below 60°C after sterilization;

[0049] S4: Subculture: After the primary culture is completed, the professional personnel will carry out subculture, wherein the sterile young shoots obtained after 2 weeks of primary culture are transferred to different subculture culture media, and the professional personnel will observe the production of adventitious shoots, and the total number of inoculated shoots is counted after 30 days of culture, and the wilting rate and the value-added rate are calculated by statistical data, and the preparation formula of the culture medium used in the subculture is MS+0.2mg / L 6-BA+0.2mg / LIBA+7g / L agar+20g / L sucrose, and the pH of the culture medium is 5.8;

[0050] S5: Heat treatment and detoxification: Professionals will perform heat treatment and detoxification on the regenerated buds, transfer the plants to a constant temperature incubator, and culture them for 7 days at 32℃ for 16 hours of light and 30℃ for 8 hours of darkness. After 7 days of culture, the temperature will be raised to 38℃ for 16 hours of light and 32℃ for 8 hours of darkness for 20 days. After 20 days of culture, professional personnel will observe and calculate the survival rate.

[0051] S6: stem apex tissue differentiation: After heat treatment, stem apex tissue differentiation is performed by professionals. When performing stem apex tissue differentiation, professionals need to peel off 1mm stem apex from the detoxified stem segment under a stereo microscope and transfer it into differentiation medium. The differentiation medium formula is MS+2.0mg / L6-BA+0.2mg / L NAA+0.5mg / L KT+30g / L sucrose, and the pH is 5.8. When peeling off the stem apex, the detoxified stem segment is first placed on sterile filter paper to absorb water, and the stem apex is cut off with a sterile surgical blade, and the cut surface is placed on the above differentiation medium;

[0052] S7: Rooting culture of regenerated buds: Professionals carry out rooting culture of regenerated buds, wherein when carrying out rooting culture of regenerated buds, seedlings of >2 cm after differentiation are selected and transferred to newly prepared rooting medium, and professional personnel observe the rooting situation and calculate the rooting rate, and the culture time for rooting culture of regenerated buds is 30 days, and after the rooting of regenerated buds is completed, professional personnel carry out virus detection, and process according to the virus detection result, wherein if the virus detection result shows that there is no virus in the grape seedlings, it is judged that the virus removal is successful, and if the virus detection result shows that there is virus in the grape seedlings, it is judged that the virus removal fails, and if the result is judged that the virus removal is successful, professional personnel will plant the virus-free grape seedlings, and if the result is judged that the virus removal fails, professional personnel will check the virus removal step and re-detoxify the grape seedlings, and at the same time professional personnel will calculate the virus removal success rate of the grape seedlings according to the virus detection result, and compare the calculated data with the existing data, wherein the preparation formula of the rooting medium is 1 / 2MS+0.2mg / L 6-BA+0.2mg / L IBA+20g / L sucrose, the pH of the culture medium is 5.8, and IBA is added when the temperature drops below 60°C after sterilization.

[0053] Embodiment 4

[0054] Reference Figure 1 , a grape detoxification culture medium method, comprising the following steps:

[0055] S1: Preliminary preparation: Preliminary preparation is performed by professionals, wherein the professionals first select grape explants from the grape field, and collect the explants selected from the grape field, and the selected explants are buds that have just protruded and have not germinated on semi-lignified branches, and the distance between two adjacent buds is not less than 3 cm, and the selected grape explants are processed by professionals at the same time, wherein during the processing, the professionals remove excess leaves and tendrils from the collected explants, wrap them with gauze, and rinse them with running water for 4 hours, pinch off the heads and tails of the washed branches, retain the middle section, and cut them into 3 cm small sections with pruning shears, wherein 2 cm is below the bud and 1 cm is above the bud;

[0056] S2: Explant disinfection: Professionals are responsible for explant disinfection, wherein the professional transfers the cleaned stem segments to a sterile beaker or a triangular flask on a clean bench, and uses 75% ethanol for preliminary disinfection for 30 seconds, washes twice with sterile water after the preliminary disinfection, and uses 0.1% mercuric chloride for secondary disinfection for 8 minutes after the washing, and after the secondary disinfection, pours out the disinfectant and washes 4 times with sterile water, and each washing time is 5 minutes, and the number of explant stem segments in the explant disinfection process is not less than 50;

[0057] S3: Primary culture: Professionals prepare rooting medium and use it to culture the explants.

[0058] S4: Subculture: After the primary culture is completed, the professional personnel will carry out subculture, wherein the sterile young shoots obtained after 2 weeks of primary culture are transferred to different subculture culture media, and the professional personnel will observe the production of adventitious shoots, and the total number of inoculated shoots is counted after 30 days of culture, and the wilting rate and the value-added rate are calculated by statistical data, and the preparation formula of the culture medium used in the subculture is MS+0.2mg / L 6-BA+0.2mg / LIBA+7g / L agar+20g / L sucrose, and the pH of the culture medium is 5.8;

[0059] S5: Heat treatment and detoxification: Professionals will perform heat treatment and detoxification on the regenerated buds, transfer the plants to a constant temperature incubator, and culture them for 7 days at 32℃ for 16 hours of light and 30℃ for 8 hours of darkness. After 7 days of culture, the temperature will be raised to 38℃ for 16 hours of light and 32℃ for 8 hours of darkness for 20 days. After 20 days of culture, professional personnel will observe and calculate the survival rate.

[0060] S6: stem apex tissue differentiation: After heat treatment, stem apex tissue differentiation is performed by professionals. When performing stem apex tissue differentiation, professionals need to peel off 1mm stem apex from the detoxified stem segment under a stereo microscope and transfer it into differentiation medium. The differentiation medium formula is MS+2.0mg / L6-BA+0.2mg / L NAA+0.5mg / L KT+30g / L sucrose, and the pH is 5.8. When peeling off the stem apex, the detoxified stem segment is first placed on sterile filter paper to absorb water, and the stem apex is cut off with a sterile surgical blade, and the cut surface is placed on the above differentiation medium;

[0061] S7: Rooting culture of regenerated buds: Professionals carry out rooting culture of regenerated buds, wherein when carrying out rooting culture of regenerated buds, seedlings of >2 cm after differentiation are selected and transferred to newly prepared rooting medium, and professional personnel observe the rooting situation and calculate the rooting rate, and the culture time for rooting culture of regenerated buds is 30 days, and after the rooting of regenerated buds is completed, professional personnel carry out virus detection, and process according to the virus detection result, wherein if the virus detection result shows that there is no virus in the grape seedlings, it is judged that the virus removal is successful, and if the virus detection result shows that there is virus in the grape seedlings, it is judged that the virus removal fails, and if the result is judged that the virus removal is successful, professional personnel will plant the virus-free grape seedlings, and if the result is judged that the virus removal fails, professional personnel will check the virus removal step and re-detoxify the grape seedlings, and at the same time professional personnel will calculate the virus removal success rate of the grape seedlings according to the virus detection result, and compare the calculated data with the existing data, wherein the preparation formula of the rooting medium is 1 / 2MS+0.2mg / L 6-BA+0.2mg / L IBA+20g / L sucrose, the pH of the culture medium is 5.8, and IBA is added when the temperature drops below 60°C after sterilization.

[0062] Embodiment 5

[0063] Reference Figure 1 , a grape detoxification culture medium method, comprising the following steps:

[0064] S1: Preliminary preparation: Preliminary preparation is performed by professionals, wherein the professionals first select grape explants from the grape field, and collect the explants selected from the grape field, and the selected explants are buds that have just protruded and have not germinated on semi-lignified branches, and the distance between two adjacent buds is not less than 3 cm, and the selected grape explants are processed by professionals at the same time, wherein during the processing, the professionals remove excess leaves and tendrils from the collected explants, wrap them with gauze, and rinse them with running water for 4 hours, pinch off the heads and tails of the washed branches, retain the middle section, and cut them into 3 cm small sections with pruning shears, wherein 2 cm is below the bud and 1 cm is above the bud;

[0065] S2: Explant disinfection: Explant disinfection is performed by professionals, wherein the professionals transfer the cleaned stem segments to a sterile beaker or a triangular flask on a clean bench, and use 75% ethanol for preliminary disinfection for 30 seconds, and then use sterile water to wash twice after the preliminary disinfection. After the washing, use 0.1% mercuric chloride for secondary disinfection for 8 minutes. After the secondary disinfection, pour out the disinfectant and wash 5 times with sterile water, and each washing time is 5 minutes. The number of explant stem segments in the explant disinfection process is not less than 50;

[0066] S3: Primary culture: Professionals prepare rooting medium and perform primary culture on explants using the rooting medium, wherein the rooting medium is prepared with a formula of MS+1.0mg / L 6-BA+0.5mg / L KT+0.2mg / L IBA+30g / L sucrose+7g / L agar+0.1% PPM, the medium pH is 5.8, and IBA and PPM are added when the temperature drops below 60°C after sterilization;

[0067] S4: Subculture: After the primary culture is completed, the professional personnel will carry out subculture, wherein the sterile young shoots obtained after 2 weeks of primary culture are transferred to different subculture culture media, and the professional personnel will observe the production of adventitious shoots, and the total number of inoculated shoots is counted after 30 days of culture, and the wilting rate and the value-added rate are calculated by statistical data, and the preparation formula of the culture medium used in the subculture is MS+0.2mg / L 6-BA+0.2mg / LIBA+7g / L agar+20g / L sucrose, and the pH of the culture medium is 5.8;

[0068] S5: Heat treatment and detoxification: Professionals will perform heat treatment and detoxification on the regenerated buds;

[0069] S6: stem apex tissue differentiation: After heat treatment, stem apex tissue differentiation is performed by professionals. When performing stem apex tissue differentiation, professionals need to peel off 1mm stem apex from the detoxified stem segment under a stereo microscope and transfer it into differentiation medium. The differentiation medium formula is MS+2.0mg / L6-BA+0.2mg / L NAA+0.5mg / L KT+30g / L sucrose, and the pH is 5.8. When peeling off the stem apex, the detoxified stem segment is first placed on sterile filter paper to absorb water, and the stem apex is cut off with a sterile surgical blade, and the cut surface is placed on the above differentiation medium;

[0070] S7: Rooting culture of regenerated buds: Professionals carry out rooting culture of regenerated buds, wherein when carrying out rooting culture of regenerated buds, seedlings of >2 cm after differentiation are selected and transferred to newly prepared rooting medium, and professional personnel observe the rooting situation and calculate the rooting rate, and the culture time for rooting culture of regenerated buds is 30 days, and after the rooting of regenerated buds is completed, professional personnel carry out virus detection, and process according to the virus detection result, wherein if the virus detection result shows that there is no virus in the grape seedlings, it is judged that the virus removal is successful, and if the virus detection result shows that there is virus in the grape seedlings, it is judged that the virus removal fails, and if the result is judged that the virus removal is successful, professional personnel will plant the virus-free grape seedlings, and if the result is judged that the virus removal fails, professional personnel will check the virus removal step and re-detoxify the grape seedlings, and at the same time professional personnel will calculate the virus removal success rate of the grape seedlings according to the virus detection result, and compare the calculated data with the existing data, wherein the preparation formula of the rooting medium is 1 / 2MS+0.2mg / L 6-BA+0.2mg / L IBA+20g / L sucrose, the pH of the culture medium is 5.8, and IBA is added when the temperature drops below 60°C after sterilization.

[0071] The grape detoxification culture medium method in Example 1, Example 2, Example 3, Example 4 and Example 5 was tested, and the results were as follows:

[0072]

[0073] Compared with the existing methods, the grape detoxification culture medium methods prepared in Example 1, Example 2, Example 3, Example 4 and Example 5 have uniform detoxification effects and significantly improved detoxification success rates, and Example 1 is the best example.

[0074] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A grape detoxification culture medium method, characterized in that: The following steps are involved: S1: Preliminary preparation: Preliminary preparation is carried out by professionals; S2: Explant disinfection: Explant disinfection is performed by professionals; S3: Primary culture: Professionals prepare rooting medium and use it to culture the explants. S4: Subculture: After the primary culture is completed, professionals will carry out subculture; S5: Heat treatment and detoxification: Professionals will perform heat treatment and detoxification on the regenerated buds; S6: Shoot tip tissue differentiation: After heat treatment, the stem tip tissue differentiation is performed by professionals; S7: Rooting culture of regenerated buds: Rooting culture of regenerated buds is carried out by professionals.

2. A grape detoxification culture medium method according to claim 1, characterized in that: In S1, preliminary preparation is performed by professionals, wherein during the preliminary preparation, the professionals first select grape explants from the grape field, and collect the explants selected from the grape field, and the selected explants are buds that have just protruded and have not germinated on semi-lignified branches, and the distance between two adjacent spores is not less than 3 cm.

3. A grape detoxification culture medium method according to claim 2, characterized in that: At the same time, professionals will process the selected grape explants. During the processing, professionals will remove the excess leaves and tendrils from the collected explants, wrap them with gauze, and rinse them with running water for 4 hours. After rinsing, the branches will be pinched off the heads and tails, leaving the middle section, and cut into 3 cm small sections with branch shears, including 2 cm below the bud and 1 cm above the bud.

4. A grape detoxification culture medium method according to claim 1, characterized in that: In the S2, the explants are disinfected by professionals, wherein the professionals transfer the cleaned stem segments to sterile beakers or flasks on a clean bench, and use 75% ethanol for preliminary disinfection for 30 seconds, and then use sterile water to wash twice after the preliminary disinfection. After the washing, use 0.1% mercuric chloride for secondary disinfection for 8 minutes, and after the secondary disinfection, pour out the disinfectant and wash 3-5 times with sterile water, and each washing time is 5 minutes. The number of explant stem segments in the explant disinfection process is not less than 50.

5. A grape detoxification culture medium method according to claim 1, characterized in that: In S3, professional personnel prepare rooting medium and perform primary culture on explants using the rooting medium, wherein the rooting medium has a formula of MS+1.0mg / L 6-BA+0.5mg / L KT+0.2mg / L IBA+30g / L sucrose+7g / L agar+0.1% PPM, the medium has a pH of 5.8, and IBA and PPM are added when the temperature drops below 60°C after sterilization.

6. A grape detoxification culture medium method according to claim 1, characterized in that: In the S4, after the primary culture is completed, the professional personnel will carry out the subculture, wherein during the subculture, the sterile young shoots obtained after 2 weeks of primary culture are transferred to different subculture culture media, and the professional personnel observe the production of adventitious buds, and the total number of inoculated buds is counted after 30 days of culture, and the wilting rate and the value-added rate are calculated by statistical data, and the preparation formula of the culture medium used in the subculture is MS+0.2mg / L 6-BA+0.2mg / LIBA+7g / L agar+20g / L sucrose, and the pH of the culture medium is 5.

8.

7. A grape detoxification culture medium method according to claim 1, characterized in that: In S5, professionals perform heat treatment to detoxify the regenerated buds, transfer the plants to a constant temperature incubator, and culture them in an environment of 32°C for 16 hours of light and 30°C for 8 hours of darkness for 7 days. After 7 days of culture, the temperature is raised to 38°C for 16 hours of light and 32°C for 8 hours of darkness for heat treatment for 20 days. After 20 days of culture, professionals observe and calculate the survival rate.

8. A grape detoxification culture medium method according to claim 1, characterized in that: In the S6, after the heat treatment, the stem apex tissue differentiation is performed by professionals, wherein the stem apex tissue differentiation is performed by professionals from the detoxified stem segment under a stereo microscope to peel off 1 mm of the stem apex and transfer it into a differentiation medium, wherein the differentiation medium formula is MS+2.0 mg / L6-BA+0.2 mg / LNAA+0.5 mg / L KT+30 g / L sucrose, and the pH is 5.8, wherein when peeling off the stem apex, the detoxified stem segment is first placed on sterile filter paper to absorb moisture, and the stem apex is cut off with a sterile surgical blade, and the cut surface is placed on the above-mentioned differentiation medium.

9. A grape detoxification culture medium method according to claim 1, characterized in that: In S7, professionals perform rooting culture of regenerated buds, wherein when performing rooting culture of regenerated buds, seedlings of >2 cm after differentiation are selected and transferred to a newly prepared rooting medium, and professionals observe the rooting situation and count the rooting rate, and the culture time for rooting culture of regenerated buds is 30 days, and after the rooting of the regenerated buds is completed, professionals perform virus detection, and process according to the virus detection results, wherein if the virus detection results show that there is no virus in the grape seedlings, it is judged that the virus removal is successful, and if the virus detection results show that there is virus in the grape seedlings, it is judged that the virus removal fails, and if the result is that the virus removal is successful, the professionals plant the virus-free grape seedlings, and if the result is that the virus removal fails, the professionals perform a virus removal step inspection, and re-detoxify the grape seedlings, and at the same time, the professionals calculate the virus removal success rate of the grape seedlings according to the virus detection results, and compare the calculated data with the existing data 。 10. A grape detoxification culture medium method according to claim 9, characterized in that: The preparation formula of the rooting medium is 1 / 2MS+0.2mg / L6-BA+0.2mg / L IBA+20g / L sucrose, the pH of the medium is 5.8, and IBA is added when the temperature drops below 60°C after sterilization.

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