Grape test tube micro-grafting seedling raising method

Abstract

The invention discloses a grape test tube micro-grafting seedling raising method, and relates to the technical field of plant biologication.The method comprises the steps that initial rootstocks resistant to at least one of drought, low temperature or saline-alkali stress in Ningxia and initial scions adapting to the local climate in Ningxia are selected; cutting one end of the to-be-treated scion into a V shape to obtain a target scion, cutting a vertical notch in one end of the to-be-treated rootstock to obtain a target rootstock, inserting the V-shaped end of the target scion into the vertical notch end of the target rootstock to obtain a grafting body, and fixing an interface of the grafting body through a silicone tube; the grafted body is placed in a B-5 culture medium to be cultured at the day and night temperature of 24-26 DEG C / 18-21 DEG C, the illumination period of 16 h / d and the light intensity of 20000 lx; the initial grafted seedlings are transplanted into a matrix with the ratio of black soil to vermiculite to perlite being 3: 1: 1, the matrix is irrigated with a 1 / 8 MS nutrient solution, and target grafted seedlings are obtained. According to the method, by optimizing stock selection, grafting steps and culture conditions, efficient breeding of the stress-resistant grape variety is achieved.

Description

Technical Field

[0001] The present application relates to the field of plant biotechnology, and in particular to a method for raising grape seedlings by micro-grafting in vitro. Background Art

[0002] Micrografting is a technique for grafting rootstocks and scions in a test tube. It is a combination of tissue culture and grafting technology, and has gradually become an important means of studying grape resistance. Micrografting technology is a combination of tissue culture and grafting technology. It has the advantages of short cycle, high survival rate, and no seasonal restrictions. However, in the current micrografting technology, grape grafting often faces problems such as low rootstock and scion affinity, callus interference, and the spread of pests and diseases, resulting in poor interface healing and abnormal growth (such as the "big and small feet" phenomenon), especially in arid, low-temperature, and salinized areas such as Ningxia. The current grafting technology has not designed a rootstock screening system for regional adversity, and the screening efficiency of resistant varieties is low. It is difficult to quickly verify the influence of the rootstock on the drought resistance, cold resistance, and salt-alkali resistance of the scion, that is, it is difficult to quickly screen stress-resistant rootstocks. Summary of the invention

[0003] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a method for raising grape seedlings by micro-grafting in vitro, which achieves efficient breeding of stress-resistant grape varieties by optimizing rootstock selection, grafting steps and culture conditions.

[0004] The present application provides a method for growing grape seedlings by micro-grafting in vitro, comprising:

[0005] Selection of rootstock and scion: Selecting the initial rootstock resistant to at least one of the drought, low temperature or salinity stress in Ningxia and the initial scion adapted to the local climate of Ningxia;

[0006] Aseptic treatment: using sterilized tools in a clean bench to cut off 1.5-2 cm stem segments with 1-2 nodes from the initial rootstock and the initial scion, respectively, and disinfecting the stem segments of the initial rootstock and the initial scion with 75% alcohol, respectively, to obtain a rootstock to be treated and a scion to be treated;

[0007] Grafting operation: in a clean bench, one end of the scion to be treated is cut into a V shape to obtain a target scion, a vertical incision is cut at one end of the stock to be treated to obtain a target stock, the V-shaped end of the target scion is inserted into the vertical incision end of the target stock to obtain a graft, and the interface of the graft is fixed by a silicone tube;

[0008] Tissue culture environment control: the grafted body is placed in a B-5 culture medium and cultured at a day and night temperature of 24-26°C / 18-21°C, a photoperiod of 16h / d, and a light intensity of 20000lx to obtain an initial grafted seedling;

[0009] Transplantation management: The initial grafted seedlings were transplanted into a matrix of black soil: vermiculite:perlite=3:1:1, and the matrix was irrigated with 1 / 8MS nutrient solution to obtain target grafted seedlings.

[0010] According to some embodiments of the present application, the formula of the B-5 culture medium is: 3.21 g / L B-5 basal culture medium + 30 g / L sucrose + 7 g / L agar.

[0011] According to some embodiments of the present application, the pH value of the B-5 culture medium is between 5.8 and 6.0.

[0012] According to some embodiments of the present application, the silicone tube is a transparent silicone tube, the outer diameter of the transparent silicone tube is 0.3 cm, and the inner diameter of the transparent silicone tube is 0.2 cm.

[0013] According to some embodiments of the present application, the length of the V-shaped end of the target scion is 0.5 cm, and the length of the cut at the vertical cut end of the target rootstock is 0.4 cm.

[0014] According to some embodiments of the present application, before the initial stock and the initial scion are respectively cut off with a sterilization tool and the stem segments of 1.5 to 2 cm with 1 to 2 nodes, and the stem segments of the initial stock and the initial scion are respectively disinfected with 75% alcohol to obtain the to-be-treated stock and the to-be-treated scion, the method includes:

[0015] A clefting tool is obtained, and the clefting tool is wrapped with tin foil. The wrapped clefting tool is placed in a high-temperature sterilizer and sterilized at a temperature of 115 to 125° C. for 20 minutes to obtain a sterilized tool.

[0016] According to some embodiments of the present application, the initial scion is one of Cabernet Sauvignon, Marselan and Chardonnay.

[0017] The beneficial effects of the present invention are as follows: a stress-resistant rootstock-scion combination system is directly constructed through regional screening, the environmental restrictions in Ningxia are specifically addressed, and the efficiency of screening stress-resistant varieties is significantly improved; the stem segments are cut by ultra-clean workbench operation and sterilized tools, combined with 75% alcohol disinfection, to minimize exogenous pollution (such as pathogens) and reduce the spread of diseases and pests; the precise docking of the V-shaped scion and the vertical incision rootstock is adopted, and the interface is fixed with a silicone tube, which not only enhances the fit of the rootstock and scion (improves affinity), but also reduces the interference of excessive callus proliferation on the healing of the interface, thereby avoiding abnormal growth phenomena such as "big and small feet"; the B-5 culture medium is adopted, combined with the standardized culture conditions of day and night temperature difference (24-26°C / 18-21°C), long light (16h / d), and high light intensity (20000lx), to promote rapid callus differentiation and growth of the grafted body and shorten the culture cycle; the black soil: vermiculite: pearl The matrix with a ratio of 3:1:1 rock provides a root environment with a balance of air permeability and water retention, and cooperates with 1 / 8MS nutrient solution to accurately control nutrient supply, ensuring a smooth transition of grafted seedlings from tissue culture environment to field adversity, and enhancing the expression of drought resistance, salt-alkali resistance and other traits; through test tube micro-grafting technology combined with standardized stress-resistant rootstock screening, drought, low temperature or salt-alkali stress can be simulated in a controlled tissue culture environment, and the resistance performance of the rootstock-scion combination can be quickly evaluated, which significantly shortens the verification cycle. The target grafted seedlings finally obtained can be directly used in Ningxia adversity field trials to accelerate the selection and promotion of stress-resistant grape varieties; standardized operations of the entire process from rootstock selection, sterile treatment to tissue culture environment can reduce human errors and improve technical repeatability; combined with the efficient characteristics of test tube micro-grafting, factory-based production of stress-resistant grape seedlings can be achieved, breaking through the bottleneck of traditional grafting technology restricted by seasons, and meeting the large-scale planting needs of Ningxia and similar adversity areas. The present application achieves efficient breeding of stress-resistant grape varieties through this method by optimizing rootstock selection, grafting steps and culture conditions.

[0018] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0020] Figure 1 A schematic diagram of the process of the grape test tube micro-grafting seedling raising method provided in the embodiment of the present application;

[0021] Figure 2 A schematic diagram of a portion of the aseptic processing process provided in an embodiment of the present application;

[0022] Figure 3 A schematic diagram of the grafting operation process provided in the embodiment of the present application;

[0023] Figure 4 A comparative schematic diagram of the grafting operation process of conventional grape seedling cultivation and the grape test tube micro-grafting seedling cultivation of the present application;

[0024] Figure 5 This is a schematic diagram of the grape test tube micro-grafted seedlings after transplanting and domestication provided in the embodiments of the present application. DETAILED DESCRIPTION

[0025] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0026] In the description of the present application, it should be understood that descriptions involving orientation, such as up, down, front, back, left, right, etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0027] In the description of this application, if there is a description of first or second, it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0028] In the description of this application, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in this application based on the specific content of the technical solution.

[0029] Micrografting is a technique for grafting rootstocks and scions in a test tube. It is a combination of tissue culture and grafting technology, and has gradually become an important means of studying grape resistance. Micrografting technology is a combination of tissue culture and grafting technology. It has the advantages of short cycle, high survival rate, and no seasonal restrictions. However, in the current micrografting technology, grape grafting often faces problems such as low rootstock and scion affinity, callus interference, and the spread of pests and diseases, resulting in poor interface healing and abnormal growth (such as the "big and small feet" phenomenon), especially in arid, low-temperature, and salinized areas such as Ningxia. The current grafting technology has not designed a rootstock screening system for regional adversity, and the screening efficiency of resistant varieties is low. It is difficult to quickly verify the influence of the rootstock on the drought resistance, cold resistance, and salt-alkali resistance of the scion, that is, it is difficult to quickly screen stress-resistant rootstocks.

[0030] In order to solve the above problems, the present application proposes a method for growing grape seedlings by micro-grafting in a test tube. The embodiments of the present application will be further described below in conjunction with the accompanying drawings.

[0031] Reference Figure 1 , Figures 3 to 5 A grape test tube micro-grafting seedling raising method comprises the following steps:

[0032] Step S100, selection of rootstock and scion: selecting an initial rootstock resistant to at least one of drought, low temperature or saline-alkali stress in Ningxia and an initial scion adapted to the local climate of Ningxia.

[0033] It should be noted that in the rootstock selection stage, it is clearly required to select rootstocks that are resistant to at least one of Ningxia's drought, low temperature or saline-alkali stress. At the same time, the scion needs to adapt to the local climate of Ningxia to directly improve the plant's ability to survive in adversity; Scion adaptation: Select scion that adapts to Ningxia's climate to ensure that the aboveground part maintains the excellent traits of local varieties (such as fruit quality and yield), and at the same time combine it with stress-resistant rootstocks to form an ideal combination of "stress-resistant rootstock + high-quality scion".

[0034] Step S200, aseptic treatment: using a sterilization tool in a clean bench to cut off 1.5-2 cm stem segments with 1-2 nodes from the initial rootstock and the initial scion, respectively, and disinfecting the stem segments of the initial rootstock and the initial scion with 75% alcohol to obtain a to-be-treated rootstock and a to-be-treated scion.

[0035] In this step, a sterile environment ensures the success rate of seedling cultivation: (1) Clean bench operation and disinfection: The stem segments are treated with 75% alcohol and sterilized with sterilized tools to effectively avoid microbial contamination and reduce the risk of fungal or bacterial infection during tissue culture; (2) Standardized cutting specifications: 1.5-2 cm stem segments with 1-2 nodes balance tissue activity and operational feasibility. The nodes provide callus differentiation sites and promote graft healing.

[0036] Step S300, grafting operation: in an ultra-clean workbench, one end of the scion to be processed is cut into a V shape to obtain a target scion, a vertical incision is cut at one end of the stock to be processed to obtain a target stock, the V-shaped end of the target scion is inserted into the vertical incision end of the target stock to obtain a graft, and the interface of the graft is fixed by a silicone tube.

[0037] In this step, grafting technology innovation improves the efficiency of interface healing: (1) V-shaped scion and vertical incision design: increase the contact area between the rootstock and the scion, promote rapid connection of the vascular bundle, and accelerate the transport of water and nutrients; (2) Silicone tube fixation: compared with traditional binding materials, silicone tube is more stable under sterile conditions, avoiding interface displacement or contamination, while maintaining moderate air permeability

[0038] It should be noted that Figure 3A represents the target rootstock, B represents the target scion, C represents the graft, and D represents the silicone tube; Figure 4 A in the figure represents the conventional grape grafting seedling raising process and the number of days required, and A represents the grape test tube micro-grafting seedling raising grafting process and the number of days required in the present application, wherein scion represents the target scion, and rootstocks represents the target rootstock.

[0039] Step S400, tissue culture environment control: the graft is placed in a B-5 culture medium and cultured at a day and night temperature of 24-26°C / 18-21°C, a light cycle of 16h / d, and a light intensity of 20000lx to obtain an initial grafted seedling.

[0040] In this step, the tissue culture environment is precisely regulated to optimize growth: (1) B-5 culture medium: rich in organic nitrogen and trace elements, suitable for callus induction and early growth of woody plants, providing balanced nutrition for the grafted body; (2) Day and night temperature difference and photoperiod: 24-26°C during the day to promote photosynthesis, and 18-21°C at night to reduce respiratory consumption; 16 hours of light (20,000 lx high light intensity) simulates the long-day conditions in summer, accelerates the accumulation of photosynthetic products, and shortens the seedling period.

[0041] In some embodiments, the graft is cultured in B-5 medium at a day and night temperature of 25°C / 20°C, a light cycle of 16h / d of light and 8h / d of darkness, and a light intensity of 20000lx to obtain an initial grafted seedling.

[0042] Step S500, transplanting management: transplanting the initial grafted seedlings into a matrix of black soil: vermiculite:perlite=3:1:1, and irrigating the matrix with 1 / 8MS nutrient solution to obtain target grafted seedlings.

[0043] In this step, transplanting management ensures adaptability in the later stage: (1) Substrate ratio (3:1:1): black soil provides water retention and nutrients, vermiculite and perlite enhance air permeability and drainage, avoid the common compaction problem of Ningxia saline-alkali soil, and promote root development; (2) 1 / 8MS nutrient solution irrigation: low-concentration nutrient solution avoids root damage in the early stage of transplanting, gradually adapts to the external environment, and improves the survival rate.

[0044] It should be noted that the test tube micro-grafting technology can be produced in factories, break through seasonal restrictions, shorten the seedling cycle, and standardize the process to reduce manpower and resource consumption. It is suitable for large-scale promotion in ecologically fragile areas such as Ningxia. Through the cultivation of stress-resistant varieties, it reduces irrigation and soil improvement inputs, and helps the sustainable development of grape cultivation in arid and saline-alkali areas. Specifically, through the four core improvements of regionalized stress-resistant rootstock screening system, sterile precision grafting operation, standardized tissue culture environment control and adaptive transplanting management, the problems of low rootstock and scion affinity, pest and disease risks, and low efficiency of stress resistance verification in the background technology are systematically solved, especially providing an efficient, stable and popularizable grape stress-resistant seedling cultivation solution for adverse areas such as Ningxia.

[0045] Exemplarily, the varieties of initial rootstocks selected for drought resistance are 140Ru and 1103P, and the variety of initial scion is Cabernet Sauvignon; the varieties of initial rootstocks selected for salt-alkali resistance are SO4 and 5BB, and the variety of initial scion is Marselan, and at the same time, 0.3% NaCl is added to the transplanting matrix to simulate saline-alkali conditions; the varieties of initial rootstocks selected for cold resistance are Beda and Vitis vinifera, and the variety of initial scion is Chardonnay, and an additional -15°C low temperature stress is added for 48 hours.

[0046] Among them, 5BB is a hybrid of winter grape and riverbank grape, SO4 is the hybrid offspring of winter grape and riverbank grape, 140Ru is a rootstock bred by hybridizing sand grape and other grape varieties, 1103P is a hybrid offspring of winter grape and sand grape, Beda is a hybrid of riverbank grape and American grape, and wild grape directly uses the native wild species wild grape or its hybrid offspring (such as the "Shanhe system" rootstock).

[0047] In some embodiments, the grape in vitro micrografting seedling method can also be performed under bacterial conditions.

[0048] In the present application, a stress-resistant rootstock-scion combination system is directly constructed through regional screening, which specifically solves the environmental restrictions in Ningxia and significantly improves the efficiency of stress-resistant variety screening; the stem segments are cut by ultra-clean workbench operation and sterilized tools, combined with 75% alcohol disinfection, to minimize exogenous pollution (such as pathogens) and reduce the spread of pests and diseases; the V-shaped scion is precisely docked with the vertical incision rootstock, and the interface is fixed with a silicone tube, which not only enhances the fit of the rootstock and scion (improves affinity), but also reduces the interference of excessive callus proliferation on the healing of the interface, thereby avoiding abnormal growth phenomena such as "big and small feet"; the B-5 culture medium is used, combined with the standardized culture conditions of day and night temperature difference (24-26℃ / 18-21℃), long light (16h / d), and high light intensity (20000lx), to promote rapid callus differentiation and growth of the grafted body and shorten the culture cycle; by black soil: vermiculite: perlite=3: The 1:1 matrix provides a root environment with a balance of air permeability and water retention, and cooperates with 1 / 8MS nutrient solution to accurately control nutrient supply, ensuring a smooth transition of grafted seedlings from tissue culture environment to field adversity, and enhancing the expression of drought resistance, salt-alkali resistance and other traits; through test tube micro-grafting technology combined with standardized stress-resistant rootstock screening, drought, low temperature or salt-alkali stress can be simulated in a controlled tissue culture environment, and the resistance performance of the rootstock-scion combination can be quickly evaluated, significantly shortening the verification cycle, and the target grafted seedlings finally obtained can be directly used in Ningxia adversity field trials to accelerate the selection and promotion of stress-resistant grape varieties; from rootstock selection, aseptic treatment to tissue culture environment, the whole process is standardized to reduce human errors and improve technical repeatability; combined with the efficient characteristics of test tube micro-grafting, the factory production of stress-resistant grape seedlings can be realized, breaking through the bottleneck of traditional grafting technology restricted by seasons, and meeting the large-scale planting needs of Ningxia and similar adversity areas. This application uses this method to optimize rootstock selection, grafting steps and culture conditions to achieve efficient breeding of stress-resistant grape varieties.

[0049] In a possible implementation, the formula of B-5 culture medium is: 3.21 g / L B-5 basal culture medium+30 g / L sucrose+7 g / L agar.

[0050] It should be noted that the other ingredients in the formula are: 30 g / L sucrose: as a carbon source, 7 g / L agar: as a coagulant.

[0051] In one possible implementation, the pH value of the B-5 medium is between 5.8 and 6.0.

[0052] It should be noted that the pH value of B-5 culture medium is between 5.8 and 6.0, which meets the weakly acidic environment requirements for plant cell culture.

[0053] In a possible implementation, 0.1 mg / L NAA is further added to the B-5 medium.

[0054] It should be noted that 0.1 mg / L NAA was added to the B-5 medium to promote callus formation.

[0055] In a possible implementation, the silicone tube is a transparent silicone tube, the outer diameter of the transparent silicone tube is 0.3 cm, and the inner diameter of the transparent silicone tube is 0.2 cm.

[0056] In a possible implementation, the length of the V-shaped end of the target scion is 0.5 cm, and the length of the cut at the vertical cut end of the target rootstock is 0.4 cm.

[0057] It should be noted that the tools needed must be sterilized at high temperature in advance. When used, they must be sprayed with 75% disinfectant alcohol and then placed in a clean bench for ultraviolet sterilization. Remove the leaves from the grape tissue culture seedlings, use 1.5-2cm stem segments for the rootstock and scion, and leave 1-2 nodes. Cut the scion into a "V" shape with a size of 0.5cm, cut a 0.4cm incision vertically on the rootstock, and graft the "V"-shaped scion onto the rootstock. Fix the grafting site with a transparent hose with an inner diameter of 0.2cm and an outer diameter of 0.3cm. After the grafting is completed, seal the bottle mouth with a transparent plastic sealing film, mark the variety and time, observe the growth status and grafting site of the grape test tube micro-grafted seedlings every 3 days, and keep records. Steps for soil culture of test tube micro-grafted seedlings: Preparation before transplanting: Wash the roots of the seedlings with ultrapure water to remove residual culture medium and mold carried by the roots. Required matrix: black soil: vermiculite: perlite

[0058] =3:1:1, stir the soil with 1 / 8MS nutrient solution; Management after transplanting: observe the growth of seedlings every 3 days, water on time, and acclimate the grape test tube micro-grafted seedlings for 7-10 days to enable them to grow normally.

[0059] It should be noted that Figure 5 This is a schematic diagram of the grape test tube micro-grafted seedlings after transplanting and domestication.

[0060] It is understandable that, referring to Figure 2 , before step S200, including but not limited to the following steps:

[0061] Step S201, obtaining a clefting tool, wrapping the clefting tool with tin foil, placing the wrapped clefting tool in a high-temperature sterilizer and sterilizing it at a temperature of 115-125° C. for 20 minutes to obtain a sterilized tool.

[0062] Specifically, the experimental preparation work: tools: tweezers, surgical scissors, No. 4 scalpel, iron rack, 15cm glass dish, 15cm filter paper, sealing film, marker pen; silicone tube with inner diameter of 0.2cm and outer diameter of 3mm; disinfection tools: alcohol lamp, high-temperature sterilizer, 75% disinfectant alcohol; high-temperature disinfection: the cleavage tools: tweezers, No. 4 scalpel, 15cm glass dish and 15cm filter paper are wrapped with tin foil and sterilized in a high-temperature sterilizer at 121℃ for 20min; clean bench: ultraviolet sterilization for 30min.

[0063] Understandably, the initial scion was one of Cabernet Sauvignon, Marselan and Chardonnay.

[0064] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0065] The above is a preferred embodiment of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications are also considered to be within the scope of protection of the present application.

[0066] Although the embodiments of the present application have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the claims and their equivalents.

Claims

1. A method for raising grape seedlings by micrografting in vitro, characterized in that: include: Selection of rootstock and scion: Selecting the initial rootstock resistant to at least one of the drought, low temperature or salinity stress in Ningxia and the initial scion adapted to the local climate of Ningxia; Aseptic treatment: using sterilized tools in a clean bench to cut off 1.5-2 cm stem segments with 1-2 nodes from the initial rootstock and the initial scion, respectively, and disinfecting the stem segments of the initial rootstock and the initial scion with 75% alcohol, respectively, to obtain a rootstock to be treated and a scion to be treated; Grafting operation: in a clean bench, one end of the scion to be treated is cut into a V shape to obtain a target scion, a vertical incision is cut at one end of the stock to be treated to obtain a target stock, the V-shaped end of the target scion is inserted into the vertical incision end of the target stock to obtain a graft, and the interface of the graft is fixed by a silicone tube; Tissue culture environment control: the grafted body is placed in a B-5 culture medium and cultured at a day and night temperature of 24-26°C / 18-21°C, a photoperiod of 16h / d, and a light intensity of 20000lx to obtain an initial grafted seedling; Transplantation management: The initial grafted seedlings were transplanted into a matrix of black soil: vermiculite:perlite=3:1:1, and the matrix was irrigated with 1 / 8MS nutrient solution to obtain target grafted seedlings.

2. The grape test tube micrografting seedling raising method according to claim 1, characterized in that: The formula of the B-5 culture medium is: 3.21 g / L B-5 basic culture medium+30 g / L sucrose+7 g / L agar.

3. The grape test tube micrografting seedling raising method according to claim 2, characterized in that: The pH value of the B-5 culture medium is between 5.8 and 6.

0.

4. The grape test tube micrografting seedling raising method according to claim 1, characterized in that: The silicone tube is a transparent silicone tube, the outer diameter of the transparent silicone tube is 0.3 cm, and the inner diameter of the transparent silicone tube is 0.2 cm.

5. The grape test tube micrografting seedling raising method according to claim 1, characterized in that: The length of the V-shaped end of the target scion is 0.5 cm, and the length of the cut at the vertical cut end of the target rootstock is 0.4 cm.

6. The grape test tube micrografting seedling raising method according to claim 1, characterized in that: Before the steps of cutting off 1.5-2 cm stem segments with 1-2 nodes from the initial rootstock and the initial scion by a sterilizing tool, and disinfecting the stem segments of the initial rootstock and the initial scion with 75% alcohol to obtain the rootstock and the scion to be treated, the method comprises: A clefting tool is obtained, and the clefting tool is wrapped with tin foil. The wrapped clefting tool is placed in a high-temperature sterilizer and sterilized at a temperature of 115 to 125° C. for 20 minutes to obtain a sterilized tool.

7. The grape test tube micrografting seedling raising method according to claim 1, characterized in that: The initial scion is one of Cabernet Sauvignon, Marselan and Chardonnay.

Images