Establishment method of cherry rootstock Gisela 6 leaf in-vitro regeneration system and transgenic application of cherry rootstock Gisela 6 leaf in-vitro regeneration system

Abstract

The invention discloses an establishment method of a cherry rootstock Gisela 6 leaf in-vitro regeneration system and transgenic application thereof, and belongs to the field of plant biotechnology and genetic engineering. According to the regeneration system established by optimizing the formula of the culture medium, the regeneration frequency of the 'Gisela 6' leaf is stabilized at a relatively high level, and the quality of the regenerated material is remarkably improved. The callus generated by induction of the method provided by the invention is compact and strong in browning resistance, and the transgenic callus capable of stably expressing RUBY is obtained by a leaf disc transformation method; meanwhile, vitrification of regenerated buds is effectively inhibited, robust plants easy to transplant are obtained, and technical support is provided for precise improvement and breeding speed.

Description

A method for establishing an in vitro leaf regeneration system for cherry rootstock 'Gisela 6' and its transgenic application. Technical Field

[0001] This invention belongs to the field of plant biotechnology and genetic engineering, specifically relating to a method for establishing an in vitro regeneration system of cherry rootstock 'Gisela 6' leaves and its transgenic application. Background Technology

[0002] Tissue culture technology is a key platform for rapid propagation, germplasm preservation, and genetic engineering breeding. Reports of in vitro regeneration systems in cherry rootstock breeding are not uncommon, and high regeneration frequencies can be achieved under certain conditions. However, a high regeneration frequency does not equate to an efficient, stable, and transformable regeneration system. In practical applications, especially in providing high-quality recipient materials for genetic transformation, current technologies still face a series of key challenges hindering industrialization and breeding efficiency: First, callus quality is the primary factor affecting the success of subsequent genetic transformation. Conventional methods often use high concentrations of cytokinin to achieve high callus induction rates, which easily leads to excessively rapid callus growth, loose texture, high water content, and severe browning. Such calluses are extremely sensitive to external stresses, with significantly reduced survival rates during transformation processes such as Agrobacterium infection and antibiotic screening. Even if regeneration occurs, it greatly limits the efficiency of obtaining transgenic positive seedlings.

[0003] Secondly, the physiological state of regenerated seedlings directly affects their suitability for subsequent research or production. Under existing systems, regenerated buds are prone to physiological problems such as vitrification, clustering, or malformation. Vitrified seedlings are weak, have difficulty rooting, and are difficult to transplant successfully; while clustered buds are not conducive to the separation of individual plants and the development of robust seedlings, making it impossible to obtain genetically uniform, robust, independent plants, thus reducing the practical application value of the regeneration system.

[0004] Therefore, for the cherry rootstock 'Gisela 6', there is still a lack of an integrated solution in the field, from the preparation of high-quality recipient materials to efficient regeneration after transformation, which affects the research progress of cherry germplasm innovation and trait improvement. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a method for establishing an in vitro regeneration system of cherry rootstock 'Gisela 6' leaves and its transgenic application. This invention can stably produce dense callus tissue with strong browning resistance to enhance transformation tolerance while ensuring a high regeneration frequency; it can effectively regulate the regeneration process, inhibit physiological abnormalities such as vitrification, and obtain robust, transplantable normal plants.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In the first aspect of the present invention, a method for establishing an in vitro regeneration system of cherry rootstock 'Gisela 6' leaves is provided, comprising the following steps: (1) Subculture: 'Gisela 6' tissue culture seedlings are inoculated into a subculture medium and cultured under light conditions to obtain robust tissue culture seedlings; (2) Callus induction: Leaves of robust tissue culture seedlings cultured for 25 days in step (1) are taken, and wounds are made by cutting the leaves vertically along the main vein with a sterile scalpel. The treated leaves are inoculated into a callus induction medium and cultured under dark conditions to induce the formation of callus tissue; (3) Regeneration bud induction: Leaves with callus tissue obtained in step (2) are transferred to a bud differentiation medium and cultured under light conditions to induce the differentiation of callus tissue into regeneration buds; (4) Regeneration seedling proliferation: Leaves with regeneration buds obtained in step (3) are transferred to a proliferation medium and cultured under light conditions to strengthen seedlings. When the regeneration seedlings grow to 3-5 cm, they are separated from the leaves and inoculated into a subculture medium to continue growing.

[0007] In step (1), the subculture medium uses MS as the basic medium and adds 30-50 g / L sucrose, 0.2-0.4 mg / L 6-BA, 0.1-0.3 mg / L IBA, 0.05-0.15 mg / L GA3, 3-5 g / L plant gel and 0.4-0.6 g / L LMES, with a pH of 5.4.

[0008] In step (2), the callus induction medium uses QL as the basic medium and adds 10-30 g / L sorbitol, 4-6 g / L soluble starch, 4-6 g / L glucose, 0.05-0.15 mg / L 6-BA, 0.5-1.5 mg / L NAA, 3-5 g / L plant gel and 0.4-0.6 g / L MES, with a pH of 5.8.

[0009] In step (3), the bud differentiation medium uses QL as the basic medium and adds 10-30 g / L sorbitol, 4-6 g / L soluble starch, 4-6 g / L glucose, 0.1-0.5 mg / L 6-BA, 0.05-0.1 mg / L NAA, 3-5 g / L plant gel and 0.4-0.6 g / L MES, with a pH of 5.8.

[0010] In step (4), the proliferation medium uses 1 / 2 MMS as the basic medium and adds 20-40 g / L sorbitol, 0.4-0.6 g / L hydrolyzed casein, 0.1-0.5 mg / L 6-BA, 0.05-0.1 mg / L NAA, 3-5 g / L plant gel and 0.4-0.6 g / L MES, with a pH of 5.8.

[0011] This invention, tailored to the growth characteristics of cherry rootstocks, formulated suitable culture media for establishing a leaf transformation system for 'Gisela 6', providing research support for cherry studies. For example, MS medium, rich in nutrients, is used during the subculturing stage; QL medium, more suitable for cell division and differentiation, is used during callus and regenerated shoot induction; and 1 / 2 MMS medium is used during regenerated shoot proliferation to reduce the negative impact of macronutrients on proliferation. Furthermore, sorbitol, more suitable for rootstocks, is used as a carbon source, while soluble starch and glucose are supplemented as multiple energy sources to ensure adequate energy supply for the plants, resulting in good and rapid growth and development.

[0012] Preferably, in step (2), the conditions for dark culture are: temperature 23±1℃ and culture time 15 days.

[0013] In steps (3) and (4), the conditions for light culture are: temperature 23±1℃, light intensity 3000 Lux, and photocycle 16h light / 8h darkness.

[0014] In a second aspect, the present invention provides the application of the above-described method in preventing callus browning in in vitro culture of cherry rootstock 'Gisela 6' leaves and / or improving the survival rate of callus after Agrobacterium infection.

[0015] In a third aspect, the present invention provides the application of the above-described method in inhibiting vitrification of regenerated buds during in vitro regeneration of cherry rootstock 'Gisela 6'.

[0016] The beneficial effects of this invention: This invention establishes an in vitro leaf regeneration system for the cherry rootstock 'Gisela 6'. Through systematic optimization of the culture medium formulation and process, it not only achieves a high frequency of adventitious bud regeneration but also significantly improves the overall quality of the regenerated material. The induced callus is dense and exhibits strong resistance to browning, greatly enhancing its tolerance to stresses such as Agrobacterium infection. Simultaneously, this system effectively inhibits vitrification of regenerated buds, resulting in robust and easily transplantable plants. Based on this high-quality regeneration system, an efficient genetic transformation platform was further constructed: on the one hand, the GFP / NptII system successfully achieved efficient transformation from callus tissue to transgenic resistant buds and complete plants; on the other hand, the RUBY / HptII system established a transformation verification scheme that allows for rapid and intuitive identification through visual observation at the callus stage. The successful application of these two different reporter and screening systems demonstrates the high reliability and broad adaptability of this regeneration system as a genetic transformation recipient, providing crucial technical support for gene function research and molecular breeding of cherry rootstocks. Attached Figure Description

[0017] Figure 1 shows the process of establishing a cherry leaf regeneration system.

[0018] Figure 2 shows the plasmid map of the plant overexpression vector containing the RUBY reporter gene used in Example 5.

[0019] Figure 3 shows the genetic transformation process based on the RUBY reporter gene; Figure A shows the state of co-culture of leaves infected with Agrobacterium, and Figure B shows the transgenic callus with a red phenotype generated at the leaf cut after selection and culture with hygromycin. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] The cherry rootstock 'Gisela 6' (Prunus cerasus × P. canescens cv. Gisela 6) has attracted much attention in my country's cherry industry development as an excellent semi-dwarfing rootstock for sweet cherries. Currently, establishing an efficient and stable in vitro regeneration system for 'Gisela 6', a hybrid tetraploid with a complex genetic background, especially a regeneration system using leaves as explants, still faces significant challenges; furthermore, conventional regeneration systems often neglect systematic control over the quality of regenerated materials.

[0022] Therefore, there is an urgent need in this field to develop a highly efficient and high-quality in vitro leaf regeneration system specifically for the cherry rootstock 'Gisela 6'. This system would achieve high-frequency regeneration while improving the quality of the regenerated material, effectively preventing browning of wound tissue, significantly enhancing its tolerance and survival rate during genetic transformation, and obtaining robust regenerated plants free from vitrification. This would lay a solid technical foundation for germplasm innovation and biotechnology breeding of this valuable rootstock.

[0023] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0024] Example 1: Method for establishing in vitro regeneration of cherry rootstock 'Gisela 6' leaves (1) Cherry rootstock tissue culture seedlings were grown into robust tissue culture seedlings in subculture medium (MS + 40g sucrose + 0.3 mg / 6-BA + 0.2mg / L IBA + 0.1mg / GA + 4 g / L plant gel + 0.5 g / L MES, pH 5.4). The growth conditions were a temperature of 23±1℃, a light intensity of 5000Lux, and a photoperiod of 16h / 8h (Figure 1-A).

[0025] (2) Cut leaves from tissue culture seedlings that have been cultured for 25 days (Figure 1-B), cut them vertically to the main vein with a sterile scalpel, and place them on callus induction medium (QL + 20g sorbitol + 5g soluble starch + 5g glucose + 0.1 mg / L 6-BA + 1 mg / L NAA + 4 g / L plant gel + 0.5 g / L MES, pH 5.8) for dedifferentiation. The growth conditions are a temperature of 23±1℃ and dark culture for about 15 days until callus tissue is produced (Figure 1-C).

[0026] (3) Leaves with callus tissue were transferred to a shoot differentiation medium (QL + 20g sorbitol + 5g soluble starch + 5g glucose + 0.1mg / L 6-BA + 0.05mg / L NAA + 4 g / L plant gel + 0.5 g / L MES, pH 5.8) for redifferentiation. The growth conditions were: temperature 23±1℃, light intensity 3000Lux, and photoperiod 16h / 8h. Culture was continued for 20 days until regenerated shoots formed from the callus tissue (Figure 1-D).

[0027] (4) Leaves with regenerated buds (full name: regenerated adventitious buds) were transferred to proliferation medium (1 / 2 MMS + 30 g / L sorbitol + 0.5 g / L hydrolyzed casein + 0.1 mg / L 6-BA + 0.05 mg / L NAA + 4 g / L plant gel + 0.5 g / L LMES, pH 5.8) for seedling growth. The light intensity was 3000 Lux, and the photoperiod was 16 h / 8 h. The seedlings were cultured for 20 days until they grew to 3-5 cm (Figure 1-E). They were then separated from the leaves and inoculated into subculture medium for continued growth (Figure 1-F).

[0028] After callus tissue differentiated into buds, the regeneration frequency of adventitious buds from leaves was statistically analyzed. Regeneration frequency = (Number of regenerated adventitious bud leaves / Number of inoculated leaves) × 100% Table 1: Statistics on the in vitro regeneration efficiency of cherry rootstock leaves. Example 2: Verification of callus quality and browning inhibition effect. To evaluate the control effect of this system on callus quality and browning, the following comparative experiment was set up: Experimental group: The callus induction culture medium described in this invention (i.e. the formula used in step (2) of Example 1 of this invention) was used.

[0029] Control group 1: Based on the experimental group's formula, sorbitol was removed.

[0030] Control group 2 (conventional high hormone control): The conventional induction medium used in this field (MS + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose).

[0031] Each group was inoculated with 30 leaf explants. Except for the difference in the culture medium formula, the other culture methods were the same as in Example 1.

[0032] Immediately after the callus induction culture stage, experiments were conducted on callus quality and browning inhibition effect. Browning rate: the percentage of callus pieces with browned area exceeding 50% of the total area. Callus texture score ranged from 1 (loose and brittle) to 5 (dense and compact), and the average value was obtained by three blind evaluations.

[0033] Agrobacterium infection tolerance: Newly induced callus blocks from each treatment were subjected to Agrobacterium tumefaciens bacterial suspension (OD200) containing acetylsuccinone. 600 =0.6) After immersion for 10 minutes and culturing for 3 days, the proportion of surviving callus that did not undergo water staining or severe browning was counted.

[0034] Each experiment was repeated three times, and the average value of the data was calculated.

[0035] Table 2: Verification of callus quality and browning inhibition effect The statistical results above show that the culture medium of the present invention can significantly reduce callus browning, improve its texture, and greatly increase its survival rate in genetic transformation.

[0036] Example 3: Verification of regenerated bud quality and vitrification inhibition. To evaluate the quality of regenerated buds, the following experiment was set up: Experimental group: The bud differentiation medium and proliferation medium described in this invention (i.e. the formula used in steps (3) and (4) of Example 1) were used for continuous culture.

[0037] Control group 1: Based on the experimental group's formula, sorbitol was removed.

[0038] Two control groups were compared (conventional high-hormone control): MS basal medium was used, supplemented with 5.0 mg / L 6-BA and 0.05 mg / L NAA.

[0039] Each group was inoculated with 20 leaf explants with callus tissue of uniform growth state induced by the method of Example 1, and the experiment was independently repeated 3 times. Except for the difference in the culture medium formula, the culture conditions (temperature, light, photoperiod) and culture time from shoot differentiation to proliferation were exactly the same as those described in Example 1.

[0040] After the regeneration bud induction culture stage, the vitrification rate was tested. Vitrification rate is the percentage of regeneration buds with transparent and edematous leaves out of the total number of regeneration buds.

[0041] Plant growth potential was measured after the regenerated seedling propagation culture stage. Plant growth potential was measured by randomly measuring the plant height and basal stem diameter of the regenerated seedlings.

[0042] Table 3: Verification of Regenerated Bud Quality and Vitrification Inhibition The statistical results above show that the system of the present invention can effectively inhibit the vitrification and clustering of regenerated buds and promote the formation of robust seedlings.

[0043] Example 4: Genetic transformation efficiency and process verification based on the GFP / NptII system. To comprehensively evaluate the efficiency and stability of the regeneration system established in this invention in practical genetic transformation applications, an Agrobacterium-mediated stable transformation experiment was conducted.

[0044] 1. Experimental Design and Grouping: The callus tissue induced in the experimental group in Example 2 was used as the recipient.

[0045] Control group: Callus tissue induced in control group 2 of Example 2 was used as the recipient. 2. Agrobacterium strain and infection were carried by Agrobacterium tumefaciens strain EHA105, which carries a binary expression vector containing a GFP reporter gene and an NptII selection marker gene. The activated Agrobacterium was resuspended in liquid infection medium (MS salt solution containing 200 μM acetylsuccinone) to OD. 600 = 0.6.

[0046] 3. Genetic transformation process (1) Pre-culture: Place the callus tissue on a solid pre-culture medium (the bud differentiation medium formula of this invention + 200μM acetylsyl syringone) and culture in the dark at 23±1℃ for 2 days.

[0047] (2) Infection and co-culture: After infecting the pre-cultured callus with Agrobacterium, the callus was transferred to a co-culture medium (the same as the pre-culture medium) and cultured in the dark at 20±1℃ for 3 days.

[0048] (3) Screening culture: After co-culture, the callus tissue was washed three times with sterile water containing 500 mg / L cephalosporin, and then transferred to the screening medium. The screening medium was based on the bud differentiation medium formula of the present invention, with the addition of 500 mg / L cephalosporin and 50 mg / L kanamycin. Subculture was performed every 3 weeks for 8 weeks.

[0049] (4) Seedling cultivation: The resistant shoots (length > 0.5 cm) produced during the screening culture are cut off and transferred to the seedling cultivation medium. The seedling cultivation medium is based on the proliferation medium formula of this invention, with the addition of 250 mg / L cephalosporin and 25 mg / L kanamycin. Cultivate until complete plants are obtained.

[0050] Resistant callus rate: After 4 weeks of screening culture, the percentage of callus pieces that can continue to grow (without browning and death) is counted out of the initial number of infected pieces.

[0051] Resistance bud acquisition rate: At the end of 8 weeks of screening culture, the percentage of explants that can produce at least one resistance bud is counted out of the initial number of explants.

[0052] PCR positivity rate: Twenty robust seedlings that had been cultured for 4 weeks were randomly selected, genomic DNA was extracted, and PCR detection was performed using NptII and GFP specific primers. The proportion of plants that amplified the expected band was counted.

[0053] 4. The average data from three independent replicate experiments are shown in Table 4: Table 4: Validation of genetic transformation efficiency Table 4 shows that both groups of resistant plants exhibited high positive rates in PCR testing (93.3% in the experimental group and 89.5% in the control group), confirming the rigorous effectiveness of the antibiotic screening system used in this experiment. The experimental group showed significantly higher rates of resistant callus and resistant shoots than the control group (approximately 2.2-fold and 2.5-fold increases, respectively). This directly demonstrates that the system of this invention can greatly reduce losses caused by material quality issues in the early stages of screening, and can generate a greater number of resistant calluses and resistant shoots from the initial explants, thereby significantly improving the overall efficiency and total yield of obtaining a transgenic positive plant.

[0054] Example 5: Rapid Transformation Validation Based on the RUBY / HptII Visual Reporter System. To provide a rapid and intuitive genetic transformation validation scheme and further verify the stability and efficiency of the regeneration system of this invention in receiving exogenous genes, this example uses a vector containing the RUBY reporter gene for Agrobacterium-mediated leaf disc transformation. The RUBY gene, when expressed in plant cells, catalyzes the synthesis of betaine, causing the transformed tissue to appear visible as a red color, thus enabling early, non-destructive identification of the transformation event.

[0055] 1. Experimental materials: Plant materials: robust tissue culture seedlings of 'Gisela 6' that had grown for 20 days, obtained using the method in Example 1, were used as explants, with their fully expanded young leaves.

[0056] Strains and vectors: Agrobacterium tumefaciens EHA105, carrying an overexpression vector containing the RUBY reporter gene and the hygromycin resistance gene (HptII) (vector map is shown in Figure 2).

[0057] 2. Main culture medium: YEB liquid medium (for Agrobacterium culture).

[0058] Modified WPM liquid medium (for preparing infection solution): 1 / 2 WPM salt + 0.5 g / L MES + 1.0 g / L polyvinylpyrrolidone, pH 5.8.

[0059] Callus induction medium: the callus induction medium described in step (2) of Example 1 of the present invention.

[0060] 3. Experimental Methods (1) Preparation of Agrobacterium infection solution: Activated Agrobacterium was inoculated into YEB liquid medium supplemented with 50 mg / L kanamycin, 25 mg / L rifampin and 50 mg / L streptomycin (rifampin was used to confirm that it was Agrobacterium tumefaciens, streptomycin was used to confirm that it was strain EHA105 of Agrobacterium tumefaciens, and kanamycin was used to confirm that it was EHA105 containing the desired vector RUBY) and cultured at 28℃ and 200 rpm until OD. 600 =0.7. Collect bacterial cells by centrifugation at 4000 rpm for 5 min, resuspend in modified WPM liquid medium, and adjust OD. 600 Set to 0.5, for later use.

[0061] (2) Leaf disc preparation and infection: Cut leaves from tissue culture seedlings and place them in a sterile petri dish. Make three cuts perpendicular to the main vein with a sterile scalpel. Immerse the cut leaves in the prepared Agrobacterium tumefaciens solution for 8 minutes.

[0062] (3) Co-culture: After the infection is completed, use sterile filter paper to absorb the excess bacterial liquid on the leaf surface, and inoculate it onto the callus induction medium. Co-culture for 2 days at 25°C in the dark (Figure 3-A).

[0063] (4) Screening culture and callus induction: After co-culture, the leaves were transferred to callus induction medium supplemented with 3 mg / L hygromycin (screening agent) and 300 mg / L termethin (antibacterial agent) and screened in the dark at 25±1℃. Observe regularly until resistant callus tissue with a typical red phenotype is visible to the naked eye at the leaf cut (Figure 3-B).

[0064] (5) Data statistics: Explants (leaf discs) that produce red callus are considered to have successfully transformed. The transformation efficiency is calculated as follows: Transformation efficiency (%) = (Number of explants that produce red callus / Total number of inoculated explants) × 100%.

[0065] 3. Experimental Results The experiment was repeated three times, with 30 leaf disc explants inoculated per group each time. The results are shown in the table below: This embodiment establishes a rapid verification method for genetic transformation of 'Gisela 6' leaves based on the RUBY visualization reporting system. Using high-quality tissue culture seedling leaves provided by the regeneration system of this invention as recipients, transgenic callus tissue with a red phenotype was obtained after Agrobacterium infection and hygromycin screening with a stable efficiency of 30.7%.

[0066] Compared with Example 4, this example used a different gene combination (RUBY / HptII) and screening agent (hygromycin), and also achieved success, proving that the regeneration system of the present invention does not depend on a specific vector or screening system, but is a highly universal and adaptable genetic transformation platform.

[0067] In summary, Examples 4 and 5 together demonstrate that, regardless of whether an instrument-detectable GFP system or a visually perceptible RUBY system is used, stable and efficient genetic transformation can be achieved based on the high-quality in vitro regeneration system established in this invention. This greatly enhances the practical value and operability of this invention in cherry rootstock gene function research and molecular breeding.

[0068] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for establishing an in vitro regeneration system for cherry rootstock 'Gisela 6' leaves, characterized in that, Includes the following steps: (1) Subculture: 'Gisela 6' tissue culture seedlings were inoculated into subculture medium and cultured under light conditions to obtain robust tissue culture seedlings; (2) Callus induction: Leaves of robust tissue culture seedlings cultured for 25 days in step (1) were taken, and wounds were made by cutting the leaves vertically along the main vein with a sterile scalpel. The treated leaves were inoculated into callus induction medium and cultured under dark conditions to induce callus formation; (3) Regeneration bud induction: Leaves with callus obtained in step (2) were transferred to bud differentiation medium and cultured under light conditions to induce callus differentiation into regeneration buds; (4) Regeneration seedling proliferation: Leaves with regeneration buds obtained in step (3) were transferred to proliferation medium and cultured under light conditions to strengthen seedlings. When the regeneration seedlings grew to 3-5 cm, they were separated from the leaves and inoculated into subculture medium to continue growing.

2. The method according to claim 1, characterized in that, In step (1), the subculture medium uses MS as the basic medium and adds 30-50 g / L sucrose, 0.2-0.4 mg / L 6-BA, 0.1-0.3 mg / L IBA, 0.05-0.15 mg / L GA3, 3-5 g / L plant gel and 0.4-0.6 g / L MES, with a pH of 5.

4.

3. The method according to claim 1, characterized in that, In step (2), the callus induction medium uses QL as the basic medium and adds 10-30 g / L sorbitol, 4-6 g / L soluble starch, 4-6 g / L glucose, 0.05-0.15 mg / L 6-BA, 0.5-1.5 mg / L NAA, 3-5 g / L plant gel and 0.4-0.6 g / L MES, with a pH of 5.

8.

4. The method according to claim 1, characterized in that, In step (3), the bud differentiation medium uses QL as the basic medium and adds 10-30 g / L sorbitol, 4-6 g / L soluble starch, 4-6 g / L glucose, 0.1-0.5 mg / L 6-BA, 0.05-0.1 mg / L NAA, 3-5 g / L plant gel and 0.4-0.6 g / L MES, with a pH of 5.

8.

5. The method according to claim 1, characterized in that, In step (4), the proliferation medium uses 1 / 2 MMS as the basic medium and adds 20-40 g / L sorbitol, 0.4-0.6 g / L hydrolyzed casein, 0.1-0.5 mg / L 6-BA, 0.05-0.1 mg / L NAA, 3-5 g / L plant gel and 0.4-0.6 g / L MES, with a pH of 5.

8.

6. The method according to claim 1, characterized in that, In step (2), the conditions for dark culture are: temperature 23±1℃ and culture time 15 days.

7. The method according to claim 1, characterized in that, In steps (3) and (4), the conditions for light culture are: temperature 23±1℃, light intensity 3000 Lux, and photocycle 16h light / 8h darkness.

8. The application of the method according to any one of claims 1-7 in preventing callus browning in in vitro culture of cherry rootstock 'Gisela 6' leaves and / or improving the survival rate of callus after Agrobacterium infection.

9. The application of the method according to any one of claims 1-7 in inhibiting vitrification of regenerated buds during in vitro regeneration of cherry rootstock 'Gisela 6'.

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