A medium combination for grape regeneration system and a culture method thereof

Abstract

The present application relates to the technical field of plant tissue culture, in particular to a medium combination for grape regeneration system and a culture method thereof. The medium combination for grape regeneration system comprises embryogenic callus induction medium, secondary embryo induction proliferation medium and plantlet formation medium. The present application achieves the technical effects of embryoid induction rate of more than 80%, continuous subculture of secondary embryos, 60-day embryoid germination rate of 80% and plantlet formation rate of more than 75%, and solves the key technical problems of difficult embryogenic maintenance, unstable secondary embryo induction and low plantlet formation rate in grape embryogenesis.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a culture medium combination and culture method for grape regeneration systems. Background Technology

[0002] As a vital economic fruit crop worldwide, grape (Vitis spp.) has long relied on traditional hybridization breeding methods for variety improvement. However, most cultivated grape varieties suffer from complex genetic backgrounds, long breeding cycles, and a lack of resistance resources, limiting the improvement of breeding efficiency. With the rapid development of plant biotechnology, utilizing genetic transformation and in vitro mutagenesis for grape germplasm innovation and new variety selection has become an important direction for breaking through traditional breeding bottlenecks. Establishing an efficient and stable in vitro regeneration system is the prerequisite and foundation for achieving grape genetic transformation.

[0003] Since Galzy's successful research on grape shoot tip culture and low-temperature in vitro preservation in 1961, grape tissue culture research has consistently been at the forefront of woody plant research. In 1969, Galzy invented the stem tip culture method and achieved grape shoot tip culture and detoxification. In the 1970s, achievements such as free protoplast culture, embryoid regeneration, and anther induction to obtain a large number of diploid plants were reported, and rapid grape propagation technology became increasingly mature. Since the 1980s, especially the 1990s, research on grape regeneration systems has entered a period of rapid development. Elisabeth, Stamp, Lu Bingzhi, Torregrosa, and other scholars obtained regenerated plants through organogenesis; Martinelli, Emershad, Reustle, Salunkhe, and Yu Xiangrong, among others, obtained regenerated plants through embryoid development. To date, regeneration systems have been successfully established for various grape species, and a small number of transgenic plants have been obtained through genetic transformation.

[0004] Grape regeneration primarily involves two pathways: organogenesis and embryoidogenesis. Organogenesis refers to the direct or callus-induced production of adventitious buds from explants, which then develop into complete plants. Embryoidogenesis, on the other hand, involves non-zygotic cells mimicking the development of zygotic embryos to form somatic embryos and eventually seedlings. Generally, embryoids are considered to originate from single cells, which is advantageous for obtaining uniform transforming clones. Furthermore, embryoids can have sufficient contact with antibiotics, reducing the number of escaped transforming cells, thus offering unique advantages in genetic transformation research.

[0005] Numerous factors influence grape regeneration, primarily categorized into external and internal factors. Among external factors, the type and concentration ratio of hormones are crucial in determining regeneration success or failure. Studies have shown that a suitable combination of cytokinins (such as BA and TDZ) and auxins (such as IAA, NAA, and IBA) is essential for inducing adventitious buds or embryoids. TDZ, as a highly physiologically active phenylurea compound, exhibits superior regeneration-promoting effects compared to 6-BA, particularly for varieties with weak regeneration capabilities. Cultivation conditions such as light and temperature also affect regeneration efficiency; appropriate shading in the early stages promotes leaf regeneration. Low light conditions favor embryoid germination, while strong light promotes robust seedlings. The cultivation temperature is generally controlled at 25±1℃. Among internal factors, genotype has the most significant impact on regeneration ability, with substantial differences in regeneration rates among different grape varieties and rootstocks. Most European grape varieties face regeneration difficulties, while round-leaf grapes and sandy-grown grapes are relatively easy to induce embryoids. Explant type and its physiological state are equally important; young tissues (such as immature embryos, anthers, and shoot tips) have a significantly higher regenerative capacity than mature tissues. Anthers are the most widely used embryoid-induced explants, while shoot tips and young leaves perform better in organogenesis.

[0006] Despite significant progress in grape regeneration research, compared to other woody fruit trees like apples and citrus, grape regeneration systems still suffer from low regeneration rates, poor reproducibility, and strong genotype dependence, severely hindering the progress of grape genetic transformation and genetic engineering breeding. Currently, most reported regeneration systems are only applicable to specific varieties or genotypes, and are complex to operate with long culture cycles, making them difficult to widely apply to different grape rootstock materials. Grape rootstocks play a crucial role in disease resistance, stress tolerance, and plant growth regulation, but their regeneration capacity is often weaker than that of some table grape varieties, and related research lags behind. Therefore, developing a regeneration system that is simple to cultivate, highly efficient, reproducible, and applicable to multiple grape rootstocks has significant theoretical and practical value for promoting grape genetic transformation, in vitro mutagenesis, and the rapid propagation of superior rootstock varieties. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a culture medium combination and its cultivation method for a grape regeneration system.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a culture medium combination for a grape regeneration system, including an embryogenic callus induction medium, a secondary embryo induction proliferation medium, and a seedling culture medium;

[0010] The embryogenic callus induction medium comprises: MS medium + 300 mg / L PVP + 1 mg / L 2,4-D + 0.2 mg / L 6-BA + 6 g / L agar + 30 g / L sucrose + 1 g / L inositol;

[0011] The secondary embryo induction and proliferation medium comprises: 1 / 2 MS medium + 0.5 g / L activated carbon + 0.2 mg / L 6-BA + 6 g / L agar + 15 g / L sucrose;

[0012] The seedling culture medium comprises: MS medium + 8 g / L agar + 30 g / L sucrose + 0.5 g / L activated carbon + 0.093 mg / L NAA + 0.346 mg / L GA3.

[0013] Preferably, the grape varieties include 101-14.

[0014] This invention also provides a method for culturing a grape regeneration system based on the culture medium combination described above, comprising the following steps:

[0015] 1) Inoculate the leaves of sterile grape seedlings onto the embryogenic callus induction medium in the culture medium combination to induce embryogenic callus and obtain embryogenic callus.

[0016] 2) The embryogenic callus tissue obtained in step 1) is inoculated into the secondary embryo induction proliferation culture in the culture medium combination to induce secondary embryos and obtain rooted embryoids;

[0017] 3) The rooting embryos described in step 2) are inoculated onto the seedling culture medium in the culture medium combination for seedling culture to obtain grape seedlings.

[0018] Preferably, the leaf in step 1) has a size of 1 cm × 1 cm.

[0019] Preferably, the conditions for inducing embryogenic callus in step 1) include: dark culture, temperature of 24~26℃, relative humidity of 22%, time of 90 days, and transfer once every 30 days.

[0020] Preferably, the conditions for inducing the secondary embryo in step 2) include: 16 h light and 8 h dark alternation culture, light intensity of 1000 lx, temperature of 24~26℃, relative humidity of 22%, transfer every 30 days, and culture time of 90 days.

[0021] Preferably, the conditions for seedling cultivation in step 3) include: 16 h light and 8 h dark alternation culture, light intensity of 12000 lx, temperature of 25~27℃, relative humidity of 45%, and time of 60 days.

[0022] The present invention also provides the application of the culture medium combination described above in a grape regeneration system.

[0023] The abbreviation for plant hormones used in this invention is as follows:

[0024] Polyvinylpyrrolidone (PVP), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl adenine (6-BA), 1-naphthaleneacetic acid (NAA), gibberellin (GA3).

[0025] The beneficial effects of this invention are:

[0026] This invention utilizes a "stage-specific culture medium combination and precise synergy of light and temperature" to solve technical problems through the following intrinsic connections: In the embryo induction stage, high concentrations of 2,4-D and 6-BA, combined with PVP anti-browning agent, continuously activate the embryogenic potential of somatic cells under dark conditions, overcoming the initial embryoid formation barrier; In the secondary embryo proliferation stage, 1 / 2 MS low-salt culture medium combined with activated carbon adsorbing endogenous inhibitors, combined with weak light and 6-BA, relieves apical dominance inhibition, promoting the repeated formation of secondary embryos on the surface of already elongated embryoids, achieving geometric expansion of embryogenic cells; In the seedling stage, precisely proportioned NAA and GA3 synergistically promote root primordia differentiation and true leaf elongation under high light intensity and diurnal temperature variation, breaking the asynchronous nature of radicle elongation and bud germination. This achieves technical effects such as an 80% embryoid induction rate, continuous subculturing of secondary embryos, an 80% embryoid germination rate after 60 days, and a seedling formation rate exceeding 75%, solving the key technical problems of difficulty in maintaining mesoembryogenicity in grape somatic embryogenesis, unstable secondary embryo induction, and low seedling formation rate. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0028] Figure 1 Induction of callus tissue in leaves 101-14; Note: CK, 1, 2, 3, 4, 5, 6, 7, 8 represent the induction status of leaf callus tissue under different concentrations of hormone treatment;

[0029] Figure 2 Induction of petiole callus from 101-14; Note: CK, 1, 2, 3, 4, 5, 6, 7, 8 represent the induction status of petiole callus under different concentrations of hormone treatment;

[0030] Figure 3 Induction of callus tissue in stem segments 101-14; Note: CK, 1, 2, 3, 4, 5, 6, 7, 8 represent the induction status of callus tissue in stem segments under different concentrations of hormone treatment;

[0031] Figure 4Callus induction rate for different varieties and explants;

[0032] Figure 5 This refers to the process of embryoid elongation, the formation of secondary embryos, and the induction of embryoid seedling formation. Note: 1: embryonic callus; 2: embryoid; 3: cotyledon stage embryoid; 4: secondary embryo; 5: embryoid elongation; 6: embryoid seedling formation. Detailed Implementation

[0033] This invention provides a culture medium combination for a grape regeneration system, comprising an embryogenic callus induction medium, a secondary embryo induction and proliferation medium, and a seedling culture medium. The embryogenic callus induction medium comprises: MS medium + 300 mg / L PVP + 1 mg / L 2,4-D + 0.2 mg / L 6-BA + 6 g / L agar + 30 g / L sucrose + 1 g / L inositol. The secondary embryo induction and proliferation medium comprises: 1 / 2 MS medium + 0.5 g / L activated carbon + 0.2 mg / L 6-BA + 6 g / L agar + 15 g / L sucrose. The seedling culture medium comprises: MS medium + 8 g / L agar + 30 g / L sucrose + 0.5 g / L activated carbon + 0.093 mg / L NAA + 0.346 mg / L GA3. This invention does not specifically limit the source of the above culture media and reagents; those skilled in the art can use commercially available alternatives. In this invention, the grape variety preferably includes 101-14.

[0034] This invention also provides a method for culturing a grape regeneration system based on the culture medium combination described above, comprising the following steps:

[0035] 1) Inoculate the leaves of sterile grape seedlings onto the embryogenic callus induction medium in the culture medium combination to induce embryogenic callus and obtain embryogenic callus.

[0036] 2) The embryogenic callus tissue obtained in step 1) is inoculated into the secondary embryo induction proliferation culture in the culture medium combination to induce secondary embryos and obtain rooted embryoids;

[0037] 3) The rooting embryos described in step 2) are inoculated onto the seedling culture medium in the culture medium combination for seedling culture to obtain grape seedlings.

[0038] This invention involves inoculating sterile grape seedling leaves onto an embryogenic callus induction medium within the aforementioned culture medium combination to induce embryogenic callus, thereby obtaining embryogenic callus. In this invention, the leaf size is preferably 1 cm × 1 cm. The preferred conditions for embryogenic callus induction include: dark culture, a temperature of 24–26°C, a relative humidity of 22%, a time of 90 days, and subculturing every 30 days.

[0039] In this invention, embryogenic callus is inoculated into the culture medium combination for secondary embryo induction and proliferation culture to obtain rooted embryoids. In this invention, the preferred conditions for secondary embryo induction include: 16 h light, 8 h dark alternation culture, light intensity of 1000 lx, temperature of 24–26 °C, relative humidity of 22%, subculturing every 30 days, and a culture time of 90 days.

[0040] In this invention, the rooting embryoids are inoculated onto the seedling culture medium in the culture medium combination for seedling cultivation to obtain grape seedlings. In this invention, the preferred conditions for seedling cultivation include: 16 h light, 8 h dark alternation culture, light intensity of 12000 lx, temperature of 25-27℃, relative humidity of 45%, and time of 60 days.

[0041] The present invention also provides the application of the culture medium combination described above in a grape regeneration system.

[0042] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] Induction of callus from different grape rootstocks

[0045] 1.1 Materials

[0046] 1.1.1 Test Materials

[0047] The test materials were grape rootstocks from the vineyard of the College of Food Science and Engineering of Gansu Agricultural University and the grape base of Wuwei Forestry Research Institute: 101-14, 520A, 1103P, 140R, and wild grape. A brief description of the materials is shown in Table 1.

[0048] Table 1 Different grape rootstock varieties

[0049]

[0050] 1.1.2 Instruments, Equipment and Medicines

[0051] Equipment: Climate chamber, high-temperature sterilizer, laminar flow hood, alcohol lamp, scissors, tweezers, Erlenmeyer flasks, petri dishes, pipettes, pipette tips, spray bottle, sealing film, markers

[0052] Chemicals: 75% alcohol, 95% alcohol, mercuric chloride, agar, sucrose, KOH, inositol, PVP (Solarbio), 2,4-D (Solarbio), 6-BA (Solarbio)

[0053] 1.2 Methods

[0054] 1.2.1 Establishment of in vitro culture system

[0055] Preparation of culture medium: When preparing 1 L of MS medium, first weigh 30 g of sucrose, 6 g of agar, and 4.43 g of MS and put them into a pot. Stir constantly to mix the medium evenly. Use a pH meter to add KOH to control the pH value between 5.8 and 6.0. Add 1 L of RO water and heat while stirring continuously to dissolve MS, sucrose, agar, etc. After boiling the medium, pour it into the prepared conical flask while hot, seal it tightly with a sealing film, and put it in an autoclave for sterilization. When the temperature of the autoclave drops to 50-60℃, take out the medium, add the appropriate plant growth regulator in a clean bench, and let it cool before use. To prepare 1 L of GS medium, first weigh 30 g of sucrose and 6 g of agar, pour them into a pot, and then use a 10 ml graduated cylinder to measure 10 mL of macro-elements, 5 mL of micro-elements, 5 mL of iron salts, 5 mL of calcium salts, and 5 mL of organic matter. Add RO water to 1 L, stirring constantly to ensure the medium is thoroughly mixed. Add KOH and use a pH meter to maintain the pH between 5.8 and 6.0. While heating, stir constantly to dissolve the sucrose and agar. After boiling, while still hot, dispense the medium into prepared Erlenmeyer flasks, seal them tightly with film, and sterilize them in an autoclave. When the temperature of the autoclave drops to 50-60℃, remove the flasks and add the appropriate plant growth regulators in a clean bench. After cooling, set aside for use.

[0056] Primary culture: Culture medium: GS medium with added agar 6 g / L, sucrose 30 g / L, IAA 0.2 mg / mL, and chlorhexidine 0.08 g / L. Culture conditions: 16 h light, 8 h dark alternation culture, light intensity of 12000 lx, constant temperature culture of 25 ± 1℃, and relative humidity of 22%.

[0057] Subculture: After approximately 30 days of initial culture, stem segments with single buds and leaves were transferred to a new proliferation medium and cultured for another 30 days to establish a sterile seedling line. Culture medium: GS medium with 6 g / L agar, 30 g / L sucrose, and 0.2 mg / mL IAA. Culture conditions: Alternating 16 h light and 8 h dark cycles, light intensity of 12000 lx, constant temperature of 25 ± 1℃, and relative humidity of 22%.

[0058] 1.2.2 Callus induction

[0059] Culture medium preparation: MS medium was used as the basal medium, with 6 g / L agar, 30 g / L sucrose, 1 g / L inositol, and 300 mg / L PVP added. Different types of hormones (2,4-D, 6-BA) and concentrations (2,4-D: 0, 0.5, 1.0 mg / L; 6-BA: 0, 0.2, 0.5, 1.0, 1.5 mg / L) were combined to design a total of 9 treatments, as shown in Table 2.

[0060] Table 2. Experimental Design for Callus Induction in Grape Leaves / Pedicels / Stem Segments

[0061]

[0062] Inside the clean bench, under aseptic conditions, the leaves of five varieties of test-tube seedlings were cut into shapes of about 1 cm × 1 cm, and the petioles and stem segments were cut into lengths of 1 cm. They were then inoculated onto induction culture dishes with different ratios. Five materials were inoculated onto each culture dish, and five culture dishes were inoculated onto each variety. After inoculation, the state of the callus tissue was observed and recorded.

[0063] Culture conditions: continuous dark culture, constant temperature of 25 ± 1 ℃, and relative humidity of 22%.

[0064] 2.2.3 Data Statistics and Analysis

[0065] Callus induction rate = (Number of callus produced / Number of explants inoculated) × 100%

[0066] The experimental data were statistically analyzed using Microsoft Office Excel 2010.

[0067] 2.3 Results and Analysis

[0068] 2.3.1 Callus induction

[0069] This experiment used MS medium as the basal medium and inoculated different organs of grape plantlets (101-14, 520A, 1103P, 140R, and wild grape) onto MS medium under nine different treatments (leaves, petioles, and stem segments). The effects of two plant growth regulators, 2,4-D and 6-BA, on callus induction in different organs of different grape plantlets were investigated. It was found that under the CK treatment, no callus was induced in leaves, petioles, or stem segments of any variety (e.g., ...). Figure 1 (CK) Figure 2 (CK) Figure 3 In the control group (CK), callus was induced in all other treatments regardless of the induction rate, and embryogenic callus was induced in the leaves of in vitro plantlets 101-14 (see...). Figure 1In the culture medium (5), the induction results of callus tissue varied greatly due to different hormone concentration ratios. Some callus tissues on the culture medium showed a dehydrated and browned state (e.g., Figure 1 Middle (1) Figure 1 (2) Figure 2 Middle (1) Figure 2 (2) Figure 3 Middle (1) Figure 3 In the middle (2), the callus induction state was better on some culture media (such as...). Figure 1 (3) Figure 1 Middle (4) Figure 2 Middle (4) Figure 3 (3)

[0070] Induction rates were calculated at 30, 60, and 90 days. The results showed that different grape varieties had varying abilities to induce callus formation, and even within the same variety, the ability to induce callus formation varied depending on the organ. Of all treatments, only the leaves of the 101-14 grape plantlets induced embryogenic callus formation; the petioles and stem segments of the 101-14, as well as the leaves, petioles, and stem segments of the 520A, 1103P, 140R, and wild grape varieties, only induced non-embryogenic callus formation in all treatments. Table 3-12 shows that no callus was induced in any organ of any variety in the blank control. However, compared to the blank control, different concentrations of 2,4-D and 6-BA promoted callus differentiation in the leaves, petioles, and stem segments of all five grape varieties, and the callus induction rate increased with increasing culture time.

[0071] 1.3.2 Induction of 101-14 grape callus

[0072] Table 3 shows that when the contents of 2,4-D and 6-BA were 1.0 mg / L and 0.2 mg / L, respectively, the final induction rate of callus in leaves of 101-14 was 80.0%, and the induced callus was embryogenic. When the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.0 mg / L, respectively, the induction rate of leaf callus was 84.0%, but the induced callus was non-embryogenic. When the contents of 2,4-D and 6-BA were 0.5 mg / L and 0.5 mg / L, respectively, the final induction rate of leaf callus was the lowest, only 32.0%. The callus induced by the other hormone concentrations was all non-embryogenic. Figure 1 and Figure 5 ).

[0073] The final induction rate of petiole callus (101-14) was highest (60.0%) when the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.0 mg / L, respectively. The final induction rate was the same (52.0%) when the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.5 mg / L, and 1.0 mg / L and 1.5 mg / L, respectively. The lowest induction rate (32.0%) was observed when the contents of 2,4-D and 6-BA were 0.5 mg / L and 0.2 mg / L, respectively. All calluses induced by the various hormone concentrations were non-embryonic. Figure 2 ).

[0074] The final induction rate of callus from stem segments 101-14 ranged from 32.0% to 52.0%, with 2,4-D and 6-BA contents of 1.0 mg / L and 0.5 mg / L, and 0.5 mg / L and 1.5 mg / L, respectively. Furthermore, all callus induced by the stem segments at all hormone concentrations were non-embryonic callus. Figure 3 ).

[0075] For the same grape variety, the ease of callus induction varies greatly depending on the hormone concentration applied to different organs, and the properties of the induced callus also differ. The callus induced by different hormone concentrations and ratios in grape 101-14 includes both non-embryonic and embryogenic callus.

[0076] Table 3. Effects of different hormone concentrations and ratios on callus induction in different organs from 101 to 14.

[0077]

[0078] 1.3.3 Induction of 520A grape callus

[0079] As shown in Table 4, for 520A, the final induction rate of leaf callus was the highest among all materials at 80.0% when the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.0 mg / L, respectively. The second highest induction rate was 68.0% when the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.5 mg / L, respectively. The lowest final induction rate of leaf callus was 32.0%, which occurred when the contents of 2,4-D and 6-BA were 0.5 mg / L and 0.5 mg / L, respectively.

[0080] When the contents of 2,4-D and 6-BA were 1.0 mg / L, 0.5 mg / L, and 1.0 mg / L, 0.2 mg / L, respectively, the final induction rate of 520A petiole callus was highest at 0.5 mg / L and 1.0 mg / L, respectively, reaching 52.0%. The second highest induction rate was 48.0% when the contents of 2,4-D and 6-BA were 0.5 mg / L, 1.5 mg / L, and 1.0 mg / L, 1.5 mg / L, respectively. The lowest induction rate was 32.0% when the contents of 2,4-D and 6-BA were 0.5 mg / L, 0.5 mg / L, and 1.0 mg / L, 0.2 mg / L, respectively.

[0081] The highest final induction rate of callus from the 520A stem segment occurred when the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.0 mg / L, respectively, with an induction rate of 48.0%. When the contents of 2,4-D and 6-BA were 0.5 mg / L, 1.5 mg / L and 1.0 mg / L, 1.5 mg / L, respectively, the final induction rate of callus from the 520A stem segment was 44.0%. The lowest final induction rate of callus from the 520A stem segment was observed when the contents of 2,4-D and 6-BA were 0.5 mg / L, 0.2 mg / L and 1.0 mg / L, 0.5 mg / L, respectively, with a value of 32.0%.

[0082] For the same grape variety, the ease of callus induction varies greatly among different organs under different concentrations of hormone treatment. Furthermore, the callus induced by 520A from leaves, petioles, and stem segments was non-embryonic callus regardless of the hormone concentration ratio.

[0083] Table 4. Effects of different hormone concentrations and ratios on callus induction in different organs of 520A.

[0084]

[0085] 1.3.4 Induction rate of 1103P callus

[0086] Table 5 shows that none of the explants from the 1103P in vitro seedlings induced embryogenic callus in any of the treatments for callus induction culture; all induced callus was non-embryogenic. For the same grape variety, the ease of callus induction varied considerably among different organs under different hormone concentrations.

[0087] Regarding the final induction of callus from leaves of 1103P in vitro seedlings, the callus induction rate was highest at 72.0% when the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.0 mg / L, respectively. This was the highest callus induction rate among all explants of 1103P in vitro seedlings. When the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.5 mg / L, the final callus induction rate was 64.0%, ranking second among all explants of 1103P in vitro seedlings. When the contents of 2,4-D and 6-BA were 0.5 mg / L and 0.5 mg / L, the final callus induction rate was the lowest among all leaf callus induction rates, at 32.0%.

[0088] The highest final induction rate of petiole callus from 1103P in vitro plantlets was 52.0%, which occurred when the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.0 mg / L, respectively. The next highest induction rate was 48.0% when the contents of 2,4-D and 6-BA were 0.5 mg / L, 1.5 mg / L, 1.0 mg / L, and 1.5 mg / L. The lowest induction rate was 24.0% when the contents of 2,4-D and 6-BA were 0.5 mg / L and 0.2 mg / L, which was the lowest among all explants of 1103P in vitro plantlets.

[0089] The highest final induction rate of callus from stem segments of 1103P in vitro seedlings was 52.0%, which occurred when the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.0 mg / L, respectively. When the contents of 2,4-D and 6-BA were 0.5 mg / L, 1.5 mg / L, 1.0 mg / L, and 1.5 mg / L, the final induction rate of callus from stem segments was 48.0%. When the contents of 2,4-D and 6-BA were 0.5 mg / L and 0.2 mg / L, the final induction rate of callus from stem segments of 1103P in vitro seedlings was 28.0%, which was the lowest value.

[0090] Table 5. Effects of different hormone concentrations and ratios on callus induction in different organs using 1103P.

[0091]

[0092] 1.3.5 Induction rate of 140R callus

[0093] The induction rate of 140R callus is shown in Table 6. It can be seen that the peak value of the final induction rate of 140R test-tube seedling leaf callus occurred when the concentration ratio of 2,4-D to 6-BA was 0.5 mg / L and 1.0 mg / L, which was 64.0%. The second highest value was when the concentration ratio of 2,4-D to 6-BA was 0.5 mg / L and 1.0 mg / L, which was 60.0%. The lowest value of the final induction rate of leaf callus was when the concentration ratio of 2,4-D to 6-BA was 0.5 mg / L and 0.2 mg / L, which was 32.0%.

[0094] The highest final induction rate of petiole callus in 140R in vitro seedlings was 48.0%, which occurred when the contents of 2,4-D and 6-BA were 0.5 mg / L, 1.0 mg / L, and 1.0 mg / L, 1.0 mg / L. The second highest induction rate was 44.0% when the concentration ratio of 2,4-D and 6-BA was 0.5 mg / L and 1.5 mg / L. The lowest induction rate of petiole callus in 140R in vitro seedlings was only 24.0% when the concentration ratio of 2,4-D and 6-BA was 0.5 mg / L and 0.2 mg / L.

[0095] The highest final induction rate of callus from stem segments of 140R in vitro seedlings was 48.0% when the contents of 2,4-D and 6-BA were 0.5 mg / L, 1.0 mg / L, 1.0 mg / L, and 1.5 mg / L. The lowest final induction rate of callus from stem segments of 140R in vitro seedlings was 28.0%, which occurred when the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.5 mg / L.

[0096] For the same grape variety, the ease of callus induction varies greatly among different organs under different concentrations of hormone treatment. Furthermore, the callus induced by leaves, petioles, and stem segments of 140R was non-embryonic callus regardless of the hormone concentration ratio.

[0097] Table 6. Effects of different hormone concentrations and ratios on callus induction in different organs of 140R.

[0098]

[0099] 1.3.6 Induction of callus from wild grape

[0100] As shown in Table 7, the final induction rate of callus tissue from *Vitis thunbergii* leaves was highest when the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.0 mg / L, respectively, with an induction rate of 88.0%. This was followed by the induction rate of 84.0% when the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.5 mg / L, respectively. The final induction rate of callus tissue from *Vitis thunbergii* leaves was generally higher than that of other tissues. The lowest induction rate was observed when the contents of 2,4-D and 6-BA were 0.5 mg / L and 0.2 mg / L, respectively, with a value of 36.0%.

[0101] The highest final callus induction rate of *Vitis thunbergii* petioles was 64.0%, achieved when the contents of 2,4-D and 6-BA were 0.5 mg / L and 1.0 mg / L, respectively. The next highest rates were 60.0% when the contents of 2,4-D and 6-BA were 0.5 mg / L, 1.0 mg / L, 1.0 mg / L, and 1.5 mg / L, respectively. The lowest callus induction rate was 36.0% when the contents of 2,4-D and 6-BA were 0.5 mg / L and 0.2 mg / L, respectively.

[0102] The two relatively high final induction rates of callus from *Vitis thunbergii* stem segments were 64.0% and 60.0%, respectively, when the contents of 2,4-D and 6-BA were 0.5 mg / L, 1.0 mg / L and 0.5 mg / L, 1.5 mg / L and 1.0 mg / L, 1.5 mg / L, respectively. The lowest final induction rate of callus from stem segments was 36.0%, when the contents of 2,4-D and 6-BA were 1.0 mg / L and 0.2 mg / L, respectively.

[0103] For the same grape variety, the ease of callus induction varies greatly depending on the organ and the concentration of hormones used. Furthermore, all callus induced by wild grapes at all hormone concentrations is non-embryonic.

[0104] Table 7. Effects of different hormone concentrations and ratios on callus induction in different organs of *Vitis thunbergii*.

[0105]

[0106] 1.3.7 Overview of callus induction rates for different varieties and explants

[0107] Table 8. Callus induction rate of different varieties and explants

[0108]

[0109] Depend on Figure 4Table 8 shows that the callus induction rate varies among different varieties using the same explant, and also among different explants of the same variety. Therefore, the overall callus induction rate varies among different varieties. The comprehensive callus induction rates for different varieties were 47.3%, 43.8%, 42.2%, 40.0%, and 51.8%, respectively. Among the five different grape varieties in vitro, *Vitis vinifera* was the easiest to induce callus, followed by 101-14, then 520A and 1103P. 140R had the lowest callus induction rate among the five.

[0110] The callus induction rate of wild grape leaves was significantly higher than that of petioles and stem segments, with induction rates of 57.5%, 49.5%, and 48.5%, respectively.

[0111] The callus induction rate of leaves of 101-14 was the highest among the three explants, at 58.0%, and the induction rate of petiole callus (43.5%) was higher than that of stem segment callus (40.5%).

[0112] The callus induction rates of each explant of 520A were similar to those of Grapevine and 101-14, with the callus induction rates showing the order of leaf > petiole > stem segment, with values ​​of 53.0%, 40.0%, and 38.5%, respectively.

[0113] The callus induction rates of the various explants of 1103P were 45.0%, 38.0%, and 38.5%, respectively. Unlike the callus induction rates of explants of Grapevine, 101-14, and 520A, the callus induction rate showed a trend of leaf > stem segment > petiole.

[0114] The callus induction rates of the 140R explants were similar to those of the 1103P explants, showing a pattern of leaf > stem segment > petiole, with callus induction rates of 45.5%, 37.0%, and 37.5%, respectively.

[0115] Table 9. Effects of different culture media on the induction of secondary embryos in '101-14' grapes.

[0116]

[0117] During secondary embryo induction, different culture medium formulations significantly affected the frequency of secondary embryo formation in '101-14' grapes. (See Table 9 and...) Figure 5It was found that at 30, 60, and 90 days post-inoculation, the secondary embryo formation rate of all treatment groups increased with the extension of culture time. Among them, the 1 / 2 MS + 0.2 mg / L 6-BA + 6 g / L agar + 15 g / L sucrose treatment group showed the best induction effect. The secondary embryo formation rates of this treatment at 30, 60, and 90 days of culture were 15%, 50%, and 80%, respectively, all significantly higher than other treatment groups, and reached the highest induction efficiency at 90 days. In contrast, the MS + 0.2 mg / L 6-BA + 6 g / L agar + 30 g / L sucrose treatment group had formation rates of 10%, 45%, and 60% at the three time points, respectively, showing the second best overall induction effect. The two treatment groups with added 1 mg / L 2,4-D (MS + 0.2 mg / L 6-BA + 1 mg / L 2,4-D + 6 g / L agar + 30 g / L sucrose and 1 / 2 MS + 0.2 mg / L 6-BA + 1 mg / L 2,4-D + 6 g / L agar + 15 g / L sucrose) showed lower secondary embryo formation rates at all time points than the corresponding treatment groups without 2,4-D. Among them, the MS + 0.2 mg / L 6-BA + 1 mg / L 2,4-D + 6 g / L agar + 30 g / L sucrose treatment group showed the worst induction effect, with a formation rate of only 55% at 90 days. These results indicate that a lower inorganic salt concentration (1 / 2 MS) combined with an appropriate concentration of 6-BA (0.2 mg / L) is beneficial for the induction of secondary embryos in '101-14' grapes; while the addition of 2,4-D showed an inhibitory effect on secondary embryo induction. Therefore, 1 / 2 MS + 0.2 mg / L 6-BA + 6 g / L agar + 15 g / L sucrose can be used as the preferred culture medium for induction of secondary embryos in '101-14' grapes.

[0118] Table 10 Effects of different culture media on grape embryoid germination and seedling formation.

[0119]

[0120] From Table 10 and Figure 5It was found that different culture medium formulations significantly affected the germination and seedling formation efficiency of '101-14' grape embryoids. At both 30 and 60 days of culture, the germination rate and seedling formation rate of all treatment groups increased significantly with prolonged culture time. The MS + 0.093 mg / L NAA + 0.346 mg / L GA3 + 8 g / L agar + 30 g / L sucrose treatment group showed the best overall performance. At 30 days of culture, the germination rate and seedling formation rate of this treatment were 45% and 35%, respectively; after 60 days of culture, the germination rate increased to 80%, and the seedling formation rate reached 75%, both the highest values ​​among all treatments. The MS + 0.2 mg / L IAA + 8 g / L agar + 30 g / L sucrose treatment group was the second best. The germination rate of this group was 50% and the seedling rate was 40% at 30 days; at 60 days, the germination rate increased to 75% and the seedling rate increased to 60%, all of which were significantly better than the MS control treatment without plant growth regulators. The MS + 0.093 mg / L NAA + 0.2 mg / L 6-BA + 8 g / L agar + 30 g / L sucrose treatment group had a germination rate and seedling rate of 40% and 30% at 30 days; at 60 days, they reached 75% and 65%, respectively, showing good germination and seedling ability, but slightly lower than the treatment group containing GA3. The MS control treatment (without any plant growth regulators) had the worst effect, with a germination rate and seedling rate of only 25.0% and 15.0% at 30 days; at 60 days, they only reached 45.0% and 30.0%, respectively, significantly lower than the treatment groups with plant growth regulators. The above results indicate that adding appropriate types and concentrations of plant growth regulators to MS basal medium can significantly promote the germination and seedling formation of '101-14' grape embryoids. Among them, the MS + 0.093 mg / L NAA + 0.346 mg / L GA3 + 8 g / L agar + 30 g / L sucrose treatment group showed the most ideal germination and seedling formation effects and can be used as the preferred culture medium for the germination and seedling formation of embryoids of this variety.

[0121] in conclusion

[0122] In this experiment, leaves, petioles, and stem segments were cultured using a combination of 6-BA and IAA. The results showed that for all explant materials from five different varieties, the callus induction rate was 0 without the addition of any hormones, indicating that plant hormones are essential for callus induction. Among the five varieties (101-14, 520A, 1103P, 140R, and *Vitis thunbergii*), leaf callus induction was superior to that from petioles and stem segments in all treatments. However, the callus induction rates from petioles and stem segments varied among different varieties. In all treatments of petioles, leaves, and stem segments of 101-14, the final callus induction rate of leaves was 80.0% when the contents of 2,4-D and 6-BA were 1.0 mg / L and 0.2 mg / L, respectively. Furthermore, the induced callus was embryogenic. Therefore, the optimal medium for callus induction in 101-14 was: MS + 1.0 mg / L 2,4-D + 0.2 mg / L 6-BA + 6 g / L agar + 30 g / L sucrose. For 520A and *Vitis vinifera*, the callus induction rate was: leaves > petioles > stem segments. For 1103P and 140R, the callus induction rate was: leaves > stem segments > petioles. The results showed that the optimal medium for callus induction in 520A, 1103P, 140R, and *Vitis vinifera* was: MS + 0.5 mg / L 2,4-D. The optimal medium for secondary embryo induction was 1 / 2 MS medium + 0.2 mg / L 6-BA + 6 g / L agar + 15 g / L sucrose. After 90 days of culture, the secondary embryo formation rate reached 80%, significantly better than other treatments. The optimal medium for embryoid germination and seedling formation was MS medium + 0.093 mg / L NAA + 0.346 mg / L GA3 + 8 g / L agar + 30 g / L sucrose. After 60 days of culture, the germination rate reached 80% and the seedling formation rate reached 75%, significantly better than the control and IAA, 6-BA, and other treatments. This invention provides key technical support for the efficient in vitro regeneration and genetic transformation of '101-14' grapes.

[0123] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A culture medium composition for a grape regeneration system, characterized in that, This includes embryogenic callus induction medium, secondary embryo induction and proliferation medium, and seedling culture medium; The embryogenic callus induction medium comprises: MS medium + 300 mg / L polyvinylpyrrolidone + 1 mg / L 2,4-D + 0.2 mg / L 6-BA + 6 g / L agar + 30 g / L sucrose + 1 g / L inositol; The secondary embryo induction and proliferation medium comprises: 1 / 2 MS medium + 0.5 g / L activated carbon + 0.2 mg / L 6-BA + 6 g / L agar + 15 g / L sucrose; The seedling culture medium comprises: MS medium + 8 g / L agar + 30 g / L sucrose + 0.5 g / L activated carbon + 0.093 mg / L NAA + 0.346 mg / L GA3.

2. The culture medium combination according to claim 1, characterized in that, The grape varieties mentioned include 101-14.

3. A method for cultivating a grape regeneration system based on the culture medium combination described in claim 1 or 2, characterized in that, Includes the following steps: 1) Inoculate the leaves of sterile grape seedlings onto the embryogenic callus induction medium in the culture medium combination to induce embryogenic callus and obtain embryogenic callus. 2) The embryogenic callus tissue obtained in step 1) is inoculated into the secondary embryo induction proliferation culture in the culture medium combination to induce secondary embryos and obtain rooted embryoids; 3) The rooting embryos described in step 2) are inoculated onto the seedling culture medium in the culture medium combination for seedling culture to obtain grape seedlings.

4. The cultivation method according to claim 3, characterized in that, Step 1) The dimensions of the leaf are 1cm × 1cm.

5. The cultivation method according to claim 3, characterized in that, Step 1) The conditions for inducing embryogenic callus include: dark culture, temperature of 24~26℃, relative humidity of 22%, time of 90 days, and subculturing once every 30 days.

6. The cultivation method according to claim 3, characterized in that, Step 2) The conditions for inducing secondary embryos include: 16 h light, 8 h dark exchange culture, light intensity of 1000 lx, temperature of 24~26℃, relative humidity of 22%, transfer every 30 days, and culture time of 90 days.

7. The cultivation method according to claim 3, characterized in that, Step 3) The conditions for seedling cultivation include: 16h light and 8h dark alternation culture, light intensity of 12000 lx, temperature of 25~27℃, relative humidity of 45%, and time of 60d.

8. The use of the culture medium combination of claim 1 in a grape regeneration system.

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