A method for rapidly establishing a grape callus regeneration system

Abstract

The patent discloses a method for quickly establishing a Cabernet Sauvignon grape pericarp callus regeneration system, and belongs to the technical field of cell engineering. The method takes the pericarp of Cabernet Sauvignon grape as an explant, realizes efficient induction and rapid regeneration of callus through three key steps of explant disinfection treatment, primary callus induction culture, callus proliferation and subculture. The method optimizes the culture medium formula, adds specific concentrations of 6-BA, KT, IAA, NAA and 2,4-D in the induction medium, and significantly improves the induction rate and growth rate of callus. Experimental results show that the stable callus regeneration system can be obtained in 40-50 days by using the method, the callus induction rate can reach more than 90%, and the survival rate of regenerated plants is high. The patent has the advantages of simple operation, good repeatability and short cycle, and provides an effective technical means for genetic improvement, gene function research and rapid propagation of wine grapes, and has important theoretical value and application prospect.

Description

Technical Field

[0001] This patent belongs to the field of plant tissue culture technology, specifically relating to a method for rapidly establishing a callus regeneration system for Cabernet Sauvignon grape skins. Background Technology

[0002] Wine grapes are an important economic crop, among which Cabernet Sauvignon is one of the world's most famous grape varieties, widely praised for its wines' deep color, rich tannins, and complex aromas. With the development of molecular biology and genetic engineering technology, breeding new wine grape varieties with superior traits (such as disease resistance, stress resistance, and quality improvement) through genetic improvement has become an important direction in grape breeding.

[0003] Establishing an efficient and stable plant callus regeneration system is fundamental for genetic transformation and gene function research. Callus refers to a cluster of thin-walled cells with meristematic capacity that forms on the surface of a wound after a plant is injured. It can serve not only as material for rapid plant propagation but also as an ideal recipient system for cell culture, protoplast culture, and genetic transformation research.

[0004] Currently, there are some reports on grape callus regeneration systems, but these mainly focus on using leaves, shoot tips, stem segments, and anthers as explants, while research on using fruit peel as explants is relatively limited. Compared with other explants, fruit peel has advantages such as convenient material acquisition, minimal damage to the mother plant, and the ability to be obtained in large quantities during the fruit development season. However, existing methods for inducing grape callus from fruit peel generally suffer from low induction rates, slow callus growth, poor differentiation ability, and long culture cycles, which limit its application in genetic improvement and gene function research.

[0005] Culture medium is one of the key factors affecting callus induction and regeneration. The type and concentration of plant growth regulators and antioxidants play a decisive role in callus formation and differentiation. 6-Benzylaminopurine (6-BA) and zeatin (KT) are commonly used cytokinins that promote cell division and adventitious shoot differentiation. Indoleacetic acid (IAA), naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) are commonly used auxins, with 2,4-D showing a significant effect on callus induction, but high concentrations can inhibit callus differentiation. Indolebutyric acid (IBA) is often used to induce rooting. Antioxidants such as ascorbic acid (AC), activated charcoal (AC), and polyvinylpyrrolidone (PVPP) readily produce phenolic secondary metabolites during tissue culture, which are easily oxidized. Adding antioxidants can alter the redox potential around the explants, thereby inhibiting phenolic oxidation and reducing browning. Optimizing the combination and concentration of these plant growth regulators and antioxidants to improve the induction rate and regeneration efficiency of Cabernet Sauvignon peel callus is a technical problem that urgently needs to be solved.

[0006] Therefore, developing a simple, short-cycle, and efficient callus regeneration system for Cabernet Sauvignon grapes is of great significance for promoting the genetic improvement and molecular biology research of Cabernet Sauvignon. Summary of the Invention

[0007] To address the problems existing in the prior art, this patent provides a method for rapidly establishing a grape callus regeneration system. This method uses Cabernet Sauvignon peel as an explant and optimizes the culture medium formula and culture conditions to achieve efficient induction and rapid regeneration of callus, providing technical support for the genetic improvement and gene function research of Cabernet Sauvignon.

[0008] 1. Technical Solution To achieve the above objectives, the present invention adopts the following technical solution: A method for rapidly establishing a grape callus regeneration system includes the following steps: (1) Selection and disinfection of explants Select young, tender fruits from healthy, pest-free Cabernet Sauvignon vines at the optimal stage. At this time, the fruits are of moderate size, with tender skin tissue, strong division ability, and good callus induction effect. After harvesting, rinse the fruits thoroughly with tap water, then disinfect them on a clean bench: remove the stems, soak in sodium hypochlorite while shaking evenly, rinse 3-5 times with sterile water, then soak in 75% ethanol, shaking continuously to ensure thorough disinfection; finally rinse with sterile water to remove residual disinfectant; cut the fruits open, carefully remove the pulp, and use the skin as explants; blot the surface moisture of the disinfected skin with sterile filter paper and set aside.

[0009] Preferably, the optimal time for fruit is when the fruit is a young fruit 40-50 days after flowering.

[0010] Preferably, the explants are all 0.5-0.5 cm Cabernet Sauvignon grape skins.

[0011] Preferably, the disinfection method is as follows: after rinsing with running tap water for 2-3 hours, surface disinfection is performed (sequentially soaking in 10% sodium hypochlorite and shaking evenly for 15 minutes, rinsing with sterile water 3-5 times, then soaking in 75% ethanol for 30 seconds, and rinsing with sterile water 3-5 times).

[0012] (2) Callus induction culture The pericarp explants treated in step (1) were inoculated onto callus induction medium, with 5-6 pieces inoculated per dish. The callus induction medium was based on MS medium, with the addition of 1.0-2.0 mg / L 6-BA, 0.1-0.5 mg / L NAA, 0.5-1.0 mg / L 2,4-D, 25-35 g / L sucrose, and 6-8 g / L agar, and the pH was adjusted to 5.8-6.0. After inoculation, the culture dishes were placed in a culture room and cultured in the dark. Then, they were cultured for 20-25 days at a temperature of 24-26℃, a light intensity of 1500-2500 lux, and a light duration of 8-16 hours / day. At this time, light purple, loose callus tissue began to form at the cut of the pericarp explants.

[0013] Preferably, the callus tissue needs to be cultured in the dark for about 21 days.

[0014] Preferably, after the callus is cultured in the dark, it is transferred to a light intensity of 1500 lux and cultured for another 202 days under a light duration of 16 hours / day to obtain callus tissue.

[0015] Preferably, the optimal formulation of the callus induction medium is: MS medium + 6-BA 1.5 mg / L + NAA 0.3 mg / L + 2,4-D 0.8 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8-0.1.

[0016] Furthermore, using this formula, the callus induction rate can reach over 90%, and the callus grows vigorously, has a loose texture, is light purple in color, and has good differentiation potential.

[0017] (3) Subculture and proliferation culture The primary callus tissue obtained in step (2) was transferred to a subculture medium for subculture, with 3-4 pieces inoculated per bottle. The subculture medium was based on MS medium, supplemented with 0.5-1.0 mg / L 6-BA, 0.05-0.2 mg / L NAA, 0.1-0.4 mg / L KT, 25-35 g / L sucrose, 6-8 g / L agar, and 0.5-1 g / L VC / PVPP / AC, with the pH adjusted to 5.8-6.0. The callus tissue was cultured for 15-20 days under conditions similar to step (2) (except for dark culture) to allow further growth and proliferation while maintaining its differentiation capacity.

[0018] Preferably, all culture media contain antioxidants, wherein the antioxidant is PVPP.

[0019] Preferably, the optimal formulation of the subculture medium is: MS medium + 6-BA 0.8 mg / L + NAA 0.1 mg / L + KT 0.3 mg / L + sucrose 30 g / L + agar 8 g / L + PVPP 0.5 g / L, pH 5.8-0.1.

[0020] Preferably, the culture conditions are a temperature of 25°C, a light intensity of 2000 lux, and a light duration of 16 hours / day for 152 days to obtain callus tissue.

[0021] Furthermore, after subculture, the callus tissue grows faster, has a looser structure, and a brighter color.

[0022] Preferably, the culture medium used in each of the above steps must be sterilized by high-pressure steam at 121°C for 20 minutes before use to ensure a sterile environment.

[0023] 2. Beneficial effects Compared with existing technologies, this patent has the following beneficial effects: (i) Novel and convenient explant selection: This patent establishes a callus regeneration system using the skin of Cabernet Sauvignon grapes as explants. The skin explants are easy to obtain, cause little damage to the mother plant, and can be obtained in large quantities during the fruit development season. This overcomes the shortcomings of traditional explants, such as seasonal limitations and significant damage to the mother plant, when using leaves, stem tips, and stem segments as explants.

[0024] (ii) High callus induction rate and fast growth rate: By optimizing the induction culture medium formula, especially the combination of 6-BA, NAA and 2,4-D, the callus induction rate can reach more than 90%, and the callus grows vigorously and has a loose texture, which lays a good foundation for subsequent differentiation culture.

[0025] (III) Short culture cycle: Using the method of this patent, it only takes 50-60 days from explant inoculation to obtaining larger callus tissue, which is 15-20 days shorter than the traditional method, and significantly improves the culture efficiency.

[0026] (iv) Stable and reproducible regeneration system: This patent ensures that the induction, subculture and proliferation of callus tissue can be carried out stably through strict control of culture conditions and culture medium formula, with good experimental reproducibility and high survival rate.

[0027] (v) Simple operation and low cost: The culture medium used in this patent has simple and readily available components, and the culture conditions are easy to control. It does not require special instruments and equipment and is suitable for large-scale promotion and application.

[0028] In summary, the method provided by this patent is simple to operate, has a short cycle time, high efficiency, and good reproducibility. It successfully establishes a rapid regeneration system for callus tissue in Cabernet Sauvignon grape skins, providing an effective technical platform for genetic transformation, gene function research, variety improvement, and rapid propagation of Cabernet Sauvignon. It has significant theoretical value and broad application prospects. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings of the embodiments will be briefly described below.

[0030] The accompanying drawings described below are only related to some embodiments of the invention and are not intended to limit the invention.

[0031] In the attached diagram: Figure 1 A schematic diagram of the Cabernet Sauvignon grape callus regeneration system of the present invention is shown; Figure 2 A schematic diagram of the 0.5-0.5 cm pericarp explant of the present invention is shown; Figure 3 A schematic diagram of callus tissue obtained by dark culture according to the present invention is shown; Figure 4 A schematic diagram of the callus tissue obtained by the induced culture of the present invention is shown; Figure 5 A schematic diagram of the callus tissue obtained by subculture according to the present invention is shown; Figure 6 The diagram shows callus tissues obtained by different antioxidants of the present invention: A: callus tissue with added vitamin C, B: callus tissue with added AC, C: callus tissue with added PVPP; Figure 7 A schematic diagram of the browned callus tissue of the present invention is shown; Figure 8 A schematic diagram of the callus tissue obtained after proliferation according to the present invention is shown. Detailed Implementation The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the content described herein. Any obvious modifications or substitutions made to the methods, steps, or conditions of the present invention derived therefrom are still within the scope of protection of the present invention.

[0032] The calculation formulas for some results of this invention are as follows: Contamination rate = Number of contaminated explants / Total number of inoculated explants × 100%; Callus formation rate = Number of explants that have formed callus / Total number of inoculated explants × 100%; Induction rate = Number of successfully induced explants / Total number of inoculated explants × 100%; Browning rate = (Number of browned explants / Number of inoculated explants) × 100% All data were processed and analyzed using software such as Excel, SPSS 26.0, DPS data processing system, and Orthogonal Design Assistant II V3.1 Professional Edition.

[0033] Example 1: Explant selection and disinfection Healthy, disease-free Cabernet Sauvignon vines were selected and marked. Young fruits, 45 days after flowering, were then collected from these marked vines. The fruits were brought back to the laboratory and rinsed with tap water for 120 minutes. They were then sterilized on a clean bench: the stems were removed with a scalpel; the fruits were placed in sterile Erlenmeyer flasks, and 2.5%, 5%, and 10% sodium hypochlorite solutions were added, followed by shaking for 15 minutes. The sodium hypochlorite was quickly removed, and the fruits were rinsed five times with sterile water. Next, the fruits were soaked in 75% ethanol for 45 seconds, with continuous shaking, and rinsed five times with sterile water for one minute each time. The fruits were then longitudinally cut, and the pulp carefully removed, using the skin as explants. The sterilized skin was blotted dry with sterile filter paper and cut into 0.5-0.5 cm pieces. These pieces were inoculated onto MS medium, with five plates per dish and three biological replicates. Contamination rates were recorded during culture.

[0034] As shown in Table 1, the results indicate that the contamination rate was only (10.60 ± 0.04)% when disinfected with 10% NaClO. However, the contamination rate remained around 20% even after disinfection with NaClO concentrations below 10%. Furthermore, NaClO concentrations above 10% caused irritation and damage to the explants, manifesting as burning and cracking of the grape pericarp, significantly impacting callus induction. Therefore, 10% NaClO disinfection is the optimal disinfection condition for explants.

[0035] Table 1: Effects of NaClO concentration on explants Processing Number NaClO concentration (%) Pollution rate (%) 1 2.5 <![CDATA[27.330.07 a ]]> 2 5 <![CDATA[19.670.03 b ]]> 3 10 <![CDATA[10.600.04 c ]]> Experimental Example 2: Callus induction culture 1. Callus Induction: Samples were collected from the Yuquanying area on the eastern foothills of Helan Mountain in Ningxia Hui Autonomous Region (Lt381425"NLn1060143"E). Healthy, disease-free fruits were selected as samples. The samples were brought back to the laboratory and first rinsed with tap water for 120 minutes, then sterilized on a clean bench: the fruit stalks were removed with a scalpel; the fruits were placed in sterile Erlenmeyer flasks, soaked in 10% sodium hypochlorite solution and shaken evenly for 15 minutes, the sodium hypochlorite solution was quickly poured out, and the fruits were rinsed 5 times with sterile water; then soaked in 75% ethanol for 45 seconds, shaking the Erlenmeyer flask continuously; and rinsed 5 times with sterile water for 1 minute each time. The sterilized explants were then inoculated. Dark culture is extremely important for callus induction. The culture medium was MS medium supplemented with 1.0 mg / L 6-BA and 0.2 mg / L 2,4-D. After dark culture for 14, 21, 28, and 35 days, the callus rate was statistically analyzed.

[0036] 2. The explants that had formed callus were transferred to new dishes and cultured on MS medium with the addition of 1.0-2.0 mg / L 6-BA, 0.1-0.5 mg / L NAA, and 0.5-1.0 mg / L 2,4-D. The pH was adjusted to 5.8-6.0, the temperature was 252℃, the light intensity was 1500 lux, and the photoperiod was 16 h / d. Three biological replicates were performed and cultured for about 20 days. The callus tissue was then analyzed.

[0037] As shown in Table 2, dark culture for an appropriate period of time promotes callus formation. The callus emergence rate reaches its highest level (95.60%) after 21 days of dark culture. As the dark culture time increases, the callus emergence rate begins to decrease and contamination begins to increase. Therefore, 21 days of dark culture is the optimal time for callus induction.

[0038] Table 2: Effect of dark culture time on callus tissue Processing Number Dark culture time (d) Recovery rate (%) 1 14 <![CDATA[84.290.07 c ]]> 2 21 <![CDATA[95.600.29 a ]]> 3 28 <![CDATA[91.330.31 b ]]> 4 35 <![CDATA[81.430.73 cd ]]> As shown in Table 3, appropriate concentrations of 6-BA, NAA, and 2,4-D can significantly improve the callus induction rate of Cabernet Sauvignon peel. Treatment 5 (6-BA 1.5 mg / L + NAA 0.3 mg / L + 2,4-D 0.8 mg / L) showed the highest callus induction rate, reaching 92.5%, and the callus was in the best condition, appearing as light purple, loose, and growing vigorously, with good differentiation potential. Therefore, this formula was determined to be the optimal formula for callus induction medium.

[0039] Table 3: Effects of plant growth regulator ratios on callus induction rate Processing Number 6-BA (mg / L) NAA (mg / L) 2,4-D (mg / L) Induction rate (%) callus state 1 1.0 0.1 0.5 72.3 It has a denser texture and grows more slowly. 2 1.0 0.3 0.8 81.5 Pale purple, loose texture, grows well 3 1.0 0.5 1.0 76.8 Darker in color, prone to browning 4 1.5 0.1 0.5 85.7 Pale purple, loosely textured, and growing vigorously. 5 1.5 0.3 0.8 92.5 Pale purple, loose texture, very vigorous growth, and great potential for differentiation. 6 1.5 0.5 1.0 83.2 Medium quality, grows well 7 2.0 0.1 0.5 80.6 It has a loose texture and grows slowly. 8 2.0 0.3 0.8 78.4 Light brown, with a relatively dense texture 9 2.0 0.5 1.0 67.2 It has a darker color, is prone to browning, and grows more slowly. Example 3: Callus subculture 1. Samples were collected from Yuquanying area (Lt381425"NLn1060143"E) on the eastern foothills of Helan Mountain in Ningxia Hui Autonomous Region. Healthy, disease-free fruits were selected as samples. The fruits were brought back to the laboratory and first rinsed with tap water for 120 minutes. Then, they were disinfected on a laminar flow hood: the fruit stalks were removed with a scalpel; the fruits were placed in sterile Erlenmeyer flasks, soaked in 10% sodium hypochlorite solution and shaken evenly for 15 minutes, the sodium hypochlorite solution was quickly poured out, and the fruits were rinsed 5 times with sterile water; then soaked in 75% ethanol for 45 seconds, shaking the Erlenmeyer flask continuously; rinsed 5 times with sterile water for 1 minute each time; the treated fruit peel pieces were inoculated onto callus induction medium.

[0040] 2. The formula for callus induction medium was determined to be MS medium + 6-BA 1.5 mg / L + NAA 0.3 mg / L + 2,4-D 0.8 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8. The above medium was dissolved by heating and then diluted to a 1000 mL Erlenmeyer flask. The flask was autoclaved at 121 °C for 21 minutes and cooled before use. The medium was then dispensed into petri dishes (approximately 40 mL per dish) in a laminar flow hood, and 5 explants were inoculated into each dish. The petri dishes were placed in a culture chamber at 25 °C and cultured in the dark for 21 days. Then, cultured for another 202 days under a light intensity of 1500 lux and a light duration of 16 hours / day.

[0041] 3. Transfer well-grown callus tissue to different culture dishes for subculture. The subculture medium is MS medium supplemented with different combinations of 6-BA (0.5-1.0 mg / L), NAA (0.05-0.2 mg / L), and KT (0.1-0.5 mg / L). Culture for approximately 20 days, and then assess the callus growth.

[0042] As shown in Table 4, appropriate concentrations of 6-BA, NAA, and KT promote callus induction, especially treatment 5. In MS medium with 0.8 mg / L 6-BA, 0.1 mg / L NAA, and 0.3 mg / L KT, the induction rate reached 89.5%. The callus exhibited the best performance, showing good growth, a 2-3 fold increase in volume, a pale yellow color, a bright luster, and a loose texture. Therefore, this medium was identified as the best subculture medium for callus.

[0043] Table 4. Effects of plant growth regulator formulation ratios on callus subculture: Processing Number 6-BA (mg / L) NAA (mg / L) KT (mg / L) Induction rate (%) callus state A 0.5 0.05 0.1 75.2 Pale yellow, relatively dense in texture, with localized browning, and grows slowly. B 0.5 0.1 0.3 80.9 Pale yellow, loose in texture, growing well C 0.5 0.2 0.5 81.6 Pale yellow, loose in texture, growing well D 0.8 0.05 0.1 83.7 Pale yellow, loose in texture, and growing vigorously. E 0.8 0.1 0.3 89.5 It grows well, increasing in size 2-3 times, is pale yellow with a bright color, and has a loose texture. F 0.8 0.2 0.5 85.8 Pale yellow, growing well, loose texture G 1.0 0.05 0.1 71.6 It has a darker color, is prone to browning, has a denser texture, and grows more slowly. H 1.0 0.1 0.3 73.6 Light brown, with a relatively dense texture I 1.0 0.2 0.5 74.4 It has a darker color, is prone to browning, and grows more slowly. Example 4: Environmental conditions for callus subculture 1. Samples were collected from Yuquanying area (Lt381425"NLn1060143"E) on the eastern foothills of Helan Mountain in Ningxia Hui Autonomous Region. Healthy, disease-free fruits were selected as samples. The fruits were brought back to the laboratory and first rinsed with tap water for 120 minutes. Then, they were disinfected on a laminar flow hood: the fruit stalks were removed with a scalpel; the fruits were placed in sterile Erlenmeyer flasks, soaked in 10% sodium hypochlorite solution and shaken evenly for 15 minutes, the sodium hypochlorite solution was quickly poured out, and the fruits were rinsed 5 times with sterile water; then soaked in 75% ethanol for 45 seconds, shaking the Erlenmeyer flask continuously; rinsed 5 times with sterile water for 1 minute each time; the treated fruit peel pieces were inoculated onto callus induction medium.

[0044] 2. The formula for callus induction medium was determined to be MS medium + 6-BA 1.5 mg / L + NAA 0.3 mg / L + 2,4-D 0.8 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8, with 5 callus inoculated per dish. The above medium was dissolved by heating and then diluted to a 1000 mL Erlenmeyer flask. The flask was then autoclaved at 121 °C for 21 minutes and cooled before use. The culture medium was then aliquoted into petri dishes, approximately 40 mL per dish, in a laminar flow hood. The petri dishes were placed in an incubator at 25 °C and incubated in the dark for 21 days. Following this, the culture was continued for 202 days under a light intensity of 1500 lux and a light duration of 16 hours / day.

[0045] 3. A basal medium of MS medium + 6-BA 0.8 mg / L + NAA 0.1 mg / L + KT 0.3 mg / L, pH 5.8, was used. Five pieces of callus in good condition were inoculated into each dish. The above medium was dissolved by heating and then brought to a final volume of 1000 mL in an Erlenmeyer flask. The flask was then autoclaved at 121°C for 21 minutes and cooled before use. The contents were then aliquoted into petri dishes (approximately 40 mL per dish) in a laminar flow hood. The petri dishes were placed in an incubator, and three antioxidants—ascorbic acid (VC), activated charcoal (AC), and polyvinylpyrrolidone (PVPP)—were added to the prepared medium, resulting in nine treatments. The culture conditions were set as follows: temperature 25°C, light intensities of 1500 lux, 2000 lux, and 2500 lux, and photoperiods of 12 h / day, 14 h / day, and 16 h / day, respectively. After approximately 20 days of culture, the browning rate and callus growth were statistically analyzed.

[0046] As shown in Table 5, localized browning began to appear in the callus tissue around 40-50 days. Without antioxidant treatment, the browning worsened over time. Therefore, adding an appropriate amount of antioxidant to the culture medium is essential. Treatment 3 showed the best performance, with a browning rate of only 38.67 ± 0.06%, indicating that the addition of antioxidant significantly improved the browning of the callus tissue and inhibited its further development. Therefore, 0.5% PVPP was determined to be the optimal formulation for callus tissue antioxidant.

[0047] Table 5: Effects of antioxidants on callus tissue Processing Number VC (g / L) AC (g / L) PVPP (g / L) Browning rate 1 0 0 0 <![CDATA[73.900.09 a ]]> 2 0.1 0 0 <![CDATA[56.330.05 b ]]> 3 0 0.5 0 <![CDATA[42.670.03 cd ]]> 4 0 0 0.5 <![CDATA[38.670.06 d ]]> As shown in Table 6, the light environment has a significant impact on the callus induction rate, especially the light intensity and duration. When the intensity is low and the duration is short, the callus clumps are small and grow slowly. However, when the light intensity is too high, some callus clumps exhibit partial vitrification. Treatment 4 (light intensity 2000 lux, light duration 16 hours / day) showed the best performance, with a callus induction rate of 85.3%, and the callus clumps grew well and were pale yellow in color. Therefore, this condition was determined to be the optimal light condition for subculture.

[0048] Table 6: Effects of light conditions on callus tissue Processing Number Light intensity (lux) Light duration (h / d) Induction rate (%) callus tissue state 1 1500 12 68.3 The clumps are smaller and grow more slowly. 2 1500 16 75.6 The clumps are of moderate size and the growth is good. 3 2000 12 82.7 The clusters are relatively large and growing well. 4 2000 16 85.3 The clumps are relatively large, growing vigorously, and pale yellow in color. 5 2500 12 72.5 Some clumps exhibited vitrification. Example 5: 1. Samples were collected from Yuquanying area (Lt381425"NLn1060143"E) on the eastern foothills of Helan Mountain in Ningxia Hui Autonomous Region. Healthy, disease-free fruits were selected as samples. The fruits were brought back to the laboratory and first rinsed with tap water for 120 minutes. Then, they were disinfected on a laminar flow hood: the fruit stalks were removed with a scalpel; the fruits were placed in sterile Erlenmeyer flasks, soaked in 10% sodium hypochlorite solution and shaken evenly for 15 minutes, the sodium hypochlorite solution was quickly poured out, and the fruits were rinsed 5 times with sterile water; then soaked in 75% ethanol for 45 seconds, shaking the Erlenmeyer flask continuously; rinsed 5 times with sterile water for 1 minute each time; the treated fruit peel pieces were inoculated onto callus induction medium.

[0049] 2. The formula for callus induction medium was determined to be MS medium + 6-BA 1.5 mg / L + NAA 0.3 mg / L + 2,4-D 0.8 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8, with 5 callus inoculated per dish. The above medium was dissolved by heating and then diluted to a 1000 mL Erlenmeyer flask. The flask was then autoclaved at 121 °C for 21 minutes and cooled before use. The culture medium was then aliquoted into petri dishes, approximately 40 mL per dish, in a laminar flow hood. The petri dishes were placed in an incubator at 25 °C and incubated in the dark for 21 days. Following this, the culture was continued for 202 days under a light intensity of 1500 lux and a light duration of 16 hours / day.

[0050] 3. The formula for the callus subculture medium was determined to be MS medium + 6-BA 0.8 mg / L + NAA 0.1 mg / L + KT 0.3 mg / L + sucrose 30 g / L + agar 8 g / L + PVPP 0.5 g / L, pH 5.8. The medium was dissolved by heating and then diluted to a 1000 mL Erlenmeyer flask. The flask was autoclaved at 121°C for 21 minutes and cooled before use. The culture medium was then aliquoted into petri dishes, approximately 40 mL per dish, in a laminar flow hood. Well-grown callus tissue was transferred to the subculture medium, 1-2 pieces per dish. The culture conditions were: temperature 25°C, light intensity 2000 lux, light duration 16 hours / day, for a further 152 days.

[0051] At this point, the obtained callus tissue has completed the pericarp callus regeneration system. From explant inoculation to obtaining larger and denser callus masses, the entire process takes 50-60 days, significantly shortening the culture cycle. For even larger callus masses, the following methods can be used for propagation.

[0052] 4. Continue proliferation culture using the same formulation as the callus subculture medium. Dissolve 0.8 mg / L 6-BA, 0.1 mg / L NAA, 0.3 mg / L KT, 30 g / L sucrose, 8 g / L agar, and 0.5 g / L PVPP in MS medium (pH 5.8) by heating, then bring the volume to a 1500 mL Erlenmeyer flask. Autoclave at 121°C for 21 minutes, cool, and set aside. Then, aliquot the solution into culture bottles (approximately 50 mL per bottle) in a laminar flow hood. Transfer healthy callus tissue to proliferation medium, inoculating 3 pieces per plate. Continue culture under the following conditions: 25°C, 2000 lux light intensity, and 16 hours / day. Control the growth time according to the desired callus size.

[0053] 5. Industrial Application Value The method for rapidly establishing a callus regeneration system for Cabernet Sauvignon grape skins provided by this patent has significant industrial application value: Accelerating the genetic improvement process of wine grapes: This regeneration system can serve as a recipient system for genetic transformation, used to introduce target genes such as disease resistance, stress resistance, and quality improvement, and rapidly cultivate new wine grape varieties.

[0054] Gene function research platform: This system enables research on gene editing, gene silencing, and other techniques, allowing for in-depth exploration of the molecular mechanisms underlying important traits such as grape growth and development, quality formation, and stress response.

[0055] Rapid propagation of superior varieties: The callus regeneration system can be used to rapidly propagate superior wine grape varieties, improve propagation efficiency, shorten the seedling cycle, and meet the industry's demand for high-quality seedlings.

[0056] Production of secondary metabolites: Callus culture can be used to produce secondary metabolites such as polyphenols and anthocyanins from grapes, which have important medicinal and health benefits, providing a new approach for the industrial production of natural products.

[0057] In conclusion, this patent not only has significant theoretical research value, but also has broad application prospects in the wine grape industry, and is expected to generate significant economic and social benefits.

Claims

1. A method for rapidly establishing a grape callus regeneration system, characterized in that, Includes the following steps: (1) Selection and disinfection of explants: Select healthy and disease-free Cabernet Sauvignon grapes, disinfect them, and then dry them with filter paper for later use. Then, cut the skin into small pieces in a clean bench to serve as explants. (2) Callus induction culture: The explants treated in step (1) were inoculated onto the callus induction culture medium, and after dark culture, they were then cultured under light to obtain callus; the callus induction culture medium was based on MS, B5, NN69 and other media, with 6-BA 1.0-4.0 mg / L, NAA 0.1-0.5 mg / L, 2,4-D 0.5-1.0 mg / L, KT 1.0-4.0 mg / L, sucrose 25-35 g / L and agar 6-8 g / L added, and the pH was adjusted to 5.8-6.0; (3) Subculture and proliferation culture: Transfer the callus obtained in step (2) to the subculture medium and culture for 15-20 days; Subculture media were based on MS, B5, NN69, etc., with the addition of 6-BA 1.0-2.0 mg / L, NAA 0.1-0.2 mg / L, 2,4-D 0.1-0.2 mg / L, KT 0.1-0.4 mg / L, sucrose 25-35 g / L, agar 6-8 g / L, and antioxidants such as PVPP, AV, and VC. The pH was adjusted to 5.8-6.

0.

2. The method according to claim 1, characterized in that, The Cabernet Sauvignon grapes mentioned in step (1) are young fruits 40-50 days after flowering, and the explants mentioned in step (1) are all 0.5-0.5cm Cabernet Sauvignon grape skins.

3. The method according to claim 1, characterized in that, The disinfection method described in step (1) is as follows: after rinsing with running tap water for 2-3 hours, surface disinfection is carried out (soaking in 10% sodium hypochlorite and shaking evenly for 15 minutes, rinsing with sterile water 3-5 times, then soaking in 75% ethanol for 30 seconds, and rinsing with sterile water 3-5 times).

4. The method according to claim 1, characterized in that, The callus tissue described in step (2) needs to be cultured in the dark for about 21 days.

5. The method according to claim 1, characterized in that, The callus tissue described in step (2) was cultured in the dark and then transferred to a light intensity of 1500 lux and cultured for 202 days under a light duration of 16 hours / day to obtain callus tissue.

6. The method according to claim 1, characterized in that, The optimal formulation of the callus induction medium in step (2) is: MS medium + 6-BA (1.5 mg / L) + NAA (0.3 mg / L) + 2,4-D (0.8 mg / L) + sucrose 30 g / L + agar 6 g / L, pH 5.8-0.

1.

7. The method according to claim 1, characterized in that, Antioxidants must be added to all culture media in step (3), and the antioxidant in step (3) is PVPP.

8. The method according to claim 1, characterized in that, The optimal formulation of the subculture medium in step (3) is: MS medium + 6-BA (0.8 mg / L) + NAA (0.1 mg / L) + KT (0.3 mg / L) + sucrose 30 g / L + agar 8 g / L + PVPP 0.5 g / L, pH 5.8-0.

1.

9. The method according to claim 1, characterized in that, The culture conditions described in step (3) are a temperature of 25°C, a light intensity of 2000 lux, and a light duration of 16 hours / day for 152 days to obtain callus tissue.

10. The method according to claim 1, characterized in that, The culture media in steps (2) and (3) must be sterilized by high-pressure steam at 121°C for 21 minutes before use.

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