Tissue culture and rapid propagation method for virus-free seedlings of grapes

Abstract

The invention provides a method for tissue culture and rapid propagation of virus-free seedlings of grapes, and belongs to the technical field of rapid propagation of plants. The grape virus-free seedling tissue culture and rapid propagation method comprises the steps of explant induced differentiation, virus-free culture, virus detection, rooting culture and acclimatization and transplantation. According to the method, explants are detoxified in a heat treatment mode, the heat treatment comprises the steps that illumination is conducted at 37-38 DEG C for 5-6 h, darkness is conducted at 32-33 DEG C for 5-6 h, circulation is conducted in sequence, and the illumination intensity is 2000-3000 Lx; according to the heat treatment method, the survival rate of the test-tube plantlets can be increased, the detoxification rate is increased, a foundation is laid for detoxification of the 'Maisseria' variety grapes, and the heat treatment method can be applied to industrial large-scale production.

Description

Technical Field

[0001] The invention relates to the technical field of plant rapid propagation, in particular to a method for rapid propagation of virus-free grape seedlings through tissue culture. Background Art

[0002] Grapes (Vitis vinifera L.) are deciduous woody vines belonging to the genus Vitis (Vitis L.) of the Vitaceae family. Grapes are highly susceptible to viruses, with over 80 types of grape viruses discovered both domestically and internationally. With the continuous increase in grapevine plantings and the replacement of older orchards, the demand for grape seedlings is also increasing. Traditional cutting propagation, while simple and easy to perform, is limited in the short term due to the limited number of branches. Furthermore, seedlings are susceptible to viruses, which can lead to severe degradation of the grape's characteristics over the long term.

[0003] Although tissue culture rapid propagation systems have been established for several grape varieties, variations between varieties severely limit the genetic transformation process. Research into factors influencing proliferation, rooting, and virus removal in wine grape varieties is needed to improve their propagation and virus removal efficiency. To increase virus removal rates, plant tissue culture techniques require inoculation of sufficiently small stem apical meristems. Different heat treatment methods and durations can also affect the effectiveness of virus removal. Currently, no methods for rapid tissue culture propagation of virus-free seedlings for Marselan grapes have been reported. Summary of the Invention

[0004] In view of this, the present invention provides a method for rapid tissue culture propagation of virus-free grape seedlings.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for rapid propagation of virus-free grape seedlings through tissue culture, comprising the following steps:

[0007] (1) Explant differentiation induction: Cut the top 1-2 mm of the grape stem tip, sterilize it, inoculate it into the initiation medium, and culture it at 23-27°C for 16-18 days;

[0008] (2) Virus-free culture: The culture temperature is raised to 30-32°C and maintained for 2-3 days; then raised to 34-36°C and maintained for 2-3 days, and then heat treated, wherein the heat treatment is 37-38°C / light for 5-6 hours + 32-33°C / dark for 5-6 hours, in a cycle, and the light intensity is 2000-3000 Lx; heat treatment is performed for 18-22 days to obtain clustered buds;

[0009] (3) Virus detection: Grape leafroll virus 1 (GLRaV-1), grape fanleaf virus (GFLV), and grape spot virus (GFkV);

[0010] (4) Proliferation culture: Cut the clustered buds with negative virus test results into single buds, inoculate them into proliferation medium, and culture them for 38 to 42 days; subculture once every 38 to 42 days;

[0011] (5) Rooting culture: During the proliferation culture process, healthy tissue culture seedlings were selected, and new shoots with a length of 2.5 to 3.0 cm and containing apical growth points were cut and inoculated into rooting medium for rooting culture for 20 to 30 days;

[0012] (6) Hardening and transplanting: Move the tissue culture seedlings outdoors for hardening, and then transplant them into the cultivation medium to obtain virus-free seedlings.

[0013] Preferably, the disinfection is as follows: disinfection with 68-72% alcohol for 20-30 seconds, followed by rinsing with sterile water 2-3 times; disinfection with 4-6% sodium hypochlorite for 7-8 minutes, followed by rinsing with sterile water 4-5 times.

[0014] Preferably, the starting culture medium is MS+IBA 0.3-0.8 mg / L+KT 1-1.5 mg / L+sucrose 30 g / L+agar 7 g / L.

[0015] Preferably, the proliferation culture medium is 1 / 2MS+6-BA 1.4-1.6 mg / L and NAA 0.3-0.5 mg / L+sucrose 30 g / L+agar 7 g / L.

[0016] Preferably, the rooting medium is 1 / 2MS+IBA 0.1-0.5 mg / L+sucrose 30 g / L+agar 7 g / L.

[0017] Preferably, the culture temperature in steps (1) (4) (5) is 23-27°C, the illumination time is 10-12 hours, and the illumination intensity is 2000-3000 Lx.

[0018] Preferably, the seedling hardening is as follows: placing the tissue culture seedlings under scattered light for 5 to 7 days, and then moving them to a place with direct light for further 5 to 7 days.

[0019] Preferably, the cultivation medium is formed by mixing peat soil, vermiculite and garden soil in a volume ratio of 1:1:1.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects:

[0021] 1. The stem tip detoxification method of the present invention uses a stem tip of 1 to 2 mm as an explant, which reduces the difficulty of operation.

[0022] 2. The present invention adopts a heat treatment method of 37-38°C / light 5-6h+32-33°C / dark 5-6h, which is circulated in sequence, thereby improving the survival rate and virus-free rate of the test tube seedlings.

[0023] 3. The initiation culture medium of the present invention adds 0.3-0.8 mg / L IBA and 1-1.5 mg / L KT, which improves the germination rate of the explant; the proliferation culture medium adds 1.4-1.6 mg / L 6-BA and 0.3-0.5 mg / L NAA, which improves the proliferation coefficient of the test tube seedlings. DETAILED DESCRIPTION

[0024] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] (1) Explant differentiation induction: Select a new grape vine with vigorous growth, cut off the top 1-2mm stem tip as the explant, wash it thoroughly with water containing detergent, and then disinfect the surface in a clean bench. The disinfection process is as follows: disinfect with 68% alcohol for 30 seconds, rinse with sterile water twice; disinfect with 4% sodium hypochlorite for 7 minutes, rinse with sterile water four times. After disinfection, inoculate it into the start-up culture medium, place it in the light for 10 hours, light intensity of 3000Lx, and culture at 23-27℃ for 18 days;

[0027] The starting culture medium was MS+IBA 0.3 mg / L+KT 1 mg / L+sucrose 30 g / L+agar 7 g / L.

[0028] (2) Virus-free culture: The culture temperature was raised to 30°C and maintained for 2 days; then raised to 34°C and maintained for 2 days, and then heat treated. The heat treatment was 37°C / light for 6 hours + 32°C / dark for 6 hours, in a cycle with a light intensity of 2000Lx. After heat treatment for 20 days, clustered buds were obtained.

[0029] (3) Virus detection: Immunoglobulins of rabbit antisera against grape leafroll virus 1 (GLRaV-1), grape fanleaf virus (GFLV), and grape spot virus (GFkV) were used to detect clustered shoots;

[0030] (4) Proliferation culture: Cut the clustered buds with negative virus test results into single buds, inoculate them into proliferation medium, place them in a light-irradiated environment with a light intensity of 3000 Lx for 10 h, and culture them at a temperature of 23-27°C for 40 days; subculture once every 40 days;

[0031] The proliferation culture medium is 1 / 2MS+6-BA 1.4 mg / L+NAA 0.3 mg / L+sucrose 30 g / L+agar 7 g / L.

[0032] (5) Rooting culture: During the proliferation culture process, healthy tissue culture seedlings were selected, and new shoots with a length of 2.5 cm and containing the apical growth point were cut and inoculated into rooting medium for rooting culture for 30 days;

[0033] The rooting medium is 1 / 2MS+IBA 0.1mg / L.

[0034] (6) Hardening and transplanting: Move the tissue culture seedlings outdoors for hardening. First, place them under scattered light for 7 days, then move them to a place with direct light and continue hardening for 7 days. Then transplant them to the cultivation medium to obtain virus-free seedlings; the cultivation medium is a mixture of peat soil, vermiculite and garden soil in a volume ratio of 1:1:1.

[0035] Example 2

[0036] (1) Explant differentiation induction: Select a new grape vine with vigorous growth, cut off the top 1-2mm stem tip as the explant, wash it thoroughly with water containing detergent, and then disinfect the surface in a clean bench. The disinfection process is as follows: disinfect with 70% alcohol for 25 seconds, rinse with sterile water 3 times; disinfect with 5% sodium hypochlorite for 8 minutes, rinse with sterile water 5 times. After disinfection, inoculate it into the start-up culture medium, place it in the light for 11 hours, light intensity of 2500Lx, and culture at 23-27℃ for 18 days;

[0037] The starting culture medium was MS+IBA 0.5 mg / L+KT 1.2 mg / L+sucrose 30 g / L+agar 7 g / L.

[0038] (2) Virus-free culture: The culture temperature was raised to 31°C and maintained for 3 days; then raised to 35°C and maintained for 2 days, and then heat treated. The heat treatment was 38°C / light for 5.5 hours + 33°C / dark for 5.5 hours, cycled in sequence, with a light intensity of 2500Lx; after heat treatment for 20 days, clustered buds were obtained.

[0039] (3) Virus detection: Immunoglobulins of rabbit antisera against grape leafroll virus 1 (GLRaV-1), grape fanleaf virus (GFLV), and grape spot virus (GFkV) were used to detect clustered shoots;

[0040] (4) Proliferation culture: Cut the clustered buds with negative virus test results into single buds, inoculate them into proliferation medium, place them in a light intensity of 2500 Lx for 11 h, and culture them at a temperature of 23-27°C for 40 days; subculture once every 40 days;

[0041] The proliferation culture medium is 1 / 2MS+6-BA 1.5mg / L+NAA 0.4mg / L.

[0042] (5) Rooting culture: During the proliferation culture process, healthy tissue culture seedlings were selected, and new shoots with a length of 2.8 cm and containing apical growth points were cut and inoculated into rooting medium for rooting culture for 30 days;

[0043] The rooting medium was 1 / 2MS + IBA 0.3 mg / L + sucrose 30 g / L + agar 7 g / L.

[0044] (6) Hardening and transplanting: Move the tissue culture seedlings outdoors for hardening. First, place them under scattered light for 7 days, then move them to a place with direct light and continue hardening for 7 days. Then transplant them to the cultivation medium to obtain virus-free seedlings; the cultivation medium is a mixture of peat soil, vermiculite and garden soil in a volume ratio of 1:1:1.

[0045] Example 3

[0046] (1) Explant differentiation induction: Select a new grape vine with vigorous growth, cut off the top 1-2mm stem tip as the explant, wash it thoroughly with water containing detergent, and then disinfect the surface in a clean bench. The disinfection process is as follows: disinfect with 72% alcohol for 20 seconds, rinse with sterile water 3 times; disinfect with 6% sodium hypochlorite for 8 minutes, rinse with sterile water 5 times. After disinfection, inoculate it into the start-up culture medium, place it in the light for 12 hours, light intensity 2000Lx, and culture at 23-27℃ for 18 days;

[0047] The starting culture medium was MS+IBA 0.8 mg / L+KT 1.5 mg / L+sucrose 30 g / L+agar 7 g / L.

[0048] (2) Virus-free culture: The culture temperature was raised to 32°C and maintained for 2 days; then raised to 34-36°C and maintained for 3 days, and then heat treated. The heat treatment was 38°C / light for 6 hours + 32°C / dark for 6 hours, in a cycle with a light intensity of 3000Lx. After heat treatment for 20 days, clustered buds were obtained.

[0049] (3) Virus detection: Immunoglobulins of rabbit antisera against grape leafroll virus 1 (GLRaV-1), grape fanleaf virus (GFLV), and grape spot virus (GFkV) were used to detect clustered shoots;

[0050] (4) Proliferation culture: Cut the clustered buds with negative virus test results into single buds, inoculate them into proliferation medium, place them in a light-irradiation environment of 12 h, 2000 Lx, and 23-27°C for 40 days, and subculture every 40 days.

[0051] The proliferation medium is 1 / 2MS+6-BA 1.6 mg / L and NAA 0.5 mg / L+sucrose 30 g / L+agar 7 g / L.

[0052] (5) Rooting culture: During the proliferation culture process, healthy tissue culture seedlings were selected, and new shoots with a length of 3.0 cm and containing apical growth points were cut and inoculated into rooting medium for rooting culture for 30 days;

[0053] The rooting medium was 1 / 2MS + IBA 0.5 mg / L + sucrose 30 g / L + agar 7 g / L.

[0054] (6) Hardening and transplanting: Move the tissue culture seedlings outdoors for hardening. First, place them under scattered light for 7 days, then move them to a place with direct light and continue hardening for 7 days. Then transplant them to the cultivation medium to obtain virus-free seedlings; the cultivation medium is a mixture of peat soil, vermiculite and garden soil in a volume ratio of 1:1:1.

[0055] Comparative Example 1

[0056] The difference from Example 1 is that the IBA in the priming medium is replaced by NAA of the same concentration.

[0057] Comparative Example 2

[0058] The difference from Example 1 is that KT in the starting medium is replaced by 6-BA of the same concentration.

[0059] Comparative Example 3

[0060] The difference from Example 1 is that the concentration of IBA in the starting culture medium is 1.2 mg / L.

[0061] Comparative Example 4

[0062] Different from Example 1, the concentration of KT in the priming medium was 0.5 mg / L.

[0063] Comparative Example 5

[0064] The difference from Example 1 is that the concentration of 6-BA in the proliferation medium is 1 mg / L.

[0065] Comparative Example 6

[0066] The difference from Example 1 is that the concentration of NAA in the proliferation medium is 0.05 mg / L.

[0067] Comparative Example 7

[0068] The difference from Example 1 is that the heat treatment method is: constant temperature of 37°C / (light for 16 hours + darkness for 8 hours).

[0069] Comparative Example 8

[0070] The difference from Example 1 is that the heat treatment method is: 37°C / light for 16 hours + 32°C / dark for 8 hours in a cycle.

[0071] Comparative Example 9

[0072] The difference from Example 1 is that the heat treatment method is: 37°C / light for 8 hours + 32°C / dark for 8 hours, in sequence.

[0073] Experimental Example 1

[0074] The experiment was divided into 7 groups. Virus-free tissue culture of grape seedlings was carried out according to the methods of Examples 1 to 3 and Comparative Examples 1 to 4, respectively. 100 explants were inoculated into the priming medium in each group and cultured at 23 to 27° C. for 18 days. The germination rate was calculated. The results are shown in Table 1.

[0075] Germination rate = number of buds in grape explants / total number of grape explants inoculated × 100%.

[0076] Table 1 Germination rate of explants in each group

[0077] Group Explant germination rate (%) Example 1 98 Example 2 98 Example 3 100 Comparative Example 1 86 Comparative Example 2 73 Comparative Example 3 77 Comparative Example 4 69

[0078] It can be seen from Examples 1 to 3 that the priming medium of the present invention can effectively promote explant germination, and the germination rate is above 98%; it can be seen from Example 1 and Comparative Examples 1 to 4 that the germination rate of explants decreases when the type or concentration of the plant growth regulator in the priming medium is changed.

[0079] Experimental Example 2

[0080] The experiment was divided into eight groups. Virus-free tissue culture of grape seedlings was performed according to the methods described in Examples 1-3 and Comparative Examples 5-9. Each group was inoculated with 100 explants. After virus-free culture, clustered shoots (test tube plantlets) were obtained. Leaves from these shoots were tested for viruses, and the survival and virus-free rates of the test tube plantlets were calculated. The virus-free clustered shoots were then cut into individual buds and inoculated into a proliferation medium. After 40 days, the proliferation efficiency was calculated. The results are shown in Table 2.

[0081] Survival rate = number of test tube seedlings / number of explants × 100%;

[0082] Virus-free rate = number of virus-free seedlings / total number of test tube seedlings × 100%;

[0083] Proliferation coefficient = number of axillary buds of grape tissue culture seedlings / total number of grape tissue culture seedlings inoculated.

[0084] Table 2 Survival rate, virus-free rate and proliferation efficiency of clustered buds in each group

[0085] Group Survival rate (%) Number of virus-free strains (strains) Detoxification rate (%) proliferation coefficient Example 1 98 94 95.92 6.3 Example 2 97 93 95.88 6.5 Example 3 95 90 94.74 6.6 Comparative Example 5 99 95 95.96 5.2 Comparative Example 6 98 93 94.90 5.8 Comparative Example 7 36 36 100.00 6.7 Comparative Example 8 78 77 98.71 6.6 Comparative Example 9 85 83 97.60 6.6

[0086] As shown in Table 2, the heat treatment method affects the growth state of the test tube seedlings. The treatment method of Comparative Example 7 is: 37°C constant temperature / (light for 16 hours + dark for 8 hours) treatment for 40 days. The survival rate of the test tube seedlings is only 36%, which is significantly lower than the temperature treatment of Examples 1 to 3 and Comparative Examples 8 and 9, indicating that constant temperature treatment is not conducive to the survival of the test tube seedlings. The heat treatment method of Comparative Example 8 is: 37°C / light for 16 hours + 32°C / dark for 8 hours, cycled sequentially; the heat treatment method of Comparative Example 9 is: 37°C / light for 8 hours + 32°C / dark for 8 hours, cycled sequentially. The survival rates of the test tube seedlings in Comparative Examples 8 and 9 are 78% and 85%, respectively, which are lower than those in Examples 1 to 3, indicating that the high temperature treatment time is too long and the survival rate of the test tube seedlings decreases. In Comparative Examples 5 and 6, the concentrations of 6-BA and NAA in the proliferation medium are changed. Compared with Examples 1 to 3, the proliferation coefficient of the clustered shoots is reduced. The virus-free method of the present invention achieves a good virus-free rate under the premise of ensuring the survival rate of the test tube seedlings, the virus-free rate is above 95%, and the proliferation coefficient is high.

[0087] As can be seen from the above examples, the present invention provides a method for rapid tissue culture propagation of virus-free grape seedlings. The heat treatment method of the present invention can improve the survival rate of grape test tube seedlings and improve the virus-free rate.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of virus-free grape seedlings through tissue culture, characterized in that: The following steps are involved: (1) Explant differentiation induction: Cut the top 1-2 mm of the grape stem tip, sterilize it, inoculate it into the initiation medium, and culture it at 23-27°C for 16-18 days; (2) Virus-free culture: The culture temperature is raised to 30-32°C and maintained for 2-3 days; then raised to 34-36°C and maintained for 2-3 days, and then heat treated, wherein the heat treatment is 37-38°C / light for 5-6 hours + 32-33°C / dark for 5-6 hours, in a cycle, and the light intensity is 2000-3000 Lx; heat treatment is performed for 18-22 days to obtain clustered buds; (3) Virus detection: detection of grape leafroll virus 1, grape fan leaf virus, and grape spot virus; (4) Proliferation culture: Cut the clustered buds with negative virus test results into single buds, inoculate them into proliferation medium, and culture them for 38 to 42 days; subculture once every 38 to 42 days; (5) Rooting culture: During the proliferation culture process, healthy tissue culture seedlings were selected, and new shoots with a length of 2.5 to 3.0 cm and containing apical growth points were cut and inoculated into rooting medium for rooting culture for 20 to 30 days; (6) Hardening and transplanting: Move the tissue culture seedlings outdoors for hardening, and then transplant them into the cultivation medium to obtain virus-free seedlings.

2. The method according to claim 1, characterized in that The disinfection is as follows: disinfection with 68-72% alcohol for 20-30 seconds, followed by rinsing with sterile water 2-3 times; disinfection with 4-6% sodium hypochlorite for 7-8 minutes, followed by rinsing with sterile water 4-5 times.

3. The method according to claim 1, characterized in that The starting culture medium is MS+IBA 0.3-0.8 mg / L+KT 1-1.5 mg / L+sucrose 30 g / L+agar 7 g / L.

4. The method according to claim 1, wherein The proliferation culture medium is 1 / 2MS+6-BA 1.4-1.6 mg / L and NAA 0.3-0.5 mg / L+sucrose 30 g / L+agar 7 g / L.

5. The method according to claim 1, wherein The rooting medium is 1 / 2MS+IBA 0.1-0.5 mg / L+sucrose 30 g / L+agar 7 g / L.

6. The method according to claim 1, characterized in that The culture temperature in steps (1) (4) (5) is 23-27°C, the illumination time is 10-12 hours, and the illumination intensity is 2000-3000 Lx.

7. The method according to claim 1, characterized in that The seedling hardening comprises the following steps: placing the tissue culture seedlings under scattered light for 5 to 7 days, and then moving them to a place with direct light for further 5 to 7 days.

8. The method according to claim 1, characterized in that The cultivation medium is formed by mixing peat soil, vermiculite and garden soil in a volume ratio of 1:1:1.