Ginseng fruit detoxification rapid propagation method and application

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a ginseng fruit detoxification rapid propagation method and application, and the method comprises the following steps: stem tip rapid growth culture, chemical treatment, high-temperature passivation, stem tip stripping, multiplication culture, repeating the steps of stem tip stripping and multiplication culture twice, and then carrying out virus detection. The high-temperature passivation temperature is 40 + / -1 DEG C, 35 + / -1 DEG C and 25 + / -2 DEG C, so that stem tips can normally grow without burning seedlings; according to the present invention, the stem tip stripping adopts the 0.8-1.0 mm, the operation difficulty is substantially reduced, the germination rate can be more than 95% in the MS + NAA 0.02-0.05 mg / L + 6-BA 0.2-0.5 mg / L + GA3 0.2-0.3 mg / L + coconut juice 40-50 ml / L culture medium, the stem tip stripping only needs three times, and the detoxification time can be substantially shortened; the four treatment modes of stem tip rapid growth culture, virazole treatment, high-temperature passivation and stem tip stripping are combined, and the detoxification rate can reach 80% or above.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for rapid propagation of ginseng fruit detoxification and application. Background Art

[0002] Ginseng fruit (Solanum muricatum), formerly known as eggplant, melon eggplant, also known as longevity fruit, phoenix fruit, and gorgeous fruit, belongs to the Solanaceae family, Solanum, and is a vegetable, fruit, and ornamental herb. The fruit is mostly heart-shaped and oval-shaped. When ripe, the peel has purple stripes and has a light and elegant fragrance. The flesh is refreshing and juicy with a unique flavor. It is high in protein, low in sugar and fat, and is also rich in vitamin C and a variety of trace elements necessary for the human body. Therefore, ginseng fruit has the functions of anti-cancer, anti-aging, lowering blood pressure, lowering blood sugar, anti-inflammatory, calcium supplementation, and beauty, and is widely planted and eaten in my country.

[0003] Conventional cultivation of ginseng fruit is mainly based on stem cutting propagation, but seedlings and adult plants are very susceptible to tobacco mosaic virus, cucumber mosaic virus and potato M virus. The virus infection in the body is serious and accumulates from generation to generation, which seriously affects its growth and development, manifested as wrinkled and smaller leaves, high mortality, low fruit setting rate, and a significant decrease in yield. Seedling detoxification and rapid propagation through plant stem tip detoxification culture is an effective way to produce virus-free seedlings. At present, the detoxification technology of ginseng fruit mainly combines heat treatment with stem tip glass. The general temperature of heat treatment is 37±1℃. When this temperature is treated for a long time, the incidence of burning tips is extremely high, resulting in very little material that can be used for stem tip peeling. At the same time, the current stem tip peeling is mainly 0.1-0.3mm in size, which is difficult to peel and the germination rate of the peeled stem tip is low, resulting in an extremely low success rate of the operation. Summary of the invention

[0004] The purpose of the invention is to provide a ginseng fruit detoxification rapid propagation method and application in view of the problems existing in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is: a ginseng fruit detoxification rapid propagation method, the steps comprising:

[0006] (1) Rapid growth culture of shoot tips: Take sterile ginseng fruit seedlings, cut them into single buds, inoculate them in a growth medium with the formula: MS + NAA 0.01-0.02 mg / L + GA3 0.1-0.2 mg / L, and culture them in complete darkness at 30°C for 10-15 days;

[0007] (2) Chemical treatment: Take the rapidly growing seedlings through the stem tip, cut off the 0.5 cm terminal buds, inoculate them into the chemical treatment medium with the formula of MS + NAA 0.01-0.02 mg / L + 6-BA 0.1-0.2 mg / L, add 10-15 mg / L of ribavirin to the medium, and culture it in environment A for 15-20 days;

[0008] The environment A is specifically as follows: the temperature is adjusted to 32±1°C and the light intensity is 2000-3000lx for 12 hours every day, and then the temperature is adjusted to 25±2°C and cultured in total darkness for 12 hours;

[0009] (3) High temperature passivation: Take the chemically treated seedlings, cut off the 0.5 cm terminal buds, inoculate them into the passivation medium with the formula of MS + NAA 0.01-0.02 mg / L + 6-BA 0.1-0.2 mg / L, and culture them in environment B for 20 days;

[0010] The environment B is specifically as follows: culture at 40±1°C in total darkness for 2 hours every day, then adjust the temperature to 35±1°C in total darkness for 10 hours, and finally adjust the temperature to 25±2°C in total darkness for 12 hours under a light intensity of 2000-3000lx;

[0011] (4) Shoot tip stripping: Take the seedlings that have been passivated by high temperature, strip the bud primordium 0.08-0.10 cm below the terminal bud, inoculate it in the shoot tip stripping medium with the formula of MS + NAA 0.02-0.05 mg / L + 6-BA 0.2-0.5 mg / L + GA3 0.2-0.3 mg / L + coconut juice 40-50 ml / L, and place it at a temperature of 25±2℃ with 12 hours of light per day for 30-35 days;

[0012] (5) Proliferation culture: The seedlings peeled from the stem tip were inoculated into a medium containing 0.1 mg / L MS+PPP333 and cultured at 25±2℃ with 12 h of light per day for 15 to 20 days.

[0013] (6) Repeat steps (4) and (5) twice;

[0014] (7) Virus detection: PCR detection method is used for detection.

[0015] Furthermore, the formula of the growth medium is: MS+NAA 0.01 mg / L + GA3 0.1 mg / L.

[0016] Furthermore, the formula of the chemical treatment medium is: MS+NAA0.01mg / L + 6-BA 0.1mg / L.

[0017] Furthermore, the formula of the passivation culture medium is: MS+NAA0.01 mg / L + 6-BA 0.1 mg / L.

[0018] Furthermore, the formula of the shoot tip stripping culture medium is: MS+NAA 0.03 mg / L +6-BA 0.3 mg / L +GA3 0.25 mg / L +coconut juice 45 ml / L.

[0019] Furthermore, the formula of the proliferation culture medium is: MS+PPP333 0.1-0.2 mg / L.

[0020] Furthermore, the PCR detection method is specifically as follows: Trizol is used to extract total RNA of the sample, and after reverse transcription to synthesize cDNA, specific primers of TMV, CMV, PVY, and PVM are used, and PCR detection is performed with cDNA as a template. The amplified products are separated by 1% agarose gel electrophoresis, CMV-CP, TMV-CP, PVY-CP, and PVM-CP are used as positive controls, and water is used as a negative control. Actin of ginseng fruit is used to detect the quality of cDNA.

[0021] The beneficial technical effects of the present invention are:

[0022] 1. At present, the detoxification technology of ginseng fruit mainly combines heat treatment with stem tip glass. The general temperature of heat treatment is 37±1℃. When the temperature is treated for a long time, the incidence of burning the tip is very high, resulting in very little material available for stem tip peeling, and the success rate of the operation is low. The high temperature passivation temperature of the present invention adopts a high temperature temperature change mode of 40±1℃, 35±1℃, and 25±2℃ for passivation, which can make the stem tip grow normally without burning the seedling.

[0023] 2. At the same time, the existing ginseng fruit stem tip peeling mostly adopts 0.1-0.3mm, which is difficult to operate and has a low germination rate after peeling. When the high temperature passivation of the existing technology is combined with the stem tip peeling, even if the stem tip is not obviously damaged by the naked eye, it is extremely weak when peeled under a stereoscope, which makes the stem tip peeling operation more difficult. At the same time, when the peeled stem tip is cultured, its germination rate is also low. The stem tip peeling of the present invention adopts 0.8-1.0mm, which greatly reduces the difficulty of operation. At the same time, in the medium of MS+NAA 0.02-0.05 mg / L +6-BA 0.2-0.5 mg / L +GA3 0.2-0.3 mg / L + coconut juice 40-50ml / L, the germination rate can be as high as 95% or more.

[0024] 3. The prior art stem tip stripping usually needs to be performed 5 to 6 times, which takes a long time, while the present invention only needs to perform the operation 3 times, which can greatly shorten the detoxification time.

[0025] 3. The present invention combines four treatment methods, namely, rapid growth culture of shoot tips, virazole treatment, high temperature passivation and shoot tip peeling, to achieve a detoxification rate of more than 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0027] Figure 1 This is a diagram of the stem tip peeling result of Example 1 of the present invention.

[0028] Figure 2 This is a diagram showing the culture results of the virus-free and proliferated seedlings of Example 1 of the present invention.

[0029] Figure 3 1 is a graph showing the virus detection results of Example 1 of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] A method for rapid propagation of ginseng fruit detoxification, comprising the following steps:

[0033] (1) Rapid growth culture of shoot tips: Take sterile ginseng fruit seedlings, cut them into single buds, inoculate them into a growth medium with the formula: MS + NAA 0.01 mg / L + GA3 0.1 mg / L, and culture them in complete darkness at 30°C for 13 days.

[0034] (2) Chemical treatment: Take the rapidly growing seedlings through the stem tip, cut off the 0.5 cm terminal buds, inoculate them into a chemical treatment medium with the formula: MS + NAA 0.01 mg / L + 6-BA 0.1 mg / L, add 13 mg / L of ribavirin to the medium, and culture it in environment A for 17 days.

[0035] Environment A is as follows: the temperature is adjusted to 32±1°C and the light intensity is 2500lx for 12 hours every day, and then the temperature is adjusted to 25±2°C and cultured in total darkness for 12 hours.

[0036] (3) High temperature passivation: Take the chemically treated seedlings, cut off the 0.5 cm apical buds, inoculate them into the passivation medium with the formula: MS + NAA 0.01 mg / L + 6-BA 0.1 mg / L, and culture them under environment B for 20 days.

[0037] Environment B is as follows: daily, the culture is cultured in total darkness at 40±1°C for 2 h, then the temperature is adjusted to 35±1°C for 10 h, and finally the temperature is adjusted to 25±2°C for 12 h under a light intensity of 2500 lx.

[0038] (4) Stem tip stripping: Take the seedlings that have been passivated by high temperature and strip 0.09 cm of the bud primordium from below the apical bud (such as Figure 1 As shown in the figure, the shoot tip stripping medium was inoculated with the formula of MS + NAA 0.03 mg / L + 6-BA 0.3 mg / L + GA3 0.25 mg / L + coconut juice 45 ml / L, and cultured at 25±2℃ with 12h of light per day for 33 days.

[0039] (5) Proliferation culture: The shoots were peeled off and inoculated into the medium containing MS+PPP333 0.1 mg / L. The culture was placed at 25±2℃ and incubated for 17 days under 12 hours of light per day. The results were as follows: Figure 2 shown.

[0040] (6) Repeat steps (4) and (5) twice.

[0041] (7) Virus detection: PCR detection was used. Total RNA of the sample was extracted with Trizol and reverse transcribed into cDNA. PCR detection was performed using specific primers for TMV, CMV, PVY, and PVM, and cDNA was used as a template. The amplified products were separated by 1% agarose gel electrophoresis. CMV-CP, TMV-CP, PVY-CP, and PVM-CP were used as positive controls, and water was used as a negative control. Actin from ginseng fruit was used as an internal reference to detect the quality of cDNA (the test results are shown in Table 1). Figure 3 as shown).

[0042] Example 2

[0043] A method for rapid propagation of ginseng fruit detoxification, comprising the following steps:

[0044] (1) Rapid growth culture of shoot tips: Take sterile ginseng fruit seedlings, cut them into single buds, inoculate them into a growth medium with the formula: MS + NAA 0.01 mg / L + GA3 0.1 mg / L, and culture them in complete darkness at 30°C for 10 days.

[0045] (2) Chemical treatment: Take the rapidly growing seedlings through the stem tip, cut off the 0.5 cm terminal buds, inoculate them into a chemical treatment medium with the formula: MS + NAA 0.01 mg / L + 6-BA 0.1 mg / L, add 10 mg / L of ribavirin to the medium, and culture it in environment B for 15 days.

[0046] Environment A is as follows: the temperature is adjusted to 32±1°C and the light intensity is 2000lx for 12 hours every day, and then the temperature is adjusted to 25±2°C and cultured in total darkness for 12 hours.

[0047] (3) High temperature passivation: Take the chemically treated seedlings, cut off the 0.5 cm apical buds, inoculate them into the passivation medium with the formula: MS + NAA 0.01 mg / L + 6-BA 0.1 mg / L, and culture them under environment B for 20 days.

[0048] Environment B is as follows: daily, the culture is cultured in total darkness at 40±1°C for 2 h, then the temperature is adjusted to 35±1°C for 10 h, and finally the temperature is adjusted to 25±2°C for 12 h under a light intensity of 2000 lx.

[0049] (4) Shoot tip stripping: Take the seedlings that have been passivated by high temperature, strip the bud primordium 0.08 cm from the bottom of the apical bud, inoculate it into the shoot tip stripping medium with the formula of MS + NAA 0.02 mg / L + 6-BA 0.2 mg / L + GA3 0.2 mg / L + coconut juice 40 ml / L, and place it at a temperature of 25 ± 2 ° C and 12 h of light per day for 30 days.

[0050] (5) Proliferation culture: The seedlings peeled from the stem tips were inoculated into a culture medium containing 0.1 mg / L MS+PPP333 and cultured at 25±2℃ with 12 h of light per day for 15 days.

[0051] (6) Repeat steps (4) and (5) twice.

[0052] (7) Virus detection: PCR detection was used for detection; total RNA of the sample was extracted using Trizol, and after reverse transcription into cDNA, PCR detection was performed using specific primers of TMV, CMV, PVY, and PVM, and cDNA was used as a template. The amplified products were separated by 1% agarose gel electrophoresis, and CMV-CP, TMV-CP, PVY-CP, and PVM-CP were used as positive controls, and water was used as a negative control. Actin from ginseng fruit was used as an internal reference gene to detect the quality of cDNA.

[0053] Example 3

[0054] A method for rapid propagation of ginseng fruit detoxification, comprising the following steps:

[0055] (1) Rapid growth culture of shoot tips: Take sterile ginseng fruit seedlings, cut them into single buds, inoculate them into a growth medium with the formula: MS + NAA 0.02 mg / L + GA3 0.2 mg / L, and culture them in complete darkness at 30°C for 15 days.

[0056] (2) Chemical treatment: Take the rapidly growing seedlings through the stem tip, cut off the 0.5 cm terminal buds, inoculate them into a chemical treatment medium with the formula: MS + NAA 0.02 mg / L + 6-BA 0.2 mg / L, add 15 mg / L of ribavirin to the medium, and culture it in environment A for 20 days.

[0057] Environment A is as follows: the temperature is adjusted to 32±1°C and the light intensity is 3000lx for 12 hours every day, and then the temperature is adjusted to 25±2°C and cultured in total darkness for 12 hours.

[0058] (3) High temperature passivation: Take the chemically treated seedlings, cut off the 0.5 cm apical buds, inoculate them into the passivation medium with the formula: MS + NAA 0.02 mg / L + 6-BA 0.2 mg / L, and culture them under environment B for 20 days.

[0059] Environment B is as follows: daily, the culture is cultured in total darkness at 40±1°C for 2 h, then the temperature is adjusted to 35±1°C for 10 h, and finally the temperature is adjusted to 25±2°C for 12 h under a light intensity of 3000 lx.

[0060] (4) Shoot tip stripping: Take the seedlings that have been passivated by high temperature, strip the 0.10 cm bud primordium from under the terminal bud, inoculate it into the shoot tip stripping medium with the formula of MS + NAA 0.05 mg / L + 6-BA 0.5 mg / L + GA3 0.3 mg / L + coconut juice 50 ml / L, and place it at a temperature of 25±2℃ with 12 hours of light per day for 35 days.

[0061] (5) Proliferation culture: The seedlings peeled from the stem tips were inoculated into a medium containing MS+PPP333 0.1 mg / L and cultured at 25±2℃ with 12 h of light per day for 20 days.

[0062] (6) Repeat steps (4) and (5) twice.

[0063] (7) Virus detection: PCR detection was used. Total RNA of the sample was extracted with Trizol and reverse transcribed into cDNA. PCR detection was then performed using specific primers of TMV, CMV, PVY, and PVM and cDNA as a template. The amplified products were separated by 1% agarose gel electrophoresis. CMV-CP, TMV-CP, PVY-CP, and PVM-CP were used as positive controls, and water was used as a negative control. Actin from ginseng fruit was used to detect the quality of cDNA.

[0064] Comparative Example 1

[0065] The treatment was carried out according to the steps (2) chemical treatment, (3) high temperature passivation, and (4) stem tip peeling in Example 1.

[0066] Comparative Example 2

[0067] The treatment was carried out according to the steps of (1) rapid growth culture of shoot tips, (3) high temperature passivation, and (4) shoot tip peeling in Example 1.

[0068] Comparative Example 3

[0069] The treatment was carried out according to the steps of (1) rapid growth culture of shoot tips, (2) chemical treatment, and (4) peeling of shoot tips in Example 1.

[0070] Comparative Example 4

[0071] The treatment is carried out according to the steps of (1) rapid growth culture of shoot tips, (2) chemical treatment, and (3) high temperature passivation in Example 1.

[0072] Comparative Example 5

[0073] The treatment was carried out according to the steps of Example 1: (1) rapid growth culture of shoot tips, (3) high temperature passivation, (2) chemical treatment, and (4) shoot tip peeling.

[0074] Comparative Example 6

[0075] The operation was performed according to the method disclosed in publication number: CN101248761A, entitled: A method for rapid virus detection in detoxified tissue culture seedlings of ginseng fruit.

[0076] Comparative Example 7

[0077] The operation was carried out according to Wang Haixin, Shi Feng, An Yuming. Ginseng fruit tissue culture detoxification technology [J]. Hebei Agricultural Science and Technology, 2007, (07): 27.

[0078] Comparative Example 8

[0079] According to Tang Ziyong, Wang Yanling, Cheng Shuyun. Ginseng fruit stem tip detoxification and test tube rapid propagation technology [J]. Inner Mongolia Agricultural Science and Technology, 1999, (S1): 18. Operation.

[0080] The operation difficulty, stem tip peeling germination rate, total detoxification time and detoxification rate in Examples 1-3 and Comparative Examples 1-8 were compared, and the results are shown in Table 1 below.

[0081] Table 1 Comparison results

[0082] Difficulty of operation Shoot tip germination rate Total time for detoxification Detoxification rate Example 1 Difficult 95.9% 7 months 86.7% Example 2 Difficult 95.1% 7 months 85.4% Example 3 Difficult 95.5% 7 months 85.8% Comparative Example 1 Difficult 96.2% 6 months 80.2% Comparative Example 2 Difficult 97.6% 6 months 64.1% Comparative Example 3 Difficult 99.7% 6 months 43.2% Comparative Example 4 Extremely simple - 2 months 11.6% Comparative Example 5 Difficult 97.4% 7 months 72.6% Comparative Example 6 Very difficult 87.4% 3 months 5.8% Comparative Example 7 Very difficult 88.3% 3 months 4.2% Comparative Example 8 Very difficult 52.4% 3 months 18.4%

[0083] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for rapid propagation of ginseng fruit detoxification, characterized in that the steps include: (1) Rapid growth culture of shoot tips: Take sterile ginseng fruit seedlings, cut them into single buds, inoculate them in a growth medium with the formula: MS + NAA 0.01-0.02 mg / L + GA3 0.1-0.2 mg / L, and culture them in complete darkness at 30°C for 10-15 days; (2) Chemical treatment: Take the rapidly growing seedlings through the stem tip, cut off the 0.5 cm terminal buds, inoculate them into the chemical treatment medium with the formula of MS + NAA 0.01-0.02 mg / L + 6-BA 0.1-0.2 mg / L, add 10-15 mg / L of ribavirin to the medium, and culture it in environment A for 15-20 days; The environment A is specifically as follows: the temperature is adjusted to 32±1°C and the light intensity is 2000-3000lx for 12 hours every day, and then the temperature is adjusted to 25±2°C and cultured in total darkness for 12 hours; (3) High temperature passivation: Take the chemically treated seedlings, cut off the 0.5 cm terminal buds, inoculate them into the passivation medium with the formula of MS + NAA 0.01-0.02 mg / L + 6-BA 0.1-0.2 mg / L, and culture them in environment B for 20 days; The environment B is specifically as follows: culture at 40±1°C in total darkness for 2 hours every day, then adjust the temperature to 35±1°C in total darkness for 10 hours, and finally adjust the temperature to 25±2°C in total darkness for 12 hours under a light intensity of 2000-3000lx; (4) Shoot tip stripping: Take the seedlings that have been passivated by high temperature, strip the bud primordium 0.08-0.10 cm below the terminal bud, inoculate it in the shoot tip stripping medium with the formula of MS + NAA 0.02-0.05 mg / L + 6-BA 0.2-0.5 mg / L + GA3 0.2-0.3 mg / L + coconut juice 40-50 ml / L, and place it at a temperature of 25±2℃ with 12 hours of light per day for 30-35 days; (5) Proliferation culture: The seedlings peeled from the stem tips were inoculated into a proliferation medium with the formula: MS+PPP333 0.1-0.2 mg / L, and cultured at a temperature of 25±2°C with 12 h of light per day for 15-20 days; (6) Repeat steps (4) and (5) twice; (7) Virus detection: PCR detection method is used for detection.

2. The method according to claim 1, characterized in that: The formula of the growth medium is: MS+NAA 0.01 mg / L + GA3 0.1 mg / L.

3. The method according to claim 1, characterized in that: The formula of the chemical treatment medium is: MS+NAA0.01mg / L + 6-BA 0.1mg / L.

4. The method according to claim 1, characterized in that: The formula of the passivation culture medium is: MS+NAA 0.01 mg / L + 6-BA 0.1 mg / L.

5. The method according to claim 1, characterized in that: The formula of the shoot tip stripping culture medium is: MS+NAA 0.03 mg / L +6-BA 0.3 mg / L +GA3 0.25 mg / L +coconut juice 45 ml / L.

6. The method according to claim 1, characterized in that: The formula of the proliferation culture medium is: MS+PPP333 0.1-0.2 mg / L.

7. The method according to claim 1, characterized in that: The PCR detection method is specifically as follows: Trizol is used to extract the total RNA of the sample, and after reverse transcription to synthesize cDNA, specific primers of TMV, CMV, PVY, and PVM are used, and PCR detection is performed with cDNA as a template. The amplified products are separated by 1% agarose gel electrophoresis, CMV-CP, TMV-CP, PVY-CP, and PVM-CP are used as positive controls, and water is used as a negative control. Actin of ginseng fruit is used to detect the quality of cDNA.

8. Application of the method according to any one of claims 1 to 7 in the detoxification and rapid propagation of ginseng fruit.

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