Zingiber officinale detoxification rapid propagation technology

Abstract

The invention discloses a ginger virus-free rapid propagation technology. Rapid propagation of small yellow ginger virus-free bacteria-free seedlings is realized through stem tip virus-free culture. The method comprises the following steps: (1) explant treatment; (2) stem tip stripping; (3) culture medium and culture condition setting; and (4) virus detection. The fresh ginger for tests is selected from the Yuzhou City eight-peak Agricultural Development Co., Ltd. According to the research, TMV and CMV viruses can be completely removed through stem apex virus-free culture, and rapid propagation of small yellow ginger virus-free and bacterium-free seedlings can be realized.

Description

Technical Field

[0001] The invention relates to the technical field of ginger planting, and in particular to a ginger detoxification and rapid propagation technology. Background Art

[0002] Ginger (Zingiber officinaie Roscoe) is a perennial herbaceous plant of the genus Zingiber in the Zingiberaceae family. It is native to southeastern Asia and is widely planted in my country. Its tubers are rich in active ingredients such as curcumin and gingerol. It is nutritious and has a unique flavor. It is a specialty agricultural product that can be used as both medicine and food, and a traditional export product. However, the current average yield of ginger in my country is only about 1,500 kg / mu, which is slightly higher than the world average, but lower than that of Malaysia, India and other countries. In addition, although there are many varieties of ginger in my country, there are few high-quality and high-yield varieties, and the uses are single, resulting in unstable ginger quality. Therefore, while expanding the ginger cultivation area, it is of great significance to improve the quality of ginger and develop ginger for multiple uses.

[0003] Ginger virus-free rapid propagation technology is a method that combines virus-free and rapid propagation to produce disease-free, high-quality ginger planting materials, ensure the healthy growth of ginger seeds, and increase yields. As a crop susceptible to pests and diseases, ginger virus-free treatment is a key link in ensuring its planting quality. The purpose of virus-free treatment is to remove viruses, bacteria, fungi and other diseases that may exist in ginger. Rapid propagation technology usually uses tissue culture and shoot tip culture to quickly propagate a large number of healthy ginger seeds. Ginger virus-free rapid propagation technology is widely used in large-scale ginger planting bases to ensure that ginger planting materials are disease-free and high-quality. This technology can increase the yield of ginger, improve its quality, reduce the occurrence of pests and diseases, and achieve sustainable development of agricultural production. Ginger virus-free rapid propagation technology helps produce healthy and high-quality ginger seedlings through scientific disease removal and efficient propagation methods, which can greatly increase the yield and quality of ginger and promote the modernization of the ginger industry. Summary of the invention

[0004] S1. Treatment of explants: Select plump, large, and brightly colored ginger pieces, rinse them, and soak them in 500 times carbendazim (80% content) for sterilization for 1 hour. After drying, cover them with imported peat soil and water them appropriately. Cultivate them at a constant temperature of 28°C and maintain the humidity at about 80%. When the ginger buds grow to 2-3 cm, high-temperature treatment is performed on the ginger buds in batches at 55°C for 5-8 min.

[0005] S2. Shoot tip peeling: Rinse the high-temperature treated ginger buds with tap water for 30 minutes, disinfect with 75% alcohol for 45 seconds, and then clean with sterile water. Then, disinfect with 0.1% mercuric chloride solution for 5 minutes, 10 minutes, 15 minutes, and 20 minutes, and then wash with sterile water for 3 to 4 times. Use a binocular dissecting microscope to peel the ginger stem tip in a clean bench, and cut the stem tip 0.1 to 0.2 mm away from the leaf primordium and inoculate it on the induction medium.

[0006] S3, culture medium and culture conditions: MS is used as the basic culture medium, and different concentrations of hormones are added as induction and subculture culture medium. Induction culture medium: ①MS+6-BA1.5mg / L+NAA0.1mg / L, ②MS+6-BA2.5mg / L+NAA0.1mg / L, ③MS+6-BA3.5mg / L+NAA0.1mg / L, ④MS+6-BA3.5mg / L+NAA0.2mg / L. Proliferation and rooting are cultured simultaneously, and the culture medium formula is: MS+6-BA3mg / L+NAA0.2mg / L. The above culture media are all added with 3.0% sucrose and 0.7% agar, the pH value is 5.8, the culture temperature is 26~28℃, and each inoculation is dark cultured for 3 days before switching to light culture, with continuous light for 14 hours per day.

[0007] S4. Virus detection: Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect cucumber mosaic virus and tobacco mosaic virus in the sample, and the detection was performed according to the kit instructions of Shanghai Keborui Biotechnology Co., Ltd. DETAILED DESCRIPTION

[0008] The technical solution of the present invention is further illustrated and described below through specific implementation modes, but the present invention is not limited thereto.

[0009] Example 1 In the practice of plant sterile tissue culture, if the explants are not disinfected for a long time, bacteria or fungi will be contaminated. If the disinfection time is too long, the disinfectant will poison the explants and cause death. In this study, ginger explants (shoot apical meristems, young leaves of terminal buds) were treated with 0.1% mercuric chloride for 5, 10, 15 and 20 minutes. After 40 days of culture, the results showed that the survival rate of the stem apex disinfected for 15 minutes was the highest, reaching 65.7%, and the explant contamination rate was significantly lower than that of the treatments of 5 minutes and 10 minutes, which was 17.1%; the survival rate of the disinfection for 10 minutes was 23.3%, and the contamination rate was as high as 58.3%; the contamination rate of the disinfection for 20 minutes was the lowest, at 8.0%, but the survival rate was only 12.0%. The disinfection treatment time of 5 minutes was too short, but the contamination rate was very high (82.0%), resulting in an increase in mortality and a survival rate of only 18.0%. Therefore, it takes at least 15 minutes to disinfect ginger buds as explants with 0.1% mercuric chloride solution. If it exceeds 20 minutes, the survival rate will be affected by mercuric chloride toxicity.

[0010] Table 1 Effects of 0.1% mercuric chloride solution disinfection on the contamination rate and survival rate of ginger stem tip meristem Example 2 After culturing ginger stem tips under sterile conditions for 4 weeks, the base of the stem tip gradually swelled to 7-8 times the original volume, and the dedifferentiated cells of the explants divided to form callus tissue, which then differentiated into green adventitious buds. The number of adventitious buds differentiated on culture media with different hormone ratios varied greatly. On the four hormone ratio culture media, the differentiation rates were 48%, 64%, 52% and 71% after 30 days of culture, respectively. Among them, the effect of adventitious bud differentiation on culture medium No. 4 was the best.

[0011] Table 2 Effects of different hormone concentrations on ginger differentiation Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to determine cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) in the specimens. In the table, 3 is a blank control, 2 and 3 are negative controls, 4 and 5 are positive controls, and 6 to 18 are the OD values ​​of ginger virus-free seedling samples. The detection limit of TMV is 0.231, that is, the OD value is less than 0.231 and it is judged as negative; the detection limit of CMV is 0.221, that is, the OD value is less than 0.221 and it is judged as negative. It can be seen from the table that the OD values ​​of TMV and CMV in the virus-free seedlings of small yellow ginger are all less than 0.231, and the OD values ​​of CMV are all less than 0.221, so they are judged as negative. It shows that these test samples do not contain these two viruses. In summary, these two viruses can be completely removed by stem tip detoxification culture.

[0012] Table 3 Virus detection results (OD value)

Claims

1. A ginger detoxification and rapid propagation technology, characterized in that: The method comprises the following steps: S1. Treatment of explants: Select plump, large, and brightly colored ginger pieces, rinse them, and soak them in 500 times carbendazim (80% content) for sterilization for 1 hour. After drying, cover them with imported peat soil and water them appropriately. Cultivate them at a constant temperature of 28°C and maintain the humidity at about 80%. When the ginger buds grow to 2-3 cm, high-temperature treatment is performed on the ginger buds in batches at 55°C for 5-8 min.

2. S2, stem tip peeling: Rinse the high temperature treated ginger buds with tap water for 30 minutes, disinfect with 75% alcohol for 45 seconds, and then clean with sterile water. Then, disinfect with 0.1% mercuric chloride solution for 5 minutes, 10 minutes, 15 minutes, and 20 minutes, and then wash with sterile water for 3 to 4 times. Use a binocular dissecting microscope to peel the ginger stem tip in the clean bench, and cut the stem tip 0.1 to 0.2 mm away from the leaf primordium and inoculate it on the induction medium. 3.S3, culture medium and culture conditions: MS is used as the basic culture medium, and different concentrations of hormones are added as induction and subculture culture medium. Induction culture medium: ①MS+6-BA1.5mg / L+NAA0.1mg / L, ②MS+6-BA2.5mg / L+NAA0.1mg / L, ③MS+6-BA3.5mg / L+NAA0.1mg / L, ④MS+6-BA3.5mg / L+NAA0.2mg / L. Proliferation and rooting are cultured simultaneously, and the culture medium formula is: MS+6-BA3mg / L+NAA0.2mg / L. The above culture media are all added with 3.0% sucrose and 0.7% agar, the pH value is 5.8, the culture temperature is 26~28℃, and each inoculation is dark cultured for 3 days before switching to light culture, with continuous light for 14 hours per day. 4.S4. Virus detection: Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to determine the cucumber mosaic virus and tobacco mosaic virus in the sample, and the detection was performed according to the kit instructions of Shanghai Keborui Biotechnology Co., Ltd.

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