Method for removing viruses and endogenous and exogenous bacteria from small yellow ginger

Abstract

The invention discloses a method for removing viruses and endogenous and exogenous bacteria from small yellow ginger, and relates to the technical field of virus-free seedling cultivation, and the method comprises the following steps: S1, selecting ginger blocks which grow robustly and fleshy and are free of damage and plant diseases and insect pests as explant materials, and placing the ginger blocks in a dark environment at 28-30 DEG C for accelerating germination; s2, when the ginger buds grow to 2-3cm, selecting healthy and full ginger buds, cutting off the ginger buds, washing the ginger buds with running water, and disinfecting the ginger buds; s3, under a sterile condition, transferring the disinfected ginger buds into a primary culture medium, screening out the ginger seedlings with bacteria by adopting a high-low temperature method, and ending culture when the ginger buds grow 1-3 coleoptiles; s4, stripping leaf coleoptiles of the screened healthy and pollution-free ginger buds, cutting off 1-3mm stem tips from growing points, and transferring the stem tips into a clumpy bud induction culture medium for multiplication culture at the culture temperature of 20-25 DEG C and the illumination intensity of 2500 LUX for 8-15 days; s5, the clustered seedlings obtained after proliferation are cut into single seedlings from the base parts, the single seedlings are placed in a rooting culture medium, and virus-free plants are obtained and cultured for 30-45 d at the culture temperature of 22-25 DEG C and the illumination intensity of 3000 LUX.

Description

Technical Field

[0001] The invention relates to the technical field of virus-free seedling cultivation, and in particular to a method for removing viruses and endogenous and exogenous bacteria from small yellow ginger. Background Art

[0002] ginger( Zingiber offcinale Roscoe) is a perennial herbaceous plant of the ginger family, also known as ginger, yellow ginger, etc. It is widely cultivated in provinces and regions in central, southeastern and southwest my country. Ginger is divided into small-seed ginger, large-seed ginger, mountain ginger (wild ginger) and other varieties. It is customary to call small-seed ginger "small yellow ginger". Small yellow ginger has unique advantages such as pure yellow cut surface, spicy and strong taste, tender meat, fragrant taste, and fine fiber, and is deeply loved by people. Tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) are the main viruses that infect ginger, and Pseudomonas subtilis is the main pathogen that infects ginger. Endogenous bacterial contamination is the microorganisms (such as endophytic bacteria, fungi, etc.) inside the plant material (intracellular or intercellular) that cannot be removed by general surface disinfection methods and are brought into the culture process with the material. For example, the endophytic bacterial contamination rate of cyclamen tuber tissue culture is as high as 83%. Viruses and pathogens accumulate in ginger, leading to the degeneration of ginger varieties, chlorosis of ginger leaves, deformity, dwarfing of plants, and other symptoms, as well as reduced resistance and quality, which has become one of the main constraints in the development of the ginger industry. This ultimately leads to a 30% reduction in ginger production, and in severe cases, a reduction of more than 45%. Continuous cropping can lead to an incidence rate of up to 80%, or even a total loss of production.

[0003] High-temperature shoot apex detoxification is a method that utilizes the protein characteristics of viruses. High-temperature treatment can passivate or inhibit the reproduction of viruses. Since plant viruses are transmitted through the vascular system, and there is no vascular system in the shoot apex meristem, there are generally no viruses or very small amounts of viruses in the apical meristem.

[0004] The three main high temperature treatment methods used by previous researchers in different plants are as follows: "Zhang Shuting. Optimization of the virus-free rapid propagation system of ginger [D]. Chengdu: Sichuan Agricultural University, 2023" The ginger buds were placed in a 50ºC water bath for 10 minutes and then the stem tips were peeled off for tissue culture; "Zhou Xiaobo, Wu Yifei, Ding Zhuodi. Research on virus-free rapid propagation technology of Lilium lily [J]. Chinese Agricultural Science Bulletin, 2012, 28(31): 201-205" The bulbils of Lilium lily were placed in a 36℃ constant temperature incubator for 10 days for pretreatment and then stem tip culture was performed. "Ma Pingxia. Virus-free and virus detection of Siberian lily [D]. Lanzhou: Northwest Normal University, 2007" Ma Pingxia placed 2~3cm Siberian lily tissue culture seedlings in an artificial incubator at 38℃ for 16h of light and 25℃ for 8h of darkness for 42 days of heat treatment. Previous researchers used water baths or constant temperature incubators for high temperature treatment, but the disadvantages are that long-term high temperature treatment in a natural environment will cause excessive consumption of explant nutrients, bud shriveling, and explant rot, resulting in secondary pollution. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for removing viruses and endogenous and exogenous bacteria from small yellow ginger.

[0006] The objective of the present invention is achieved through the following technical solutions: S1. Select ginger pieces that are healthy and thick, without damage, diseases or insect pests as explant materials, and place them in a dark environment at 28-30℃ for germination; S2. When the ginger sprouts grow to 2-3 cm, select healthy and plump ginger sprouts, cut them, rinse them with running water, place them on a clean bench for disinfection, use 0.1% mercuric chloride to disinfect for 30 min, and rinse them with sterile water 3 times; S3. Under sterile conditions, transfer the disinfected ginger sprouts to the primary culture medium, use the high and low temperature detoxification method to screen out the infected ginger seedlings, and terminate the culture when the ginger sprouts grow 1-3 coleoptiles; the culture medium formula is: MS+0.2mg / LBA+0.02mg / LNAA+30g / L sucrose+8.5g / L carrageenan, pH6.0-6.4; high and low temperature detoxification is specifically: high temperature 35-40℃15d, low temperature 15-18℃10d (temperature control is adjusted by the air conditioner in the culture room), light intensity 1500LUX; Note: After the high and low temperature culture at this stage, three situations will occur in the culture medium: (1) Explant contamination due to incomplete explant disinfection or improper operation. Its characteristics are that a large number of microorganisms and fungi grow on the explants and culture medium. The source of contamination comes from external bacteria. Figure 1 .

[0007] (2) Contamination caused by endogenous bacteria, exogenous bacteria and viruses in the explants themselves is characterized by pink, blue, gray, decay, bubbles in the culture medium or turbidity in the culture medium (different types of endogenous bacteria and exogenous bacteria in the ginger sprouts will cause different phenomena). The source of contamination is the endogenous bacteria and exogenous bacteria in the ginger sprouts themselves. These explants will also be screened out. See Figure 2 .

[0008] (3) The ginger sprouts are well developed and free of any contamination. The culture medium is clean, clear and transparent. This type of ginger sprout is a healthy explant that does not contain endogenous and exogenous bacteria and is used for the next step of stem tip peeling. Figure 3 .

[0009] S4. Under sterile conditions, the healthy ginger buds after the initial screening were stripped of leaf bud sheaths, 1-3 mm stem tips were cut from the growth point, and transferred to cluster bud induction medium for proliferation culture at a temperature of 20-25°C and a light intensity of 2500 LUX for 8-15 days; the medium formula was MS+3 mg / L BA+0.2 mg / L NAA+30 g / L sucrose+8.5 g / L carrageenan, pH 6.0-6.4; S5. Cut the clustered seedlings obtained after proliferation into single seedlings from the base and place them in rooting medium. The virus-free plants were cultured at 22-25°C, with a light intensity of 3000 LUX for 30-45 days. The culture medium formula was MS + 0.02 mg / L NAA + 30 g / L sucrose + 8.5 g / L carrageenan, with a pH of 6.0-6.4.

[0010] The larger ginger sprouts in S3 were cut into halves and the culture medium was packaged in bags.

[0011] In the primary culture medium, healthy ginger sprouts without endogenous bacteria are screened out through high and low temperature treatment, and carrageenan is used as the solidifying agent in the culture medium.

[0012] The present invention adopts a high and low temperature treatment method + a stem tip detoxification method to obtain sterile seedlings. The specific method is to place the sterilized explant (2-3 cm ginger bud) in a primary culture medium, culture it in a sterile tissue culture room, control the indoor temperature at 35-40°C for 15 days through air conditioning, and then adjust it to 15-18°C for 10 days. Take a healthy ginger bud and peel off the stem tip growth point 1-3mm in a sterile environment for culture. This method is obviously different from other methods and has not been reported so far.

[0013] Beneficial effects: (1) The present invention operates under a sterile environment throughout the entire process, thereby reducing cross contamination between explants and lowering the contamination rate during callus induction and proliferation culture; (2) When treated at 35-40℃ for 15 days in the primary culture medium (MS+0.2mg / LBA+0.02mg / LNAA+30g / L sucrose+8.5g / L carrageenan, pH6.0-6.4), the culture medium provided nutrients to promote cell division and elongation at the apical growth point of ginger sprouts. When cultured at 15-18℃ for 10 days, the apical growth point grew too fast and became stronger. The length of the apical growth point could be cut to 1-3mm (conventional operations can only take 0.1-0.3mm of the growth point), which greatly improved the work efficiency and the survival rate of the apical growth point tissue culture. Previous studies have found that the smaller the apical growth point, the lower the survival rate.

[0014] (3) High temperature treatment can, on the one hand, inactivate and inhibit the reproduction of viruses, and on the other hand, screen out a large number of explant materials containing endogenous and exogenous bacteria. The principle is that the endogenous and exogenous bacteria in the explants multiply in large numbers, consume the nutrients in the ginger sprouts, and produce a large amount of excrement. Subsequently, the excrement is released into the culture medium through low temperature culture at 15-18°C for 10 days. Explants that appear pink, yellow-green, gray, halo-like, bubbles, and turbid culture medium in the culture medium all contain endogenous and exogenous bacteria and will be screened out at this stage (explants containing endogenous and exogenous bacteria often have reduced proliferation efficiency, slowed culture growth, and vitrified seedlings in the early stage of tissue culture, which will lead to difficulties in transplanting test tube seedlings and death in the later stage, and will also cause genetic variation in the culture).

[0015] (4) After high and low temperature treatment, ginger buds with strong growth, transparent culture medium and no pollution can be screened out for the peeling of stem tip growth points. Since the infected explants were screened out in the early stage, the peeled growth points formed more callus tissue and grew faster. The later contamination rate, browning of tissue culture seedlings and vitrification of seedlings were significantly controlled (the browning rate can be reduced by high or low temperature treatment), and the reproduction coefficient was also significantly improved, reaching 10.64. In previous studies on ginger tissue culture, the reproduction coefficient of ginger was between 3 and 5. Some researchers have also reported that the average reproduction coefficient of ginger tissue culture seedlings reached 20.6, but the growth state of tissue culture seedlings was poor and not suitable for further expansion and reproduction.

[0016] (5) Previous researchers have chosen agar as a culture medium solidifier in tissue culture research. The culture medium solidifier used in this application is carrageenan. Carrageenan has the characteristics of high transparency, strong viscosity and low cost. Agar is light milky white, has poor transparency, poor screening effect and high cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a manifestation of contamination caused by incomplete disinfection of explants or improper operation; Figure 2 It is the performance of explants carrying endogenous and exogenous bacterial culture media; Figure 3 For the health of explants and the performance of contamination-free culture medium; Figure 4 The invention discloses high-quality small yellow ginger seedlings obtained by adopting the inventive method of the invention. DETAILED DESCRIPTION

[0018] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. Example

[0019] Cultivation of virus-free seedlings of ginger 1. Ginger seed selection and germination: Select ginger pieces that are healthy and thick, without damage, disease or insect pests as explant materials, and place them in a dark environment at 28-30℃ for germination.

[0020] 2. Explant collection and disinfection: When the ginger sprouts grow to 2-3cm, select healthy and plump ginger sprouts, cut them and rinse them in running water for 1-2 hours, place them on a clean workbench, disinfect them with 0.1% mercuric chloride for 30 minutes, rinse them with sterile water 3 times, and soak them for the last time for 5 minutes.

[0021] 3. Explant detoxification: Under sterile conditions, transfer the sterilized ginger sprouts (cut large ginger sprouts into two halves) to bagged primary culture medium (MS+0.2mg / LBA+0.02mg / LNAA+30g / L sucrose+8.5g / L carrageenan, pH6.0-6.4), and use high and low temperature detoxification method (high temperature 35-40℃ for 15d, low temperature 15-18℃ for 10d, light intensity 1500LUX) to screen out the infected ginger seedlings. End the culture when the ginger sprouts develop 1-3 coleoptiles.

[0022] 4. Callus induction and cluster seedling formation: Peel off the leaf bud sheaths of the virus-free ginger seedlings that do not contain endogenous or exogenous bacteria, find the growth point, cut off 0.1-0.3mm stem tips, and transfer them to cluster bud induction medium (MS+3mg / LBA+0.2mg / LNAA+30g / L sucrose+8.5g / L carrageenan, pH6.0-6.4) for proliferation culture. The culture temperature is 20-25℃, the light intensity is 2500LUX, and the culture is 8-15d. A large number of callus tissues and cluster seedlings can be induced in this culture medium, and the reproduction coefficient reaches 10.64.

[0023] The virus-free seedlings of turmeric were cultivated through explant screening on primary culture medium and stem tip detoxification. 20 plants were randomly selected for detoxification testing, and the detoxification rate reached 95%.

[0024] 5. Rooting culture: The clustered seedlings obtained after proliferation are cut into single seedlings from the base and placed in rooting medium (MS + 0.02 mg / L NAA + 30 g / L sucrose + 8.5 g / L carrageenan, pH 6.0-6.4), culture temperature 22-25 ° C, light intensity 3000 LUX, culture time 30-45 days to obtain virus-free seedlings.

[0025] 6. Training and cultivation of seedlings: Wash the culture medium on the rooted virus-free seedlings, dry the moisture on the root surface, and transplant them into a 48-hole plug tray. Fill the plug tray with a soilless matrix that is a uniform mixture of humus, peat soil and perlite in the ratio of 6:3:1. Place it in a greenhouse with a small arch shed inside. After 30 days of cultivation, high-quality seedlings that can be transplanted can be obtained.

[0026] Comparative Example The difference is that the high and low temperature virus-free culture in step 3 of the embodiment is replaced by virus-free culture at room temperature.

[0027] The test results of the virus-free seedlings obtained in the comparative examples and the examples are shown in Table 1.

[0028] Table 1 Treatment Contamination rate Tissue culture seedling quality Proliferation ability Virus removal rate High and low temperature treatment + stem tip peeling virus-free culture ＜5% robust 10.64 ＞95% Shoot tip peeling virus-free culture ＞30% generally 5.67 About 80% The invention adds a step before the detoxification of the stem tip, that is, after germination, the ginger block with 2-3 cm buds is washed and disinfected, and then placed in a primary culture medium for culture, and high and low detoxification treatment is adopted. After the explants with bacteria are treated with high and low temperatures, they will be manifested in the culture medium, for example, some culture mediums turn pink, green, blue, etc., some explants rot, the culture medium becomes turbid and unclear, some bubbles are generated on the culture medium, and some "halo-shaped objects" are formed around the explants, etc. These phenomena are all caused by the bacteria in the explants, such as Figure 1-2 The culture medium of the uninfected ginger sprouts is clean, clear and transparent. Figure 3 After the culture is completed, the explants with various endogenous bacteria, exogenous bacteria and viruses can be screened out by naked eyes. After the obtained virus-free seedlings are trained, high-quality seedlings that can be cultivated can be obtained, such as Figure 4 shown.

[0029] After adding this step, most of the explants with endogenous and exogenous bacteria (including bacteria, fungi and viruses) can be screened out, which can greatly reduce the contamination rate in the later stage. At the same time, high-quality and robust explants can be screened out to improve the seedling rate, thereby achieving the effect of reducing costs and increasing efficiency. The selected robust explants without endogenous and exogenous bacteria are used for stem tip peeling, which reduces labor costs and improves the detoxification rate. After the explant buds grow in sterile culture medium for nearly 1 month, the ginger buds can develop 1-3 coleoptiles, and the stem tip growth point is elongated, which makes it easier to operate during peeling, and reduces the contamination rate caused by the operation. The culture medium used uses carrageenan as a curing agent. Compared with agarose, carrageenan has the characteristics of high transparency, strong viscosity, low cost, and better screening effect.

[0030] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. A method for removing viruses and endogenous and exogenous bacteria from small yellow ginger, characterized in that: The following steps are involved: S1. Select ginger pieces that are healthy and thick, without damage, diseases or insect pests as explant materials, and place them in a dark environment at 28-30℃ for germination; S2. When the ginger sprouts grow to 2-3 cm, select healthy and plump ginger sprouts, cut them, rinse them with running water, place them on a clean bench for disinfection, use 0.1% mercuric chloride to disinfect for 30 min, and rinse them with sterile water 3 times; S3. Under sterile conditions, transfer the disinfected ginger buds to the primary culture medium, use the high and low temperature detoxification method to screen out the infected ginger seedlings, and end the culture when the ginger buds grow 1-3 coleoptiles; the culture medium formula is: MS+0.2mg / LBA+0.02mg / LNAA+30g / L sucrose+8.5g / L carrageenan, pH6.0-6.4; high and low temperature detoxification is specifically: high temperature 35-40℃15d, low temperature 15-18℃10d, light intensity 1500LUX; S4. Peel off the leaf bud sheaths on the virus-free ginger seedlings, cut off 1-3 mm stem tips from the growth point, and transfer them to cluster bud induction medium for proliferation culture at a temperature of 20-25°C and a light intensity of 2500 LUX for 8-15 days; the medium formula is MS+3mg / LBA+0.2mg / LNAA+30g / L sucrose+8.5g / L carrageenan, pH 6.0-6.4; S5. Cut the clustered seedlings obtained after proliferation into single seedlings from the base and place them in rooting medium to induce rooting. The culture temperature is 22-25℃, the light intensity is 3000 LUX, and the culture period is 30-45 days. The culture medium formula is MS+0.02 mg / L NAA+30g / L sucrose+8.5g / L carrageenan, pH 6.0-6.

4.

2. The method for removing viruses and endogenous and exogenous bacteria from small yellow ginger according to claim 1, characterized in that: In the primary culture medium, healthy ginger sprouts without endogenous bacteria are screened out through high and low temperature treatment, and carrageenan is used as the solidifying agent in the culture medium.

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