CN112616665A - Method for effectively removing SPFMMV and SPLCV viruses of sweet potatoes - Google Patents
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Abstract
The invention discloses a method for effectively removing SPFMMV and SPLCV viruses of sweet potatoes, and belongs to the technical field of plant detoxification in plant tissue culture technology. The method for effectively removing the SPFMMV and SPLCV viruses of the sweet potatoes comprises the following steps: the stem tip culture detoxification, virus detection and high temperature treatment or antiviral agent treatment. The invention has the following advantages: the invention efficiently and rapidly removes two main viruses SPFMMV and SPLCV of the sweet potatoes by utilizing a stem tip culture and high-temperature detoxification or medicament treatment detoxification method, thereby providing technical support for mass production of subsequent detoxified sweet potato seedlings.
Description
Technical Field
The invention belongs to the technical field of plant detoxification in plant tissue culture technology, and particularly relates to a method for effectively removing SPFMMV and SPLCV viruses of sweet potatoes.
Background
Sweet potatoes (Ipomoea batatas Lam) are one of important food crops, are important industrial raw materials and feed crops, and are widely planted worldwide. The sweet potato is also named sweet potato, red sweet potato, sweet potato and sweet potato, is a perennial or perennial trailing herbaceous plant in Convolvulaceae, is rich in protein, starch, pectin, cellulose and various mineral substances, and has the reputation of 'longevity food'.
The sweet potato virus is a worldwide sweet potato disease, widely exists in sweet potato production areas in the world, and causes great harm to the quality and the yield of sweet potatoes. Sweet potato has multiple virus types, multiple new viruses and serious compound infection phenomenon, the infection proportion of single virus is only 31.9 percent, most of the single virus is infected by 3 to 5 viruses, and sweet potato is a vegetative propagation crop and mainly utilizes root tubers and stems and tendrils for propagation to cause the generation and the generation of the virus in plants and even cause serious hazards such as degeneration of species properties, loss of commodity properties and the like. Among them, Sweet Potato Feathery Mottle Virus (SPFMV) and Sweet potato leafroll virus (SPLCV) are two main viruses infecting Sweet potatoes in China, and in recent years, they occur seriously in the main Sweet potato production areas of China, causing serious damage to Sweet potato production. In order to prevent viral diseases, plant protection scientists have sought solutions from various aspects over the years, but since viral replication is closely related to plant metabolic processes, there has been no drug that can selectively inhibit viruses without harming plants so far. People find that the virus elimination of the plants can not only reduce virus harm and increase yield, but also do no harm to the plants.
The detoxification technology of the plants is widely applied at present, and the detoxification technology comprises three types: heat treatment, stem tip culture, and antiviral agent.
1. Heat treatment method
The heat treatment is to make use of the difference of tolerance of the virus and the host plant to high temperature, so that the growth rate of the plant exceeds the diffusion rate of the virus, generally, the higher the temperature is, the longer the time is, the better the detoxification effect is, but at the same time, the survival rate of the plant is in a descending trend. The main disadvantages of the heat treatment detoxification method are long detoxification time and incomplete detoxification.
2. Stem tip culture detoxification method
The shoot apical meristem of the plant is composed of meristematic cells, there is no vascular bundle in the meristematic region, the intercellular connective filaments of the cell wall are not developed, and viruses are difficult to enter, so that the growing point often contains no or very little viruses, and the rapid propagation of virus-free or virus-removed plants can be realized by adopting the shoot apical meristem culture technology.
When the stem tip is cut too large during the culture, complete removal of the virus cannot be guaranteed, and the smaller the stem tip is, the less virus is carried. Considering the detoxification effect and improving the survival rate, the apical meristem with 1-2 leaf margin bases is generally taken for culture, the stem tips of different plants are different in stripping difficulty, auxin is probably synthesized in the second pair of young leaf primordia in dicotyledonous plants, and the auxin of the meristem of the stem tips cannot be self-supplied, so that the culture of the young stem tips is more difficult than that of general buds, and exogenous auxin and cytokinin with proper concentration must be provided. Studies have shown that the virus removal effect is inversely proportional to the size of the stem tip, but the stem tip is too small to survive. The stem tip culture detoxification method has high detoxification rate and high detoxification speed, can obtain a plurality of stock propagation materials in a short time, but has the defect of low survival rate of plants.
3. Antiviral agent method
The chemically synthesized micromolecule plant virus inhibitor comprises the following components: organic acids such as carboxylic acids aspirin, dodecylacetic acid, acetylsalicylic acid and Polyacrylic Acid (PA) have good inhibitory effect on TMV, and their antiviral mechanism is to induce plants to obtain systemic disease resistance and generate pathogenesis-related protein (PR-P). The antiviral medicament is convenient to use, has a strong inhibiting effect on plant viruses and can reduce the occurrence of virus diseases, but the inhibiting effect of the antiviral medicament has pertinence on the viruses and can not effectively remove various viruses at one time; if the concentration is too high, the plant growth is also adversely affected.
In addition to the above detoxification methods, anther culture, callus culture, ovule culture may also be used for detoxification.
Disclosure of Invention
The invention aims to disclose a method for effectively removing SPFMMV and SPLCV viruses of sweet potatoes.
The purpose of the invention is realized by the following technical scheme:
a method for effectively removing SPFMMV and SPLCV viruses from sweet potatoes, the method comprising the steps of:
(1) and culturing and detoxifying the stem tip:
cutting stem sections with the length of 2-3 cm at the top of the sweet potato bud end, cutting off unfolded leaves, pouring washing powder, washing with a brush, and washing with running water for 30 min; soaking in 70% ethanol for 30s on a sterile operating table, sterilizing with 2% sodium hypochlorite for 5min, and washing with sterile water for 3 times to obtain sterilized bud segments;
carrying out stem tip stripping treatment on the sterilized bud segments under a dissecting mirror, cutting off small undeveloped leaves and large leaf primordia, cutting off growth points with 1-2 leaf primordia, wherein the length is 0.2-0.3mm, picking the stem tips into a triangular flask filled with a culture medium by using a dissecting needle for stem tip culture, and replacing a scalpel and the dissecting needle once every stem tip is inoculated so as to reduce pollution; the culture medium for stem tip culture is MS minimal medium added with 1.0mg/L, IAA 0.5.5 mg/L of 6-BA, 30g/L of sucrose and 6g/L of agar, and the pH value is 5.8; the culture conditions comprise 25 deg.C, illumination intensity of 2000Lx, and illumination time of 12 h/d; culturing for 30 days, and then transferring to MSO culture medium to differentiate into seedlings;
(2) and virus detection:
when the stem tip seedlings cultured in the step (1) are differentiated into more than two seedlings, detecting the feather mottle virus and the sweet potato leaf curl virus of the sweet potatoes by adopting RT-PCR (reverse transcription-polymerase chain reaction) on 4-6 picked leaves; the procedure of RT-PCR is: pre-denaturation: 5min at 94 ℃; denaturation: 30s at 94 ℃; renaturation: 30s at 60 ℃; extension: 60s at 72 ℃; 30 cycles; and finally, extension: 10min at 72 ℃;
the virus-free seedlings obtained by detection are directly used for producing tissue culture seedlings; subjecting the tissue culture seedling with toxicity obtained by detection to high temperature treatment and antiviral agent treatment;
(3) high temperature treatment or antiviral agent treatment:
carrying out high-temperature treatment or antiviral agent treatment on the tissue culture seedlings with toxicity obtained by detection in the step (2);
the high-temperature treatment comprises the following specific steps: after being treated for 2 to 8 hours at the temperature of 40 ℃, virus detection is carried out according to the step (2) to obtain tissue culture seedlings with the sweet potato SPFMMV and SPLCV virus removed;
the antiviral agent treatment comprises the following specific steps: respectively adding 50-200 mg/L of Dufulin, Altailing, neooctoxin or Ningnanmycin into a culture medium of the tissue culture seedling with the virus for culturing for 1-5 months, and carrying out virus detection according to the step (2) to obtain the tissue culture seedling without the SPFMFV and SPLCV viruses of the sweet potato.
The method for effectively removing the SPFMMV and SPLCV viruses of the sweet potatoes in the technical scheme comprises the inoculation material processing step before the stem tip culture detoxification step in the step (1), and the specific steps are as follows: carrying out pot culture on the germinated seedlings or the cutting seedlings of the sweet potato blocks, wherein a pot culture substrate is prepared by mixing turf and vermiculite in a ratio of 1: 1; when the sweet potato seedlings reach 20 cm-30 cm, sampling and stem tip culture are started.
The method for effectively removing SPFMMV and SPLCV viruses of sweet potatoes in the technical scheme comprises the following steps of (1) detecting primers by RT-PCR in the step (2):
SPFMV F 5-GGATTAYGGTGTTGACGAC-3，
SPFMV R 5-TCGGGACTGAARGAYACGAATTTAA-3；
SPLCV F 5-CCCCKGTGCGWRAATCCAT-3，
SPLCV R 5-ATCCVAAYWTYCAGGGAGCTAA-3。
the invention has the following beneficial effects:
sweet potato yield and quality are long-term affected by viruses. Due to the reproductive characteristics of the sweet potatoes, a large amount of viruses are accumulated in the plant bodies, and the properties of the sweet potatoes are seriously degraded. The invention efficiently and rapidly removes two main viruses SPFMMV and SPLCV of the sweet potatoes by utilizing a detoxification method of stem tip culture, high-temperature detoxification and medicament treatment, and provides technical support for mass production of subsequent detoxified sweet potato seedlings.
Description of the drawings:
1. FIG. 1 is a flow chart of sweet potato stem tip culture detoxification.
2. FIG. 2 shows the detoxification of tissue culture seedlings of sweet potatoes by high temperature treatment.
3. FIG. 3 shows the detoxification of sweet potato tissue culture seedlings by antiviral agent treatment.
The specific implementation mode is as follows:
in order to facilitate understanding of the technical scheme of the present invention, the method for effectively removing SPFMMV and SPLCV viruses of sweet potato according to the present invention will be further described with reference to the following specific examples.
Example 1:a method for effectively removing SPFMMV and SPLCV viruses of sweet potatoes comprises the following steps:
sweet Potato Feathery Mottle Virus (SPFMV) and Sweet Potato Leaf Curl Virus (SPLCV) are two major viruses infecting Sweet potatoes in China. According to the study on the parasitic characteristics and the propagation approach of the virus disease in the sweet potato body, the virus is difficult to infect and propagate at the stem tip part with intense metabolism of the meristematic cells of the sweet potato, so that the method for controlling the sweet potato virus is realized by cutting the stem tip with the diameter of 0.2-0.3mm at the top end of the sweet potato and culturing the stem tip into a regenerated seedling for detoxification.
One, materials and methods:
1. test materials:
12 sweet potato varieties are used in the test, and are respectively as follows: the sweet potato chips comprise sweet potato 9, Jishu potato 26, Jing 6, Shandong potato 19, 138, Yanshu 25, yellow rose, Xinxiang, Pushu 32, 28-1 (Nongda 28), 28-3 (Nongda white) and C16-2 (yellow banana), wherein test materials are provided by Beijing agricultural technology promotion station and Chinese agriculture university.
2. And (3) stem tip culture detoxification:
respectively cutting stem sections with the length of 2-3 cm at the top of each variety bud end, cutting off unfolded leaves, putting the stem sections into a beaker, pouring washing powder, washing with a brush, and washing with running water for 30 min. Soaking in 70% ethanol for 30s on a sterile operating table, sterilizing with 2% sodium hypochlorite for 5min, and washing with sterile water for 3 times.
Carrying out stem tip stripping treatment on the sterilized bud segments under a dissecting mirror, cutting off small undeployed leaves and large leaf primordia, cutting off growth points with 1-2 leaf primordia, wherein the length is 0.2-0.3mm, picking the stem tips into a triangular flask filled with a culture medium by using a dissecting needle for stem tip culture, inoculating 3-5 explants in each flask, and replacing a scalpel and the dissecting needle once per stem tip to reduce pollution; the culture medium for stem tip culture is MS minimal medium added with 1.0mg/L, IAA 0.5.5 mg/L of 6-BA, 30g/L of sucrose and 6g/L of agar, and the pH value is 5.8; the culture conditions were 25 deg.C, illumination intensity 2000Lx, and illumination time 12 h/d. Culturing for 30 days, transferring to MSO culture medium, and differentiating into seedling. The flow chart of the stem tip culture detoxification is shown in figure 1.
3. And (3) virus detection:
the shoot tip seedling is generally differentiated into more than two shoots, and 4-6 leaves can be picked for virus detection. Entrusted Beijing Fuyun Biotechnology Limited company adopts RT-PCR technology to detect sweet potato virus, the detected virus is sweet potato feathery mottle virus (SPFMMV) and Sweet Potato Leaf Curl Virus (SPLCV);
the procedure for RT-PCR was: pre-denaturation: 5min at 94 ℃; denaturation: 30s at 94 ℃; renaturation: 30s at 60 ℃; extension: 60s at 72 ℃; 30 cycles; and finally, extension: 10min at 72 ℃;
the primers for RT-PCR were:
SPFMV F 5-GGATTAYGGTGTTGACGAC-3，
SPFMV R 5-TCGGGACTGAARGAYACGAATTTAA-3；
SPLCV F 5-CCCCKGTGCGWRAATCCAT-3，
SPLCV R 5-ATCCVAAYWTYCAGGGAGCTAA-3；
4. high-temperature treatment and detoxification of the stem tip cultured seedlings with the viruses:
the tobacco potato 25 and the Beijing 6 stem tip culture seedlings which still carry SPFMMV and SPLCV viruses after the stem tips are stripped and subjected to virus detection are respectively treated in a constant temperature incubator at 40 ℃ for 2h, 4h and 8h, and then virus detection is carried out. For virus detection, refer to step 4. The sweet potato high-temperature treatment is shown in figure 2.
5. Treating and detoxifying the virus-carrying stem tip cultured seedlings by using an antiviral agent:
carrying out detoxification tests by taking tobacco potato 25 still carrying SPFMMV virus after stem tip peeling through virus detection, and Jing 6 and Longshu 9 tissue culture seedlings simultaneously carrying SPFMMV and SPLCV virus as materials, wherein treatment agents are Dufulin, Altailing, neooglandcin or ningnanmycin; the treatment concentration is 50mg/L, 100mg/L and 200 mg/L; transferring the sweet potato tissue culture seedlings into a culture medium added with a medicament. After treatment for 1 month, 2 months, 3 months, 4 months and 5 months respectively, the cells are transferred into an MSO culture medium without adding medicaments for culture and then virus detection is carried out. For virus detection, refer to step 4. Sweet potato antiviral agent treatment is shown in fig. 3.
Secondly, result and analysis:
1. the stem tip culture survival and detoxification conditions of different sweet potato varieties are as follows:
test results show that different varieties of stem tips have different seedling emergence rates, the seedling emergence rate of the nicotiana batatas 25 is the highest and reaches 80.2%, the seedling emergence rates of the nicotiana batatas 25 and 138 yellow roses respectively reach 73.9% and 61.2%, the seedling emergence rate is the same as that of the mercurius batatas 19, only 10.7%, and the others are all between 40% and 50%; the mortality rates of the stem tips of the varieties are different, the mortality rates of the stem tips of the three varieties of the potatoes 25 and 138 with the highest emergence rate and the yellow roses are all lower than 10 percent, the mortality rate of the commercial potatoes 19 with the lowest emergence rate is up to 22.5 percent.
The single stem tip has poor detoxification effect by culture, and the detoxification rate is more than 10-30%.
2. High-temperature treatment and detoxification of the stem tip cultured seedlings with the viruses:
test results show that the high-temperature treatment detoxification results are shown in table 1, and the removal rates of SPFMMV viruses are 100% after the tobacco potato 25 and the Jing 6 are treated for 2 hours, 4 hours and 8 hours at the temperature of 40 ℃; SPLCV virus carried by the tobacco potato 25 can be removed by 100% after being treated for 2 hours and 8 hours, but can not be completely removed after being treated for 4 hours, and the detoxification rate is 66.7%; the SPLCV carried by Jing 6 is treated for 2h, 4h and 8h at 40 ℃, and the detoxification rate is 85.7 percent.
TABLE 1 influence of high-temperature treatment of shoot-tip cultured seedlings on the detoxification rates of different sweet potatoes (%)
3. Treating and detoxifying the virus-carrying stem tip cultured seedlings by using an antiviral agent:
test results show that the tobacco potato 25 carrying the SPFMMV virus, the Jing 6 and Longshu 9 tissue culture seedlings carrying the SPFMMV virus and the SPLCV virus simultaneously can be effectively removed from the SPFMMV and the SPLCV virus after being cultured for 1 to 5 months in a culture medium added with 50 to 200mg/L of antiviral medicament (the antiviral medicament is selected from one of Dufulin, Alternate, Neoorigamine or Ningnanmycin). However, antiviral agents can have an adverse effect on the growth of tissue culture plantlets.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims; meanwhile, any equivalent changes, modifications and variations of the above embodiments according to the essential technology of the present invention are within the scope of the technical solution of the present invention.
Claims (3)
1. A method for effectively removing SPFMMV and SPLCV viruses from sweet potatoes, the method comprising the steps of:
(1) and culturing and detoxifying the stem tip:
cutting stem sections with the length of 2-3 cm at the top of the sweet potato bud end, cutting off unfolded leaves, pouring washing powder, washing with a brush, and washing with running water for 30 min; soaking in 70% ethanol for 30s on a sterile operating table, sterilizing with 2% sodium hypochlorite for 5min, and washing with sterile water for 3 times to obtain sterilized bud segments;
carrying out stem tip stripping treatment on the sterilized bud segments under a dissecting mirror, cutting off small undeveloped leaves and large leaf primordia, cutting off growth points with 1-2 leaf primordia, wherein the length is 0.2-0.3mm, picking the stem tips into a triangular flask filled with a culture medium by using a dissecting needle for stem tip culture, and replacing a scalpel and the dissecting needle once every stem tip is inoculated so as to reduce pollution; the culture medium for stem tip culture is MS minimal medium added with 1.0mg/L, IAA 0.5.5 mg/L of 6-BA, 30g/L of sucrose and 6g/L of agar, and the pH value is 5.8; the culture conditions comprise 25 deg.C, illumination intensity of 2000Lx, and illumination time of 12 h/d; culturing for 30 days, and then transferring to MSO culture medium to differentiate into seedlings;
(2) and virus detection:
when the stem tip seedlings cultured in the step (1) are differentiated into more than two seedlings, detecting the feather mottle virus and the sweet potato leaf curl virus of the sweet potatoes by adopting RT-PCR (reverse transcription-polymerase chain reaction) on 4-6 picked leaves; the procedure of RT-PCR is: pre-denaturation: 5min at 94 ℃; denaturation: 30s at 94 ℃; renaturation: 30s at 60 ℃; extension: 60s at 72 ℃; 30 cycles; and finally, extension: 10min at 72 ℃;
the virus-free seedlings obtained by detection are directly used for producing tissue culture seedlings; subjecting the tissue culture seedling with toxicity obtained by detection to high temperature treatment and antiviral agent treatment;
(3) high temperature treatment or antiviral agent treatment:
carrying out high-temperature treatment or antiviral agent treatment on the tissue culture seedlings with toxicity obtained by detection in the step (2);
the high-temperature treatment comprises the following specific steps: after being treated for 2 to 8 hours at the temperature of 40 ℃, virus detection is carried out according to the step (2) to obtain tissue culture seedlings with the sweet potato SPFMMV and SPLCV virus removed;
the antiviral agent treatment comprises the following specific steps: respectively adding 50-200 mg/L of Dufulin, Altailing, neooctoxin or Ningnanmycin into a culture medium of the tissue culture seedling with the virus for culturing for 1-5 months, and carrying out virus detection according to the step (2) to obtain the tissue culture seedling without the SPFMFV and SPLCV viruses of the sweet potato.
2. The method for effectively removing SPFMMV and SPLCV viruses from sweet potatoes according to claim 1, wherein the method further comprises a step of inoculation material treatment before the step of stem tip culture detoxification in step (1), and the method comprises the following specific steps: carrying out pot culture on the germinated seedlings or the cutting seedlings of the sweet potato blocks, wherein a pot culture substrate is prepared by mixing turf and vermiculite in a ratio of 1: 1; when the sweet potato seedlings reach 20 cm-30 cm, sampling and stem tip culture are started.
3. The method for effectively removing sweet potato SPCMV and SPLCV viruses according to claim 1, wherein the primers for RT-PCR detection in the step (2) are as follows:
SPFMV F 5-GGATTAYGGTGTTGACGAC-3，
SPFMV R 5-TCGGGACTGAARGAYACGAATTTAA-3；
SPLCV F 5-CCCCKGTGCGWRAATCCAT-3，
SPLCV R 5-ATCCVAAYWTYCAGGGAGCTAA-3。
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| CN113317320A (en) * | 2021-04-26 | 2021-08-31 | 山东省农业科学院作物研究所 | Biological agent for improving virus-free rate of sweet potatoes and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113317320A (en) * | 2021-04-26 | 2021-08-31 | 山东省农业科学院作物研究所 | Biological agent for improving virus-free rate of sweet potatoes and application thereof |
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