CN109042331B - Method for removing cymbidium mosaic virus and cymbidium ringspot virus at ultralow temperature - Google Patents
Method for removing cymbidium mosaic virus and cymbidium ringspot virus at ultralow temperature Download PDFInfo
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Abstract
The invention discloses a method for removing cymbidium mosaic virus and cymbidium ringspot virus, which comprises the following steps: taking a stem tip under a stereoscopic microscope by taking a phalaenopsis tissue culture seedling infected with a cymbidium mosaic virus and a odontoid ring spot virus as an object; putting the stem tip into a pre-culture medium for pre-culture; loading the stem tip with a loading liquid; treating the stem tip with a frozen protective solution; putting the stem tip treated by the frozen protective solution into liquid nitrogen for ultralow temperature treatment; putting the stem tip after ultralow temperature treatment into a liquid unloading carrier for unloading; putting the treated stem tip into a regeneration culture medium, and culturing under normal illumination; after 3 months, the stem tip can grow into seedlings smoothly. The invention can improve the survival rate and the regeneration rate of the stem tip.
Description
Technical Field
The invention belongs to the technical field of detoxification, and particularly relates to a method for removing cymbidium mosaic virus and cymbidium ringspot virus at ultralow temperature.
Background
There are 25 types of viruses reported to infect moth orchids, of which Cymbidium mosaic virus (CymMV) and odontoid ringspot virus (ORSV) occur most commonly and are most harmful. The long-term asexual propagation causes the virus in the butterfly orchid to be continuously accumulated, and the butterfly orchid is generally infected with the virus due to the lack of an effective infection and transmission method for preventing and controlling the two viruses, which becomes one of bottleneck problems influencing the quality of butterfly orchid seedlings. The situation that the phalaenopsis seedlings are infected by two viruses is reported in various countries in the world including China and the prevalence of the phalaenopsis seedlings is high. The cymbidium mosaic virus causes the leaves and petals of the phalaenopsis to generate chlorotic stripes or necrotic spots, and inhibits the growth of plants; the odontoglossum ringspot virus causes the leaves of the phalaenopsis plant to generate yellow stripe spots or yellow-green mottled embedded grains, petals cannot be unfolded or pigment is uneven, and local necrosis. The two viruses are often infected in a compound way, so that the harm is more serious, the growth condition of the phalaenopsis is seriously influenced, and the market value of the phalaenopsis is reduced. The serial effects of the two viruses have been reported to have serious impact on the butterfly orchid industry in taiwan province in China. The harm of the two viruses is recognized internationally, and most countries have carried out a seedling quarantine certificate system, and strict regulations that imported phalaenopsis cannot carry the two viruses result in that the export of considerable number of phalaenopsis seedlings is blocked.
Once the plant is infected with virus, the plant cannot be radically cured, so that the cultivation of the nontoxic seedling is an effective mode for preventing and treating the virus in the current production, and the production of the detoxified seedling is an effective mode for preventing and treating the virus in the current production, and is of great importance to the butterfly orchid industry. The detoxification research of the cymbidium mosaic virus and the cymbidium ringspot virus adopts the traditional detoxification method at present, wherein the widely adopted method is the combination of chemical agents and meristem culture. The method has the following three disadvantages: chemical agents have a toxic effect on plants. Research results show that the concentration of the medicament is lower, the treatment time is shorter, the toxic action on plants is not great, but the virus removal efficiency is low; the concentration of the medicament is higher, the treatment time is longer, the virus detoxification efficiency is high, but great toxic action is generated on plants and even the plants die. ② the chemical agent is easy to cause the genetic variation of the germchit. The existing research shows that the addition of chemical agents in a culture medium is easy to cause genetic variation of plant seedlings, and the related genetic stability identification of virus-free seedlings is lacked in the practical production, so that great risk exists in virus removal, and irreparable loss is caused for the industry. ③ the meristem is too small, which can easily cause browning. To avoid poisoning of the plant by the agent, researchers often reduce the concentration of the chemical agent and strip the smaller meristems. It is well known that meristem size is positively correlated with regeneration rate and negatively correlated with detoxification rate. For phalaenopsis, the problem that the browning of the taken meristem is easily caused is faced, and the browning rate and the death rate of the meristem which is less than 1mm are both 100%.
The ultra-low temperature detoxification technology is a novel detoxification technology which is extended from the ultra-low temperature preservation technology and is developed rapidly at present, and is used for removing pathogens (viruses, phytoplasmas and bacteria) from materials infected with the pathogens after transient treatment by liquid nitrogen. According to the reports of scholars at home and abroad, the ultra-low temperature detoxification technology is applied to 13 plants, and 23 viruses, 3 phytoplasmas and 1 bacterium are successfully removed. The ultra-low temperature detoxification technology only uses liquid nitrogen as a treatment means and does not use any chemical agent, thereby avoiding the problem of toxicity to plants, the liquid nitrogen preservation is proved to be genetically stable as a conventional germ plasm resource preservation means, and is different from the conventional meristem detoxification, the detoxification efficiency of the ultra-low temperature detoxification technology is unrelated to the size of the meristem, so that the small meristem is not needed to be taken.
At present, the ultra-low temperature detoxification technology is not applied to cymbidium mosaic virus and cymbidium ringspot virus.
Disclosure of Invention
In view of the above, the invention provides a method for removing the cymbidium mosaic virus and the odontoglossum ringspot virus by taking safe and effective ultra-low temperature therapy as a technical core aiming at the defects that the stem tips stripped from the cymbidium mosaic virus and the odontoglossum ringspot virus are too small and easy to brown, chemical agents have toxic action on plants and are easy to cause variation and the like in the conventional detoxification technology.
In order to solve the technical problem, the invention discloses a method for removing cymbidium mosaic virus and cymbidium ringspot virus, which comprises the following steps:
step 1, taking a stem tip with the size of 1.0-1.5cm under a stereoscopic microscope under the condition of an ultra-clean workbench by taking a phalaenopsis tissue culture seedling infected with a cymbidium mosaic virus and a odontoid ring spot virus as an object; putting the stem tip into a pre-culture medium for pre-culture;
step 2, loading the stem tip with a loading solution at room temperature;
step 3, treating the stem tips with PVS2 vitrification solution at 0 ℃, sucking PVS2 solution with a liquid transfer gun to make small drops on the aluminum-foil paper after the treatment is finished, putting the stem tips into the small drops one by one, clamping the aluminum-foil paper carrying the stem tips with tweezers, and putting the aluminum-foil paper into liquid nitrogen for ultralow-temperature treatment;
step 4, clamping the stem tip after ultralow temperature treatment, and unloading by putting in unloading liquid; putting the treated stem tip into a regeneration culture medium, culturing for 7 days in dark, and culturing under normal illumination; after 3 months, the stem tip can grow into seedlings smoothly.
Optionally, the pre-culture medium in the step 1 is BM culture medium +0.6M sucrose; the number of days of the pre-culture is 1-3 days, and the pre-culture is in a dark condition; the seedling age of the butterfly orchid tissue culture seedling is 6 weeks.
Optionally, said BM medium of step 1 comprises the following components per liter: 3g of Huabao 1, 10ml of ethylene diamine tetraacetic acid ferric sodium, 10ml of organic matter, 1.5g of peptone, 30g of sucrose and 7g of agar, and the pH value is 6.0.
Optionally, the sodium iron ethylenediaminetetraacetate in the step 1 is prepared by the following method: dissolving 3.67g of ethylenediaminetetraacetic acid ferric sodium salt in 1000ml of distilled water to prepare an ethylenediaminetetraacetic acid ferric sodium salt solution; the organic matter is prepared by the following method: 0.1-0.3g of glycine, 0.8-1.2g of thiamine, 0.05-0.15g of pyridoxine hydrochloride, 0.05-0.15g of calcium pantothenate and 8-12g of inositol are mixed and dissolved in 1000ml to prepare the organic matter.
Optionally, the loading solution in step 2 is BM +2M glycerol +0.4M sucrose; the dosage of the loading liquid is 20-30ml, and the time for loading the stem tip by the loading liquid is 20-30 minutes.
Optionally, the PVS2 vitrification solution in step 3 is: MS + 30% glycerol + 15% ethylene glycol + 15% DMSO +0.4M sucrose; the processing time of the frozen protective solution for processing the stem tip is 40-50 minutes; the amount of the PVS2 vitrification solution is 20-30 ml.
Optionally, the stem tip treatment temperature in the step 3 is 0 ℃, and the treatment time is 60-90 minutes.
Optionally, the ultra-low temperature treatment time in the step 3 is 25 to 35 minutes.
Optionally, the unloading liquid in the step 4 is BM +1.2M sucrose; the unloading time is 25-35 min.
Optionally, the regeneration medium in step 4 is BM + GA32.0 mg/L。
Compared with the prior art, the invention can obtain the following technical effects:
1) the invention does not need to strip the undersized stem tip, thereby reducing the operation difficulty and the risk of browning. Through screening three key factors of sucrose concentration in preculture, preculture days and PVS2 treatment time, the stem tip can survive and regenerate after ultralow temperature. The surviving part is observed to be cells around the shoot apical meristem. Thus, there is no need to manually strip off the small stem tips, and the liquid nitrogen already acts like a 'scalpel', narrowing the range of viable cells.
2) The invention does not adopt chemical agents and has no toxic action on plant tissues. The invention avoids using agents such as ribavirin for removing virus, only uses ultra-low temperature and the reagents required by tissue culture, observes the regenerated seedling in the later period, and ensures that the regenerated seedling grows normally without toxic phenomenon.
3) During detoxification, ultralow temperature preservation of the plants can be carried out. The detoxification is carried out by the ultra-low temperature therapy, an ultra-low temperature system must be established firstly, and the system can be simultaneously applied to detoxification and preservation, so the technology can also be used for the ultra-low temperature preservation research of phalaenopsis.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a process diagram of removing the cymbidium mosaic virus and the odontoid ring spot virus by the ultra-low temperature technology, wherein a is a butterfly orchid stem tip (1.0-1.5mm) infected with the cymbidium mosaic virus and the odontoid ring spot virus, b is droplets made on an aluminum foil strip in the ultra-low temperature process, each droplet contains one stem tip, c is the stem tip 7 days after the ultra-low temperature process, a black part dies, a green part survives and grows, and d is the stem tip successfully germinates and seedlings after the ultra-low temperature treatment for 3-4 months;
FIG. 2 shows the detection of the sword-leaved cymbidium mosaic virus and the odontoid ringspot virus by RT-PCR according to the present invention, wherein a. the situation of infecting the sword-leaved cymbidium mosaic virus with an untreated phalaenopsis plant; b. the stem tip regeneration plant after ultra-low temperature treatment is infected with cymbidium mosaic virus; c. the condition of an untreated phalaenopsis plant infected with odontoglossum ringspot virus; d. the condition that stem tip regeneration plants are infected with the odontoglossum ringspot virus after ultralow temperature treatment; m. marker, DL 2000; p. positive control; n. negative control; 1-8, detecting the number of samples;
FIG. 3 is a graph showing the effect of the treatment time of the PVS2 solution on the survival rate of the stem tips according to the present invention;
FIG. 4 is a graph of the effect of PVS2 solution treatment time on regeneration rate according to the present invention.
Detailed Description
The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.
The invention discloses a method for removing cymbidium mosaic virus and cymbidium ringspot virus at ultralow temperature, which comprises the following steps:
step 1, taking a phalaenopsis tissue culture seedling (seedling age: 6 weeks) infected with cymbidium mosaic virus and odontoid ringspot virus as an object, and taking a stem tip with the size of 1.0-1.5cm under a stereoscopic microscope under the condition of an ultra-clean workbench; putting the stem tip into a pre-culture medium (BM +0.6M sucrose) to perform pre-culture for 1-3 days (dark condition);
wherein, each liter of BM culture medium comprises the following components: huabao No. 13 g, ethylenediaminetetraacetic acid ferric sodium salt 10ml, organic matter 10ml, peptone 1.5g, sucrose 30g, agar 7g, pH 6.0.
The ethylenediaminetetraacetic acid ferric sodium salt is prepared by the following method: 3.67g of sodium iron ethylenediaminetetraacetate was dissolved in 1000ml of distilled water to prepare a 100-fold ferric salt mother liquor.
The organic matter is prepared by the following method: a100-fold organic mother liquor was prepared by mixing and dissolving 0.2g of glycine, 1g of methionine, 0.1g of pyridoxine hydrochloride, 0.1g of calcium pantothenate, and 10g of inositol in 1000ml of distilled water.
Step 2, pouring 20-30ml of a carrier liquid (BM +2M glycerol +0.4M sucrose) into a culture dish with the diameter of 9cm at room temperature, and clamping stem tips into the carrier liquid one by one for treating for 20-30 minutes;
step 3, taking out PVS2 vitrification solution (MS + 30% glycerol + 15% ethylene glycol + 15% DMSO +0.4M sucrose) stored in a refrigerator at 4 ℃, pouring 20-30ml of PVS2 vitrification solution into a culture dish with the diameter of 9cm, clamping stem tips into the PVS2 solution one by one, placing the culture dish on ice, and treating the stem tips for 60-90 minutes at 0 ℃; then sucking PVS2 solution at 0 ℃ by using a pipette gun, forming droplets (the droplets are about 2.5ul) on an aluminum foil strip (2x0.8cm), and putting stem tips into the droplets one by one; clamping the aluminum foil strips by using tweezers, and putting the aluminum foil strips into liquid nitrogen for ultralow temperature treatment for 25-35 minutes;
step 4, clamping the aluminum foil strips, and unloading the aluminum foil strips by putting in a unloading liquid (BM +1.2M sucrose) for 25-35 minutes; the treated stem tip is put into a regeneration medium (BM + GA)32.0 mg/L), dark culturing for 7 days, and culturing under normal illumination; after 3-4 months, the stem tip can be successfully grown.
Step 5, virus detection of the regeneration plant: after the regeneration plant grows to three leaves, detecting the cymbidium mosaic virus and the odontoglossum ringspot virus of the ultralow temperature regeneration seedling by a molecular biological method, wherein the negative result is a sample without the virus, and the detoxification rate is the number of samples with the negative result/the total number of samples is 100 percent; as shown in fig. 2, 8 plants infected with cymbidium mosaic virus (fig. 2a) and 6 plants infected with odontoglossum ringspot virus (fig. 2c) were treated with the ultra-low temperature therapy, and then cymbidium mosaic virus was detected in 2 regenerated plants, 6 plants were negative (fig. 2b), the detoxification rate of cymbidium mosaic virus was 75.0%, odontoglossum ringspot virus was detected in 1 regenerated plant, 5 plants were negative (fig. 2d), and the detoxification rate of odontoglossum ringspot virus was 83.3%. After the other two varieties of 'Mantianhong' and 'Diamond Huangqiang' are treated by the ultra-low temperature therapy, the detoxification rates of the mosaic disease of cymbidium ensifolium are respectively 62.5% and 50.0%, and the detoxification rates of the ringspot virus of odontalgia chinensis are respectively 66.7% and 75%. The detoxification rates of the ultralow temperature therapy on the cymbidium mosaic virus and the odontoid ring spot virus of three phalaenopsis varieties are 50.0-75.0% and 66.7-83.3% respectively.
Butterfly orchid stem tips (1.0-1.5mm) infected with the cymbidium mosaic virus and the odontoid ringspot virus are shown in figure 1a, droplets are made on an aluminum foil strip in an ultralow temperature process, each droplet comprises a stem tip shown in figure 1b, as shown in figure 1c, the stem tips 7 days after the ultralow temperature process die at black parts, and green parts survive and grow; as shown in FIG. 1d, after 3-4 months of ultralow temperature treatment, the stem tip successfully germinates into seedlings.
In the above process, sucrose plays an important role in the pretreatment stage, loading and unloading: the pretreatment can improve the dehydration resistance and the freezing resistance of the stem tip, and is a key step for successful cryopreservation, sucrose is the most commonly used osmotic protective agent for pretreatment, and the stem tip is pre-cultured on a culture medium containing high-concentration sucrose, so that the content of free water in cells can be effectively reduced, and the low-temperature stress resistance of the cells is improved; the treatment of the added carrier liquid can effectively improve the adaptability of the stem tip to a vitrification solution and improve the survival rate of the stem tip, and the addition of sucrose in the added carrier liquid is necessary, so that researches show that if the added carrier liquid is not added with sucrose, the stem tip after being stored at ultralow temperature does not survive; the effect of the liquid unloading agent is to remove residual vitrification solution in cells and reduce the poison to stem tips, and cane sugar is the main component.
Glycerol, ethylene glycol, dimethyl sulfoxide and sucrose are main components forming the PVS2 vitrification solution, the PVS2 solution is most commonly used in ultralow temperature storage, after being treated by the PVS2 solution, free water in cells is directly converted into glassy amorphous bodies from liquid states, cell viscosity is improved, ice crystal formation is avoided, and the cells are effectively protected from freezing injury.
The key parameters of the method are three of pre-culture sucrose concentration, pre-culture time and PVS2 time. The value ranges are that the concentration of pre-culture cane sugar is 0.6M, and the pre-culture time is 1-3 days; the PVS2 time was 60-90 minutes. In the experiments, the data show that sucrose concentration, preculture time and treatment time of PVS2 have a significant effect on the survival and regeneration of phalaenopsis stem tips. When the sucrose concentration in the pre-culture medium is 0.6M, the regeneration rate of the stem tip is 57 percent, which is obviously higher than the regeneration rate of the stem tip after the sucrose concentration is 0.3M, 0.8M and 1.0M; when the number of days of pre-culture is 1-3 days, the survival rate (67-77%) and the regeneration rate (50-63%) of the stem tip after the ultra-low temperature treatment are not obviously different, but are obviously higher than the survival rate (53%) and the regeneration rate (13%) of the stem tip after the pre-culture is 4 days, and the two ranges of 60-90min of PVS2 time selection have the advantages that higher survival rate and regeneration rate can be obtained, so that more regenerated seedlings are obtained; when the treatment time of the PVS2 is 60-90min, the survival rate (73-80%) and the regeneration rate (57-63%) of the stem tip after the ultralow temperature treatment reach the highest values, and both the survival rate and the regeneration rate of the stem tip are obviously reduced when the survival rate is less than 60min and when the regeneration rate is more than 90 min.
Example 1
Takes the phalaenopsis tissue culture seedling (seedling age: 6 weeks) infected with the cymbidium mosaic virus and the odontoid ringspot virus as the objectTaking 1.0-1.5cm stem tip under a stereomicroscope under the condition of a workbench; the stem tip is put into a pre-culture medium (BM +0.6M sucrose) to be pre-cultured for 2 days (dark condition); wherein the BM culture medium (per liter) comprises the following components: huabao No. 1g + 10ml of ethylenediamine tetraacetic acid ferric sodium salt (mother liquor is 3.67g dissolved in 1000ml) + 10ml of organic matter (mother liquor is 0.2g of glycine + 1.0g of thiamine + 0.1g of pyridoxine hydrochloride + 0.1g of calcium pantothenate + 10g of inositol mixed solution in 1000ml) + 1.5g of peptone; loading the stem tip with a loading solution (BM +2M glycerol +0.4M sucrose) at room temperature for 20 minutes; treating the stem tip with a frozen protective solution (PVS2 solution) at 0 deg.C for 40-50 min; sucking PVS2 solution by using a pipette, forming droplets (the droplet size is about 2.5ul) on an aluminum foil strip (2x0.8cm), and putting stem tips into the droplets one by one; clamping the aluminum foil strips by using tweezers, and putting the aluminum foil strips into liquid nitrogen for ultralow temperature treatment for 30 minutes; clamping the aluminum foil strips, and unloading the aluminum foil strips in a unloading liquid (BM +1.2M sucrose) for 30 minutes; the treated stem tip is put into a regeneration medium (BM + GA)32.0 mg/L), dark culturing for 7 days, and culturing under normal illumination; after 3 months, the stem tip can grow into seedlings smoothly.
Example 2
Taking a phalaenopsis tissue culture seedling variety 'paradise' (seedling age: 6 weeks) infected with the cymbidium mosaic virus and the odontoid ringspot virus as an object, and taking a stem tip with the size of 1.5cm under a stereoscopic microscope under the condition of an ultra-clean workbench; putting the stem tip into a pre-culture medium (BM +0.6M sucrose) for pre-culture for 1 day; wherein the BM culture medium (per liter) comprises the following components: huabao No. 1g + 10ml of sodium ferric ethylenediamine tetraacetate (3.67 g of 100 times mother liquor dissolved in 1000ml of distilled water) + 10ml of organic matter (0.2 g of glycine + 1.0g of thiamine + 0.1g of pyridoxine hydrochloride + 0.1g of calcium pantothenate + 10g of inositol mixed solution in 1000ml of distilled water) + 1.5g of peptone +30 g of sucrose + 7g of agar, pH 6.0; loading the stem tip with a loading solution (BM +2M glycerol +0.4M sucrose) for 30 minutes at room temperature; taking out PVS2 vitrification solution (MS + 30% glycerol + 15% ethylene glycol + 15% DMSO +0.4M sucrose) stored in a refrigerator at 4 ℃, pouring 20ml of PVS2 solution into a culture dish with the diameter of 9cm, clamping stem tips into the PVS2 solution one by one, placing the culture dish on ice, and treating the stem tips for 90 minutes at 0 ℃; then sucking the PVS2 solution at 0 ℃ by using a pipette gun, and placing the solution on an aluminum foilThe individual droplets (droplet size of about 2.5ul) were formed on the bar (2x0.8cm) and the stem tips were placed one by one in the droplets; clamping the aluminum foil strips by using tweezers, and putting the aluminum foil strips into liquid nitrogen for ultralow temperature treatment for 35 minutes; clamping the aluminum foil strips, and unloading the aluminum foil strips in a unloading liquid (BM +1.2M sucrose) for 30 minutes; the treated stem tip is put into a regeneration medium (BM + GA)32.0 mg/L), dark culturing for 7 days, and culturing under normal illumination; after 4 months, the stem tip can grow seedlings smoothly; after the regeneration plant grows to three leaves, the leaves are taken to carry out RT-PCR for detecting two viruses, the virus-free rate of the cymbidium mosaic virus is 62.5 percent, and the virus-free rate of the cymbidium goeringii ring spot virus is 83.3 percent.
Example 3
Taking a phalaenopsis tissue culture seedling variety 'diamond queen' (seedling age: 6 weeks) infected with the cymbidium mosaic virus and the odontoid ringspot virus as an object, and taking a stem tip with the size of 1.0cm under a stereoscopic microscope under the condition of an ultra-clean workbench; putting the stem tip into a pre-culture medium (BM +0.6M sucrose) for pre-culture for 1 day; wherein the BM culture medium (per liter) comprises the following components: huabao No. 1g + 10ml of sodium ferric ethylenediamine tetraacetate (3.67 g of 100 times mother liquor dissolved in 1000ml of distilled water) + 10ml of organic matter (0.2 g of glycine + 1.0g of thiamine + 0.1g of pyridoxine hydrochloride + 0.1g of calcium pantothenate + 10g of inositol mixed solution in 1000ml of distilled water) + 1.5g of peptone +30 g of sucrose + 7g of agar, pH 6.0; loading the stem tip with loading solution (BM +2M glycerol +0.4M sucrose) at room temperature for 25 min; taking out PVS2 vitrification solution (MS + 30% glycerol + 15% ethylene glycol + 15% DMSO +0.4M sucrose) stored in a refrigerator at 4 ℃, pouring 20ml of PVS2 solution into a culture dish with the diameter of 9cm, clamping stem tips into the PVS2 solution one by one, placing the culture dish on ice, and treating the stem tips for 60 minutes at 0 ℃; then sucking PVS2 solution at 0 ℃ by using a pipette gun, forming droplets (the droplet size is about 2.5ul) on an aluminum foil strip (2x0.8cm), and putting stem tips into the droplets one by one; clamping the aluminum foil strips by using tweezers, and putting the aluminum foil strips into liquid nitrogen for ultralow temperature treatment for 35 minutes; clamping the aluminum foil strips, and unloading the aluminum foil strips in a unloading liquid (BM +1.2M sucrose) for 30 minutes; the treated stem tip is put into a regeneration medium (BM + GA)32.0 mg/L), dark culturing for 7 days, and culturing under normal illumination; after 3 months, the stem tip is smoothSeedling formation; after the regeneration plant grows to three leaves, the leaves are taken to carry out RT-PCR for detecting two viruses, the virus-free rate of the cymbidium ensifolium leaf is 50 percent, and the virus-free rate of the cymbidium goeringii ring spot virus is 75 percent.
The influence of the pretreated sucrose concentration and the preculture time on the survival rate and the regeneration rate of the stem tip is shown in tables 1 and 2; from tables 1 and 2, it can be concluded that the survival rate of the stem tips after the treatment with the ultra-low temperature therapy was 67-77% at the sucrose concentration of 0.6-0.8M, and there was no significant difference, but the regeneration rate of the stem tips was significantly higher than 0.6M and 1.0M, and 57% at the sucrose concentration of 0.8M, and thus the sucrose concentration of 0.8M was the optimum pre-culture sucrose concentration. After the stem tip is pre-cultured by sucrose for 1-3 days, the stem tip is treated by the ultra-low temperature therapy, the survival rate and the regeneration rate have no obvious difference, but the survival rate and the regeneration rate are obviously reduced when the stem tip is pre-cultured for 4 days.
TABLE 1 Effect of pre-culture sucrose concentration on shoot tip survival and regeneration rates
TABLE 2 influence of sucrose preculture time on the survival and regeneration rate of shoot tips
The influence of the PVS2 solution treatment time on the stem tip survival rate and the regeneration rate is shown in fig. 3 and fig. 4, the PVS2 solution treatment time is 30-90min, the stem tip survival rate is 67-80%, and is significantly higher than 120min (survival rate 53%) and 150min (survival rate 23%), the stem tip regeneration rate is highest and 63% when the PVS2 solution is treated for 60min, and then the PVS2 solution is treated for 90min, the stem tip regeneration rate is 57%, the two are not significantly different, and the regeneration rates of other treatment times are significantly lower than 60min and 90 min.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (2)
1. A method for removing cymbidium mosaic virus and odontoglossum ringspot virus is characterized by comprising the following steps:
step 1, taking a stem tip with the size of 1.0-1.5cm under a stereoscopic microscope under the condition of an ultra-clean workbench by taking a phalaenopsis tissue culture seedling with the seedling age of 6 weeks infected with a cymbidium mosaic virus and a odontoid ring spot virus as an object; putting the stem tip into a pre-culture medium for pre-culturing for 1-3 days under a dark condition, wherein the pre-culture medium is BM culture medium +0.6M sucrose, and each liter of BM culture medium comprises the following components: the preparation method comprises the following steps of 1g of Huabao, 3g of ferric EDTA, 10ml of organic matter, 1.5g of peptone, 30g of sucrose and 7g of agar, wherein the pH value is 6.0, and the ferric EDTA is prepared by the following method: dissolving 3.67g of ethylenediaminetetraacetic acid ferric sodium salt in 1000ml of distilled water to prepare an ethylenediaminetetraacetic acid ferric sodium salt solution; the organic matter is prepared by the following method: dissolving 0.1-0.3g of glycine, 0.8-1.2g of thiamine, 0.05-0.15g of pyridoxine hydrochloride, 0.05-0.15g of calcium pantothenate and 8-12g of inositol in 1000ml to obtain an organic substance;
step 2, loading the stem tip with a loading liquid at room temperature, wherein the loading liquid is BM +2M glycerol +0.4M sucrose; the dosage of the loading liquid is 20-30ml, and the time for loading the stem tip by the loading liquid is 20-30 minutes;
step 3, treating the stem tips with PVS2 vitrification solution at 0 ℃ for 40-50 minutes, sucking PVS2 solution with a pipette after the treatment is finished to make small drops on aluminum foil paper, putting the stem tips into the small drops one by one, clamping the aluminum foil paper carrying the stem tips with tweezers, putting the aluminum foil paper into liquid nitrogen for ultralow temperature treatment, wherein the ultralow temperature treatment time is 25-35 minutes, and the PVS2 vitrification solution is as follows: MS + 30% glycerol + 15% ethylene glycol + 15% DMSO +0.4M sucrose; the using amount of the PVS2 vitrification solution is 20-30 ml;
step 4, clamping the stem tip after ultralow temperature treatment, and unloading by putting in unloading liquid; putting the treated stem tip into a regeneration culture medium, culturing for 7 days in dark, and culturing under normal illumination; after 3 months, the stem tip successfully grows into seedlings, and the regeneration culture medium is BM + GA32.0 mg/L。
2. The method of claim 1, wherein the unloading liquid in step 4 is BM +1.2M sucrose; the unloading time is 25-35 min.
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