CN115191353A - Sugarcane tissue culture detoxification method - Google Patents
Sugarcane tissue culture detoxification method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention provides a method for tissue culture detoxification of sugarcane, which reduces the virus carrying amount of sugarcane original seed seedlings by soaking in antiviral agents, effectively reduces the virus carrying rate in the detoxified seedlings by combining with the traditional warm water detoxification, and simultaneously detoxifies the stem tips of the tissue culture seedlings for multiple times, thereby greatly reducing the using amount of the seed stems in the detoxification process, greatly achieving the effect of multiple times of detoxification better than that of one time of detoxification, achieving the detoxification efficiency of linear mosaic disease up to 20 percent, shortening the detoxification period by about 50 percent, and shortening the seedling forming time by about 60 days. The method improves the situation that the stem tips of the sugarcanes are difficult to detoxify and form seedlings, and breaks through the technical bottleneck that the streak mosaic disease and the perennial root dwarf disease are difficult to remove by the conventional sugarcane detoxification method.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a sugarcane tissue culture detoxification method.
Background
Sugarcane mosaic virus (SCMV) and Raton Stunting Disease (RSD) are important worldwide Sugarcane diseases. Is commonly found in the major sugarcane areas of Fujian, guangdong, guangxi, sichuan and Zhejiang in China. The variety degeneration caused by sugarcane mosaic disease and perennial root dwarfing disease in the planting process of sugarcane directly influences the quality and yield of sugarcane and seriously hinders the production and utilization of sugarcane. The tissue culture detoxification technology of sugarcane is a main technical measure for solving the sexual degeneration of sugarcane. Therefore, the sugarcane virus-free seedlings are more and more popular with the vast sugarcane cultivators. China also obtains a plurality of research results in the aspect of tissue culture and detoxification of sugarcane stem tips, and establishes a breeding industrialization technical system of tissue culture and detoxification seedlings. The tissue culture detoxification technology of the sugarcane stem tips is the basis of the industrialization of sugarcane detoxification seedlings, and the tissue culture detoxification technology of the high-quality and rapid stem tips wins time and benefits for the production of new sugarcane species detoxification seedlings and detoxification seedlings. Establishing a detoxified seedling production system through a tissue culture approach is an important method for effectively controlling sugarcane mosaic disease and perennial root dwarf disease at present.
At present, few antiviral agents are applied and researched in the aspect of sugarcane tissue culture detoxification, and one-time stem tip detoxification is commonly used in a sugarcane seedling detoxification method, but the cut stem tip is small, the survival rate is low, and the effect of one-time detoxification success is difficult to achieve. The method results in large amount of used raw materials of seedlings, low detoxification efficiency, large workload and low detoxification efficiency. Meanwhile, a differentiation culture method is mostly used in the stem tip detoxification process, and 1-3mm stem tips are in a differentiation culture medium, so that the seedling emergence speed is low, and the survival rate is low.
The process therefore proposes on the one hand an early enhancement of the nutritional management of the sugar cane material and on the other hand the use of antiviral agents to reduce the toxic load of the material. Meanwhile, the stem tip detoxification treatment is carried out for a plurality of times in the tissue culture seedling stage, so that the detoxification efficiency of the sugarcane tissue culture seedlings is greatly improved, and the use amount of original seedlings is greatly reduced
Disclosure of Invention
The invention aims to provide a sugarcane stem tip detoxification method with high sugarcane tissue culture stem tip detoxification efficiency and high seedling rate and detoxification efficiency.
In order to solve the technical problems, the invention adopts the following technical scheme: a sugarcane tissue culture detoxification method is characterized by comprising the following steps: the method comprises the following steps:
(1): planting the raw materials of the sugarcane seeds for detoxification in a land with good water and fertilizer conditions, enhancing water and fertilizer management, fully growing the raw materials of the sugarcane seeds, selecting strong sugarcane plants from the field, and selecting mature stem nodes at the middle section as detoxification materials;
(2): selecting the stem nodes in the step (1) as materials for detoxification, selecting mature and plump buds as buds of the stem nodes, cutting the stem nodes into sections of three to five buds, and placing the sections in a constant-temperature water bath kettle;
(3): cutting the sugarcane seed stems treated by the warm water into single buds, soaking the single buds in an anti-disease bacteria liquid for 10 minutes, and accelerating germination at the constant temperature of 38-42 ℃ for 1 week;
(4): carrying out constant-temperature germination acceleration in the step (3) to obtain 10cm buds, cutting 1-3mm stem tips in a sterile super clean workbench, inoculating the stem tips in an MS liquid culture medium, culturing callus, and differentiating test-tube plantlets;
(5): after 10-30 buds grow out by differentiation of the test-tube plantlet in the step (4), transferring the test-tube plantlet into a rooting culture medium for culturing for 15 days, cutting a stem tip meristem again, inoculating the stem tip meristem into a liquid culture medium, culturing a callus, and differentiating a new test-tube plantlet;
(6): step 5, repeatedly carrying out stem tip detoxification culture on the test-tube plantlet with the toxicity according to the variety and the PCR detection result; taking leaves of the test-tube plantlets obtained in the steps (4) and (5) to detect viruses; through the detection of sugarcane mosaic disease and perennial root dwarfing disease, a single plant of healthy tissue culture seedlings without virus is separated, and a large number of healthy sugarcane seedlings are bred through a tissue culture and rapid propagation method, so that the method is popularized and applied to sugarcane planting areas.
Further, the method comprises the following steps of; in the step (2), the mixture is placed in a constant-temperature water bath kettle at the temperature of 52-58 ℃ and soaked in warm water for 30-120min.
Further, the method comprises the following steps of; in the step (3), the anti-bacteria liquid medicine consists of 2.5% fludioxonil suspension 1200 times liquid, 8% ningnanmycin water solution 1200 times liquid, 5% amino-oligosaccharin water solution 2000 times liquid, 30% moroxydine hydrochloride 1500 times liquid and chelating zinc fertilizer 1000 times liquid.
Further, the method comprises the following steps of; in the step (5), the shoot apical meristem is cut to a length of 1 to 3mm.
Further, the method comprises the following steps of; in the steps (4) and (5), the liquid medium contains 1-3mg/L of 2,4-D MS.
Compared with the prior art, the invention has at least one of the following beneficial effects:
firstly, the virus-free test-tube seedlings of the sugarcane can be obtained in a relatively short period; specifically, the method for obtaining the differentiated test-tube plantlet of the stem tip tissue by using the callus induction method has the advantages of high survival rate, high differentiation culture efficiency due to multiple detoxification and short time.
Secondly, the obtained seedlings have high detoxification efficiency; specifically, the common stem tip detoxification method is to use a large amount of sugarcane seed stems, treat the seed stems with warm water at 52 ℃ for 2 hours, take stem tip tissues once after germination acceleration, screen out non-toxic seedlings through PCR detection after differentiation and germination, and quickly propagate the seedlings for production through genetic identification. The invention improves the vegetative growth of plants, uses antiviral medicaments, and uses detoxified tissue culture seedlings for multiple detoxifications, thereby reducing the using amount of seedlings, shortening the period of detoxication and improving the efficiency of detoxication.
Thirdly, the method reduces the virus carrying amount of the sugarcane stock seedlings through soaking of antiviral agents, effectively reduces the virus carrying rate in virus-free seedlings by combining with traditional warm water virus removal, simultaneously removes the virus from the stem tips of the tissue culture seedlings for many times, can greatly reduce the using amount of the stock stems in the virus removal process, has the effect of removing the virus for many times which is greatly superior to that of removing the virus for one time, can achieve the virus removal efficiency of the streak mosaic disease by 20 percent, shortens the virus removal period by about 50 percent, and shortens the seedling forming time by about 60 days. Improves the situation that the stem tip of the sugarcane is difficult to detoxify and form seedlings, and breaks through the technical bottleneck that the linear mosaic disease and the perennial root dwarf disease are difficult to remove by the conventional sugarcane detoxification method.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A sugarcane tissue culture detoxification method comprises the following steps:
(1): planting the raw materials of the sugarcane seeds for detoxification in a land with good water and fertilizer conditions, enhancing water and fertilizer management, wherein the raw materials of the sugarcane seeds comprise organic fertilizer, compound fertilizer and the like, the raw materials of the sugarcane seeds grow fully, selecting strong sugarcane plants from the field, and selecting mature stem nodes in the middle section as detoxification materials; in particular, sugar cane has an increased toxic load in drought and nutrient-deficient conditions. The water and fertilizer conditions of the virus-free raw materials of the sugarcane are increased, the immunocompetence of the sugarcane can be improved, and the toxic amount of plants is reduced. Under the same growth condition, the toxicity carrying rate of the robust plants is lower than that of the robust plants, so that the conditions for planting sugarcane materials are good, and the robust plants are selected as the materials for detoxification.
(2): selecting the stem nodes in the step (1) as materials for detoxification, selecting mature and plump buds as buds of the stem nodes, cutting the stem nodes into sections of three to five buds, and placing the sections in a constant-temperature water bath kettle; the stem nodes are cut into three to five bud sections, when the sugarcane is soaked in warm water at 52 ℃, because the cut tissues of the sugarcane are necrotized under the action of the warm water, the death rate of the buds at the two ends is high, and therefore, the stem nodes of the sugarcane are cut into long sections, which is beneficial to the survival of the buds at the later stage.
(3): cutting the sugarcane seed stems treated by warm water into single buds, soaking the single buds in an anti-disease bacteria liquid for 10 minutes, and accelerating germination at the constant temperature of 38 ℃ for 1 week; specifically, under the action of the anti-bacteria liquid medicine, the sugarcane has a good anti-bacteria effect on one hand, further necrosis of tissues can be reduced, and the germination rate of the sugarcane after warm water treatment is improved. On the other hand, ningnanmycin, amino-oligosaccharin and moroxydine hydrochloride can inactivate viruses and reduce the action of virus activity, and the chelated zinc fertilizer can improve the immunity of plants.
(4): carrying out constant-temperature germination acceleration in the step (3) to obtain 10cm buds, cutting 1mm stem tips in a sterile super clean workbench, inoculating the cut stems tips in an MS liquid culture medium, culturing callus, and differentiating test-tube plantlets;
(5): after 10 buds grow out by differentiation of the test-tube plantlet in the step (4), transferring the test-tube plantlet into a rooting culture medium for culturing for 15 days, cutting a stem tip meristem again, inoculating the stem tip meristem into a liquid culture medium, culturing a callus, and differentiating a new test-tube plantlet;
(6): step 5, repeatedly carrying out stem tip detoxification culture on the test-tube plantlet with the virus according to the variety and the PCR detection result; taking leaves of the test-tube plantlets obtained in the steps (4) and (5) to detect viruses; through the detection of sugarcane mosaic disease and perennial root dwarfing disease, a single plant of healthy tissue culture seedlings without virus is separated, and a large amount of healthy sugarcane seedlings are bred through a tissue culture and rapid propagation method, so that the method is popularized and applied to sugarcane planting areas;
in the step (2), the mixture is placed in a constant-temperature water bath kettle at the temperature of 52 ℃ and is soaked in warm water for 120min;
in the step (3), the anti-bacteria liquid medicine consists of 2.5 percent of fludioxonil suspending agent 1200-fold liquid, 8 percent of ningnanmycin aqueous solution 1200-fold liquid, 5 percent of amino-oligosaccharin aqueous solution 2000-fold liquid, 30 percent of moroxydine hydrochloride 1500-fold liquid and chelating zinc fertilizer 1000-fold liquid;
in the step (5), the length of the shoot apical meristem is cut to be 1mm;
in the steps (4) and (5), the liquid medium contains 1-3mg/L of 2,4-D MS. The stem tip of the tissue culture seedling is utilized to detoxify to obtain a new detoxified test tube seedling, the use amount of the original seedling material can be greatly reduced, the steps of warm water detoxification are reduced, the amount of virus carried by the material subjected to stem tip detoxification once is relatively less, and in addition, the growth speed of the test tube seedling in a test tube is high, and the toxic amount is relatively less.
Example 2
On the basis of embodiment 1, the sugarcane tissue culture detoxification method comprises the following steps:
(1): planting sugarcane seed raw materials for detoxification in a land with good water and fertilizer conditions, enhancing water and fertilizer management, wherein the sugarcane seed raw materials comprise organic fertilizer, compound fertilizer and the like, fully grow, selecting strong sugarcane plants from the field, and selecting middle-section mature stem nodes as materials for detoxification; in particular, sugar cane has an increased toxic load in drought and nutrient-deficient conditions. The water and fertilizer conditions of the sugarcane detoxification raw materials are increased, the immunity of the sugarcane can be improved, and the toxic amount of plants is reduced. Under the same growth conditions, the toxicity carrying rate of the robust plants is lower than that of the robust plants, so that the conditions for planting sugarcane materials are good, and the robust plants are selected as the toxicity-free materials.
(2): selecting the stem nodes in the step (1) as materials for detoxification, selecting mature and plump buds as buds of the stem nodes, cutting the stem nodes into sections of three to five buds, and placing the sections in a constant-temperature water bath kettle; the stem nodes are cut into three to five bud sections, when the sugarcane is soaked in warm water at the temperature of 58 ℃, because the cut tissues of the sugarcane are necrotized under the action of the warm water, the death rate of the buds at the two ends is high, and therefore, the stem nodes of the sugarcane are cut into long sections, which is beneficial to the survival of the buds at the later stage.
(3): cutting the sugarcane seed stems treated by the warm water into single buds, soaking the single buds in an anti-disease bacteria liquid for 10 minutes, and accelerating germination at the constant temperature of 42 ℃ for 1 week; specifically, under the action of the anti-bacteria liquid medicine, the sugarcane has a good anti-bacteria effect on one hand, further necrosis of tissues can be reduced, and the germination rate of the sugarcane after warm water treatment is improved. On the other hand, ningnanmycin, amino-oligosaccharin and moroxydine hydrochloride can inactivate viruses and reduce the action of virus activity, and the chelated zinc fertilizer can improve the immunity of plants.
(4)): carrying out constant-temperature germination acceleration in the step (3) to obtain 10cm buds, cutting 3mm stem tips in a sterile super clean workbench, inoculating the stem tips in an MS liquid culture medium, culturing callus, and differentiating test-tube plantlets;
(5): after 30 buds grow out by differentiation of the test-tube plantlet in the step (4), transferring the test-tube plantlet into a rooting culture medium for culturing for 15 days, cutting a stem tip meristem again, inoculating the stem tip meristem into a liquid culture medium, culturing a callus, and differentiating a new test-tube plantlet;
(6): step 5, repeatedly carrying out stem tip detoxification culture on the test-tube plantlet with the virus according to the variety and the PCR detection result; taking leaves of the test-tube plantlets obtained in the steps (4) and (5) to detect viruses; through the detection of sugarcane mosaic disease and perennial root dwarfing disease, a single plant of healthy tissue culture seedlings without virus is separated, and a large amount of healthy sugarcane seedlings are bred through a tissue culture and rapid propagation method, so that the method is popularized and applied to sugarcane planting areas;
in the step (2), the mixture is placed in a constant-temperature water bath kettle at the temperature of 58 ℃ and soaked in warm water for 30min;
in the step (3), the anti-bacteria liquid medicine consists of 2.5 percent of fludioxonil suspending agent 1200-fold liquid, 8 percent of ningnanmycin aqueous solution 1200-fold liquid, 5 percent of amino-oligosaccharin aqueous solution 2000-fold liquid, 30 percent of moroxydine hydrochloride 1500-fold liquid and chelating zinc fertilizer 1000-fold liquid;
in the step (5), the length of the shoot apical meristem is cut to be 3mm;
in the steps (4) and (5), the liquid culture medium contains 3mg/L of 2,4-D MS;
the stem tip of the tissue culture seedling is utilized to detoxify to obtain a new detoxified test tube seedling, the use amount of the original seedling material can be greatly reduced, the steps of warm water detoxification are reduced, the amount of virus carried by the material subjected to stem tip detoxification once is relatively less, and in addition, the growth speed of the test tube seedling in a test tube is high, and the toxic amount is relatively less.
Example 3
On the basis of the embodiment 1-2 and on the basis of the embodiment 1, the sugarcane tissue culture detoxification method comprises the following steps:
(1): planting sugarcane seed raw materials for detoxification in a land with good water and fertilizer conditions, enhancing water and fertilizer management, wherein the sugarcane seed raw materials comprise organic fertilizer, compound fertilizer and the like, fully grow, selecting strong sugarcane plants from the field, and selecting middle-section mature stem nodes as materials for detoxification; in particular, sugar cane has an increased toxic load in drought and nutrient-deficient conditions. The water and fertilizer conditions of the virus-free raw materials of the sugarcane are increased, the immunocompetence of the sugarcane can be improved, and the toxic amount of plants is reduced. Under the same growth condition, the toxicity carrying rate of the robust plants is lower than that of the robust plants, so that the conditions for planting sugarcane materials are good, and the robust plants are selected as the materials for detoxification.
(2): selecting the stem nodes in the step (1) as materials for detoxification, selecting mature and plump buds as buds of the stem nodes, cutting the stem nodes into sections of three to five buds, and placing the sections in a constant-temperature water bath kettle; the stem nodes are cut into three to five bud sections, when the sugarcane is soaked in warm water at 55 ℃, because the cut tissues of the sugarcane are necrotized under the action of the warm water, the death rate of the buds at the two ends is high, and therefore, the stem nodes of the sugarcane are cut into long sections, which is beneficial to the survival of the buds at the later stage.
(3): cutting the sugarcane seed stems treated by the warm water into single buds, soaking the single buds in an anti-disease bacteria liquid for 10 minutes, and accelerating germination at the constant temperature of 40 ℃ for 1 week; specifically, under the action of the anti-bacteria liquid medicine, the sugarcane has a good anti-bacteria effect on one hand, further necrosis of tissues can be reduced, and the germination rate of the sugarcane after warm water treatment is improved. On the other hand, ningnanmycin, amino-oligosaccharin and moroxydine hydrochloride can inactivate viruses and reduce the action of virus activity, and the chelated zinc fertilizer can improve the immunity of plants.
(4): carrying out constant-temperature germination acceleration in the step (3) to obtain 10cm buds, cutting 2mm stem tips in a sterile super clean workbench, inoculating the stem tips in an MS liquid culture medium, culturing callus, and differentiating test-tube plantlets;
(5): after 20 buds grow out from the test-tube plantlet in the step (4) through differentiation, transferring the test-tube plantlet into a rooting culture medium for culturing for 15 days, cutting a stem tip meristem again, inoculating the stem tip meristem into a liquid culture medium, culturing a callus, and differentiating a new test-tube plantlet;
(6): step 5, repeatedly carrying out stem tip detoxification culture on the test-tube plantlet with the virus according to the variety and the PCR detection result; taking leaves of the test-tube plantlets obtained in the steps (4) and (5) to detect viruses; through the detection of sugarcane mosaic disease and perennial root dwarf disease, a single plant of healthy tissue culture seedlings without virus is separated, and a large amount of healthy sugarcane seedlings are bred through a tissue culture and rapid propagation method, so that the method is popularized and applied to sugarcane planting areas;
in the step (2), the mixture is placed in a constant-temperature water bath kettle at the temperature of 55 ℃ and soaked in warm water for 75min;
in the step (3), the anti-bacteria liquid medicine consists of 2.5 percent of fludioxonil suspending agent 1200-fold liquid, 8 percent of ningnanmycin aqueous solution 1200-fold liquid, 5 percent of amino-oligosaccharin aqueous solution 2000-fold liquid, 30 percent of moroxydine hydrochloride 1500-fold liquid and chelating zinc fertilizer 1000-fold liquid;
in the step (5), the length of the shoot apical meristem is cut to be 1-3mm;
in the steps (4) and (5), the liquid culture medium contains 1-3mg/L of 2,4-D MS;
the stem tip of the tissue culture seedling is utilized to detoxify to obtain a new detoxified test-tube seedling, the using amount of original seedling materials can be greatly reduced, the steps of warm water detoxification are reduced, simultaneously, the amount of virus carried by the material subjected to stem tip detoxification in one time is relatively less, and in addition, the growth speed of the test-tube seedling in a test tube is high, and the amount of virus carried is relatively less.
The method simplifies the step of stem tip stripping, remarkably reduces the technical difficulty of stem tip stripping, changes the traditional multi-step seedling formation into one-step seedling formation, shortens the period of detoxification by about 61.2 to 70.6 percent, and realizes the spanning of the period of shortening the period from the sugarcane stem to the period of detoxification of the seedling; the seedling rate is increased by more than 30.6 percent, and the current situation that the stem tip is difficult to peel off and form seedlings is improved; the detoxification rate is improved by over 65.5 percent, and the technical bottleneck that the sugarcane mosaic disease and the perennial root dwarf disease are difficult to remove by the conventional sugarcane detoxification method is broken through.
The technology starts from two links of shortening the detoxification period and ensuring the detoxification efficiency of the sugarcane test-tube plantlet, the seed stems of the sugarcane are subjected to heat treatment and chemical treatment by soaking in the anti-germ liquid medicine, the stem tips are stripped in a sterile environment, the seed stems are transferred into an optimized callus-induced liquid culture medium, virus detection is carried out on the test-tube plantlet which becomes a seedling, a healthy detoxification test-tube plantlet is obtained, and the aim of efficiently producing the detoxification seedling is fulfilled.
The method reduces the complicated steps of warm water detoxification through multiple stem tip culture detoxification.
In conclusion, the method combines two treatment methods of chemical treatment and multi-time stem tip detoxification, and improves the detoxification efficiency of the sugarcane seedling mosaic disease and the perennial root dwarf disease.
Although the invention has been described herein with reference to a number of illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More specifically, various variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure and claims of this application. In addition to variations and modifications in the component parts and/or arrangements, other uses will also be apparent to those skilled in the art.
Claims (5)
1. A sugarcane tissue culture detoxification method is characterized in that: the method comprises the following steps:
(1): planting the sugarcane seed raw materials for detoxification in a plot with good water and fertilizer conditions, enhancing water and fertilizer management, fully growing the sugarcane seed raw materials, selecting strong sugarcane plants from the field, and selecting middle-section mature stem nodes as detoxification materials;
(2): selecting the stem nodes in the step (1) as materials for detoxification, selecting mature and plump buds as buds of the stem nodes, cutting the stem nodes into sections of three to five buds, and placing the sections in a constant-temperature water bath kettle;
(3): cutting the sugarcane seed stems treated by the warm water into single buds, soaking the single buds in an anti-disease bacteria liquid for 10 minutes, and accelerating germination at the constant temperature of 38-42 ℃ for 1 week;
(4): carrying out constant-temperature germination acceleration in the step (3) to obtain 10cm buds, cutting 1-3mm stem tips in a sterile ultra-clean workbench, inoculating the stem tips into an MS liquid culture medium, culturing callus, and differentiating test-tube seedlings;
(5): after 10-30 buds grow out by differentiation of the test-tube plantlet in the step (4), transferring the test-tube plantlet into a rooting culture medium for culturing for 15 days, cutting a stem tip meristem again, inoculating the stem tip meristem into a liquid culture medium, culturing a callus, and differentiating a new test-tube plantlet;
(6): step 5, repeatedly carrying out stem tip detoxification culture on the test-tube plantlet with the virus according to the variety and the PCR detection result; taking leaves of the test-tube plantlets obtained in the steps (4) and (5) to detect viruses; through the detection of sugarcane mosaic disease and perennial root dwarfing disease, a single plant of healthy tissue culture seedlings without virus is separated, and a large amount of healthy sugarcane seedlings are bred through a tissue culture and rapid propagation method, so that the method is popularized and applied to sugarcane planting areas.
2. The method for tissue culture detoxification of sugar cane according to claim 1, wherein: in the step (2), the mixture is placed in a constant-temperature water bath kettle at the temperature of 52-58 ℃ and soaked in warm water for 30-120min.
3. The method for tissue culture detoxification of sugarcane according to claim 1, wherein the method comprises the following steps: in the step (3), the anti-bacteria liquid medicine consists of 2.5% fludioxonil suspension 1200 times liquid, 8% ningnanmycin water solution 1200 times liquid, 5% amino-oligosaccharin water solution 2000 times liquid, 30% moroxydine hydrochloride 1500 times liquid and chelating zinc fertilizer 1000 times liquid.
4. The method for tissue culture detoxification of sugarcane according to claim 1, wherein the method comprises the following steps: in the step (5), the shoot apical meristem is cut to a length of 1 to 3mm.
5. The method for tissue culture detoxification of sugar cane according to claim 1, wherein: in the steps (4) and (5), the liquid medium contains 1-3mg/L of 2,4-D MS.
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