CN111713406A - Embedding vitrification ultra-low temperature detoxification method for Huaiyoshan high-mountain potatoes - Google Patents
Embedding vitrification ultra-low temperature detoxification method for Huaiyoshan high-mountain potatoes Download PDFInfo
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Abstract
The invention discloses a method for ultra-low temperature detoxification of Huaiyoshan high-mountain potatoes by an embedding vitrification method, belonging to the technical field of biology. Firstly embedding the stem tips of the Huaiyoshan high-mountain potatoes to prepare embedded beads, then performing two pre-culture and two loading treatments on the obtained embedded beads, dehydrating, performing liquid nitrogen ultralow temperature preservation treatment, then thawing, performing dark culture and then illumination culture on the obtained stem tips to obtain the virus-free plants of the Huaiyoshan high-mountain potatoes. The Huaiyshan mountain potato virus-free plant cultivated by the method has the advantages of good growth state, high survival rate and obviously improved virus-free rate, and meanwhile, the method has the characteristics of simple and feasible preparation method and short culture period, and can realize batch production.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for detoxifying the stem tip of a Huaiyoshan high-mountain potato.
Background
The Huaiyishan high-mountain potatoes are produced in Huaiyanshan county northwest 60 kilometers in Yushan county of Jiangxi province and are national geographical sign agricultural products, but are susceptible to virus to cause degeneration in the planting process of the Huaiyishan high-mountain potatoes, so that the quality and the yield are directly influenced, and the production and the utilization of the Huaiyishan high-mountain potatoes are seriously hindered.
The potato detoxification technology is a main technical measure for solving the problem of potato seed degeneration. Therefore, the virus-free seed potatoes are more and more popular with the majority of potato cultivators. China also obtains a plurality of research results in the aspect of potato stem tip tissue culture, and establishes virus-free seed potato breeding systems in various places. The tissue culture of the potato stem tip virus-free seedlings is the basis of the potato virus-free seed potato industry, and the high-quality and rapid stem tip culture gains time and benefits for the production of the virus-free seedlings and virus-free potatoes. Establishing a virus-free production system through a tissue culture way is a very effective way for obtaining virus-free plants at present.
The conventional stem tip culture and the heat treatment plus stem tip culture are the two most traditional methods for solving the potato seed degeneration problem at present, but the methods have the limitations of time consumption, browning, difficult plant regeneration, poor detoxification effect and the like (Gaofuiqin et al 2010, Shanxi university of agriculture, 30(6) 528-plus 532). The method has the advantages that the stem tip is simply cut, the operation is convenient, the experimental period is short, the seedling rate and the detoxification rate of the stem tip tissue of the potato are improved, the method is successfully used for removing several plant viruses, a new way is opened up for removing the plant viruses, and the culture effect of the embedding vitrification method ultra-low temperature therapy is the best. However, the existing embedding vitrification method ultra-low temperature therapy for cultivating the detoxified potato stem tips still has the technical problems of low efficiency and still needs to improve the seedling rate and the detoxication rate, so that the improvement of the embedding vitrification method ultra-low temperature therapy for the potato stem tips is very necessary.
Disclosure of Invention
The invention aims to provide a method for ultra-low temperature detoxification of Huaiyoshan high-mountain potatoes by an embedding vitrification method, which solves the problems in the prior art and ensures that the stem tip tissue culture efficiency of potatoes is high, and the detoxification rate and the seedling rate of the detoxified seedlings obtained by culture are high.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for ultra-low temperature detoxification of Huaiyoshan high mountain potatoes by an embedding vitrification method, which comprises the following steps:
1. a method for ultra-low temperature detoxification of Huaiyoshan high mountain potatoes by an embedding vitrification method is characterized by comprising the following steps:
(1) under the aseptic condition, uniformly mixing the stem tip of the Huaiyoshan high-mountain potato with a calcium-ion-free modified MS culture solution containing sodium alginate and sucrose to obtain a mixed solution;
(2) embedding: under the aseptic condition, the mixed solution obtained in the step (1) is dripped into a solution containing chitosan oligosaccharide, graphene quantum dots, carbon nano tubes and CaCl2Embedding the mixture and a sucrose MS culture solution to obtain a Huaiyoshan high-mountain potato stem tip embedded bead;
(3) primary pre-culture: under the aseptic condition, carrying out primary pre-culture on the stem tip embedded beads of the Huaiyoshan high-mountain potatoes by using MS culture solution containing chitosan oligosaccharide and graphene quantum dots;
(4) secondary pre-culture: under the aseptic condition, carrying out secondary pre-culture on the stem tip embedded beads of the Huaiyoshan high-mountain potatoes subjected to primary pre-culture treatment by using MS culture solution containing chitosan oligosaccharide and carbon nano tubes;
(5) primary loading: under the aseptic condition, loading the stem tip embedded beads of the Huaiyoshan high-mountain potatoes pre-cultured twice with MS culture solution containing chitosan oligosaccharide and graphene quantum dots;
(6) secondary loading: under the aseptic condition, carrying out secondary loading on the stem tip embedded beads of the Huaiyoshan high-mountain potatoes obtained in the step (5) by using MS culture solution containing chitosan oligosaccharide and carbon nano tubes;
(7) and (3) dehydration treatment: under the aseptic condition, dehydrating the stem tip embedded beads of the Huaiyoshan high-mountain potatoes obtained in the step (6) by using an MS culture solution containing 25% of glycerol, 20% of ethylene glycol, 15% of chitosan oligosaccharide, 0.4M of graphene quantum dots and 0.4M of carbon nano tubes;
(8) and (3) under the aseptic condition, placing the stem tip embedded beads of the Huaiyoshan high-mountain potatoes subjected to dehydration treatment in the step (7) in liquid nitrogen for preservation, then thawing, taking out the stem tips from the thawed stem tip embedded beads of the Huaiyoshan high-mountain potatoes, washing the stem tips in an MS liquid culture medium containing 1.2M sucrose, MS, 0.05g/L chitosan oligosaccharide, 0.03-0.05g/L graphene quantum dots and 0.03-0.05g/L carbon nano tubes, performing dark culture on the washed stem tips, and then culturing under the illumination culture condition to obtain the detoxified plants of the Huaiyoshan high-mountain potatoes.
Preferably, the mass-volume ratio of the sodium alginate to the calcium ion-free modified MS culture solution in the step (1) is 3%, and the sucrose concentration is 0.2 mol/L; the length of the potato stem tip is 0.5-1 cm.
Preferably, in the step (2), the concentration of the chitosan oligosaccharide is 0.05g/L, the concentration of the graphene quantum dots is 0.03-0.05g/L, and the concentration of the carbon nanotubes is 0.03-0.05g/L, CaCl2The concentration is 0.1mol/L, and the sucrose concentration is 0.2 mol/L.
Preferably, the embedding time in the step (2) is 38-42min, and the particle size of the embedded beads of the stem tips of the Huaiyoshan high mountain potatoes is 1.8-2 cm.
Preferably, in the step (3), the concentration of the chitosan oligosaccharide is 0.05g/L, the concentration of the graphene quantum dots is 0.3-0.5g/L, and the pre-culture time is 24 h;
preferably, in the step (4), the concentration of the chitosan oligosaccharide is 0.05g/L, the concentration of the carbon nano tube is 0.3-0.5g/L, and the pre-culture time is 24 h.
Preferably, in the step (5), the concentration of the chitosan oligosaccharide is 2M, the concentration of the graphene quantum dots is 0.4M, and the loading time is 1 h; in the step (6), the concentration of the chitosan oligosaccharide is 2M, the concentration of the carbon nano tube is 0.4M, and the loading time is 1 h; the dehydration time in the step (7) is 1 h.
Preferably, the thawing temperature in step (8) is 37 + -1 deg.C, and the dark culture temperature is 25 + -2 deg.C.
Preferably, the illumination culture conditions in the step (8) are 14h/d of illumination time, 1500 + 2000lx of light intensity and 25 +/-2 ℃ of culture temperature.
Preferably, the thickness of the graphene quantum is 1-2nm, and the particle size is 6-7 nm.
The invention discloses the following technical effects:
according to the method, firstly, the stem tip of the Huaiyoshan high-mountain potato is suspended on an MS liquid culture medium, then the stem tip is prepared into embedded beads, and then the obtained stem tip embedded beads are subjected to two-time pre-culture and two-time loading treatment by utilizing the MS culture medium containing chitosan oligosaccharide and graphene quantum dots and containing chitosan oligosaccharide and carbon nano tubes, wherein the abundant structures of the graphene quantum dots and the carbon nano tubes can increase the dehydration effect and absorb nutrition, and the stem tip of the Huaiyoshan high-mountain potato can finally realize high seedling rate and high detoxification rate after two-time pre-culture and two-time loading treatment.
The method carries out dark culture and then illumination culture on the treated stem tip, and controls illumination conditions and time, thereby repairing the damage caused by ultralow-temperature preservation and improving the survival rate of the frozen stem tip.
The detoxification plants of the Huaiyshan mountain potatoes cultured by the method have the advantages of good growth state, high survival rate, high detoxification rate, simple and easy preparation method and short culture period, and can be used for batch production.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The "parts" in the present invention are all parts by mass unless otherwise specified.
The thickness of the graphene quantum dots used in the embodiment of the invention is 1-2nm, and the particle size is 6-7 nm.
Example 1
(1) Under the aseptic condition, taking the stem tip of the Huaiyoshan high mountain potato with the length of 0.5cm, and uniformly mixing the stem tip with a calcium ion-free modified MS culture solution containing 3% (w/v) of sodium alginate and 0.2mol/L of cane sugar to obtain a mixed solution;
(2) embedding: slowly dripping the mixed solution prepared in the step (1) into a solution containing 0.05g/L of chitosan oligosaccharide, 0.03g/L of graphene quantum dots, 0.03g/L of carbon nano tube and 0.1mol/LCaCl under the aseptic condition2Embedding for 38min in MS culture solution containing 0.2mol/L sucrose to obtain embedded beads of stem tip of rhizoma Dioscoreae Solani Tuber osi with particle size of 1.8 cm;
(3) primary pre-culture: under the aseptic condition, putting the stem tip embedded beads of the Huaiyoshan high-mountain potatoes into MS culture solution containing 0.05g/L chitosan oligosaccharide and 0.3g/L graphene quantum dots for culturing for 24 hours;
(4) secondary pre-culture: under the aseptic condition, carrying out secondary pre-culture on the embedded beads of the stem tips of the Huaiyoshan high mountain potatoes subjected to the primary pre-culture treatment in the step (3) by using an MS culture solution containing 0.05g/L chitosan oligosaccharide and 0.5g/L carbon nano tube for 24 h;
(5) primary loading: under the aseptic condition, putting the stem tip embedded beads of the Huaiyoshan high-mountain potatoes pre-cultured twice into MS culture solution containing 2M chitosan oligosaccharide and 0.4M graphene quantum dots for loading for 1 h;
(6) secondary loading: under the aseptic condition, carrying out secondary loading on the stem tip embedded beads of the Huaiyoshan high-mountain potatoes obtained in the step (5) by using an MS culture solution containing 2M chitosan oligosaccharide and 0.4M carbon nano tubes, wherein the loading time is 1 h;
(7) and (3) dehydration treatment: under the aseptic condition, dehydrating the stem tip embedded beads of the Huaiyoshan high-mountain potatoes obtained in the step (6) for 1h by using an MS culture solution containing 25% of glycerol, 20% of ethylene glycol, 15% of chitosan oligosaccharide, 0.4M of graphene quantum dots and 0.4M of carbon nano tubes;
(8) and (3) under an aseptic condition, placing the dehydrated Huaiyoshan high-mountain potato stem tip embedded beads subjected to dehydration treatment in the step (7) in liquid nitrogen for 1d, then thawing at 37 ℃, taking out the stem tips from the thawed Huaiyoshan high-mountain potato stem tip embedded beads, and washing for 3 times, 10min each time, in an MS liquid culture medium containing 1.2M sucrose MS +0.05g/L chitosan oligosaccharide +0.03g/L graphene quantum dots +0.05g/L carbon nanotubes. And (3) performing dark culture on the washed stem tips at 27 ℃ for 5 days, and then performing culture on an MS solid culture medium at 25 ℃ for 28 days under the illumination culture conditions of 1500lx light intensity and 14h/d illumination time to obtain the virus-free plants of the Huaiyoshan high mountain potatoes.
The new leaves of the Huaiyshan mountain potato virus-free plant obtained in example 1 are good in development state, and the seedling rate is 93%.
Example 2
(1) Under the aseptic condition, taking 1cm long stems and tips of the Huaiyoshan high mountain potatoes, and uniformly mixing the stems and tips with a calcium ion-free modified MS culture solution containing 3% (w/v) of sodium alginate and 0.2mol/L of cane sugar to obtain a mixed solution;
(2) embedding: slowly dripping the mixed solution prepared in the step (1) into a solution containing 0.05g/L of chitosan oligosaccharide, 0.05g/L of graphene quantum dots, 0.05g/L of carbon nano tubes and 0.1mol/LCaCl under the aseptic condition2Embedding for 42min in MS culture solution containing 0.2mol/L sucrose to obtain embedded beads of 2 cm-sized stem tips of the Huaiyishan high-mountain potatoes;
(3) primary pre-culture: under the aseptic condition, putting the stem tip embedded beads of the Huaiyoshan high-mountain potatoes into MS culture solution containing 0.05g/L chitosan oligosaccharide and 0.5g/L graphene quantum dots for culturing for 24 hours;
(4) secondary pre-culture: under the aseptic condition, carrying out secondary pre-culture on the embedded beads of the stem tips of the Huaiyoshan high mountain potatoes subjected to the primary pre-culture treatment in the step (3) by using an MS culture solution containing 0.05g/L chitosan oligosaccharide and 0.3g/L carbon nano tube for 24 h;
(5) primary loading: under the aseptic condition, putting the stem tip embedded beads of the Huaiyoshan high-mountain potatoes pre-cultured twice into MS culture solution containing 2M chitosan oligosaccharide and 0.4M graphene quantum dots for loading for 1 h;
(6) secondary loading: under the aseptic condition, carrying out secondary loading on the stem tip embedded beads of the Huaiyoshan high-mountain potatoes obtained in the step (5) by using an MS culture solution containing 2M chitosan oligosaccharide and 0.4M carbon nano tubes, wherein the loading time is 1 h;
(7) and (3) dehydration treatment: under the aseptic condition, dehydrating the stem tip embedded beads of the Huaiyoshan high-mountain potatoes obtained in the step (6) for 1h by using an MS culture solution containing 25% of glycerol, 20% of ethylene glycol, 15% of chitosan oligosaccharide, 0.4M of graphene quantum dots and 0.4M of carbon nano tubes;
(8) and (3) under an aseptic condition, placing the dehydrated Huaiyoshan high-mountain potato stem tip embedded beads subjected to dehydration treatment in the step (7) in liquid nitrogen for preservation for 1d, then performing thawing at the temperature of 38 ℃, taking the stem tips out of the thawed Huaiyoshan high-mountain potato stem tip embedded beads, and washing for 3 times, 10min each time, in an MS liquid culture medium containing 1.2M sucrose, MS +0.05g/L chitosan oligosaccharide +0.05g/L graphene quantum dots +0.05g/L carbon nano tubes. And (3) culturing the washed stem tips in the dark at 23 ℃ for 5 days, and then culturing the stem tips on an MS solid culture medium at 27 ℃ for 32 days under the illumination culture conditions of 2000lx light intensity and 14h/d illumination time to obtain the virus-free plants of the Huaiyoshan high mountain potatoes.
The new leaves of the virus-free plants of the mountains and the mountains of the Huaiyan mountains obtained in the example 2 have good development state, and the seedling rate is 93.3%.
Example 3
(1) Under the aseptic condition, taking the stem tip of the Huaiyoshan high mountain potato with the length of 0.8cm, and uniformly mixing the stem tip with a calcium ion-free modified MS culture solution containing 3% (w/v) of sodium alginate and 0.2mol/L of cane sugar to obtain a mixed solution;
(2) embedding: slowly dripping the mixed solution prepared in the step (1) into a solution containing 0.05g/L of chitosan oligosaccharide, 0.04g/L of graphene quantum dots, 0.04g/L of carbon nano tubes and 0.1mol/LCaCl under the aseptic condition2Embedding for 40min in MS culture solution containing 0.2mol/L sucrose to obtain embedded beads of stem tip of rhizoma Dioscoreae Solani Tuber osi with particle size of 1.9 cm;
(3) primary pre-culture: under the aseptic condition, putting the stem tip embedded beads of the Huaiyoshan high-mountain potatoes into MS culture solution containing 0.05g/L chitosan oligosaccharide and 0.5g/L graphene quantum dots for culturing for 24 hours;
(4) secondary pre-culture: under the aseptic condition, carrying out secondary pre-culture on the embedded beads of the stem tips of the Huaiyoshan high mountain potatoes subjected to the primary pre-culture treatment in the step (3) by using an MS culture solution containing 0.05g/L chitosan oligosaccharide and 0.3g/L carbon nano tube for 24 h;
(5) primary loading: under the aseptic condition, putting the stem tip embedded beads of the Huaiyoshan high-mountain potatoes pre-cultured twice into MS culture solution containing 2M chitosan oligosaccharide and 0.4M graphene quantum dots for loading for 1 h;
(6) secondary loading: under the aseptic condition, carrying out secondary loading on the stem tip embedded beads of the Huaiyoshan high-mountain potatoes obtained in the step (5) by using an MS culture solution containing 2M chitosan oligosaccharide and 0.4M carbon nano tubes, wherein the loading time is 1 h;
(7) and (3) dehydration treatment: under the aseptic condition, dehydrating the stem tip embedded beads of the Huaiyoshan high-mountain potatoes obtained in the step (6) for 1h by using an MS culture solution containing 25% of glycerol, 20% of ethylene glycol, 15% of chitosan oligosaccharide, 0.4M of graphene quantum dots and 0.4M of carbon nano tubes;
(8) and (3) under an aseptic condition, placing the dehydrated Huaiyoshan high-mountain potato stem tip embedded beads subjected to dehydration treatment in the step (7) in liquid nitrogen for 1d, then performing thawing at the temperature of 36 ℃, taking out the stem tips from the thawed Huaiyoshan high-mountain potato stem tip embedded beads, and washing for 3 times, 10min each time, in an MS liquid culture medium containing 1.2M sucrose MS +0.05g/L chitosan oligosaccharide +0.04g/L graphene quantum dots +0.03g/L carbon nanotubes. And (3) culturing the washed stem tips in the dark at 25 ℃ for 5 days, and then culturing the stem tips on an MS solid culture medium at 23 ℃ for 30 days under the illumination culture conditions of 1600lx light intensity and 14h/d illumination time to obtain the virus-free plants of the Huaiyoshan high mountain potatoes.
The new leaves of the Huaiyshan mountain potato virus-free plants obtained in example 3 have good development state, and the seedling rate is 93.5%.
Example 4
(1) Under the aseptic condition, taking the stem tip of the Huaiyoshan high mountain potato with the length of 0.6cm, and uniformly mixing the stem tip with a calcium ion-free modified MS culture solution containing 3% (w/v) of sodium alginate and 0.2mol/L of cane sugar to obtain a mixed solution;
(2) embedding: under the aseptic condition, the mixed liquid prepared in the step (1) is mixedSlowly dripping into solution containing 0.05g/L chitosan oligosaccharide, 0.035/L graphene quantum dot, 0.04g/L carbon nanotube, and 0.1mol/LCaCl2Embedding for 40min in MS culture solution containing 0.2mol/L sucrose to obtain embedded beads of 2 cm-diameter stem tips of the Huaiyishan high-mountain potatoes;
(3) primary pre-culture: under the aseptic condition, putting the stem tip embedded beads of the Huaiyoshan high-mountain potatoes into MS culture solution containing 0.05g/L chitosan oligosaccharide and 0.45g/L graphene quantum dots for culturing for 24 hours;
(4) secondary pre-culture: under the aseptic condition, carrying out secondary pre-culture on the stem tip embedded beads of the Huaiyoshan high mountain potatoes subjected to the primary pre-culture treatment in the step (3) by using an MS culture solution containing 0.05g/L chitosan oligosaccharide and 0.35g/L carbon nano tube for 24 h;
(5) primary loading: under the aseptic condition, putting the stem tip embedded beads of the Huaiyoshan high-mountain potatoes pre-cultured twice into MS culture solution containing 2M chitosan oligosaccharide and 0.4M graphene quantum dots for loading for 1 h;
(6) secondary loading: under the aseptic condition, carrying out secondary loading on the stem tip embedded beads of the Huaiyoshan high-mountain potatoes obtained in the step (5) by using an MS culture solution containing 2M chitosan oligosaccharide and 0.4M carbon nano tubes, wherein the loading time is 1 h;
(7) and (3) dehydration treatment: under the aseptic condition, dehydrating the stem tip embedded beads of the Huaiyoshan high-mountain potatoes obtained in the step (6) for 1h by using an MS culture solution containing 25% of glycerol, 20% of ethylene glycol, 15% of chitosan oligosaccharide, 0.4M of graphene quantum dots and 0.4M of carbon nano tubes;
(8) and (3) under an aseptic condition, placing the dehydrated Huaiyoshan high-mountain potato stem tip embedded beads subjected to dehydration treatment in the step (7) in liquid nitrogen for 1d, then performing thawing at the temperature of 37 ℃, taking the stem tips out of the thawed Huaiyoshan high-mountain potato stem tip embedded beads, and washing for 3 times, 10min each time, in an MS liquid culture medium containing 1.2M sucrose, MS +0.05g/L chitosan oligosaccharide +0.04g/L graphene quantum dot +0.035g/L carbon nanotube. And (3) dark culturing the washed stem tips at 23 ℃ for 5 days, and then culturing on an MS solid culture medium at 25 ℃ for 29 days under the illumination culture conditions of light intensity of 1800lx and illumination time of 14h/d to obtain the virus-free plants of the Huaiyoshan high mountain potatoes.
The new leaves of the Huaiyshan mountain potato virus-free plants obtained in example 4 have good development state, and the seedling rate is 93.1%.
Comparative example 1
The difference from example 1 is that the primary preculture process of step (3) is not performed, and the rest is the same as example 1.
The new leaves of the virus-free plants of the Huaiyshan high-mountain potatoes cultured are small, and the seedling rate is 68.5%.
Comparative example 2
The difference from example 1 is that the secondary preculture process of step (4) is not performed, and the rest is the same as example 1.
The new leaves of the virus-free plant of the Huaiyshan mountain high-mountain potato obtained by culture are small, and the seedling rate is 67.9%.
Comparative example 3
The difference from example 1 is that the one-time loading process of step (5) is not performed, and the rest is the same as example 1.
The detoxified plant of the Huaiyshan mountain potatoes cultured has small leaves, yellow leaves and a seedling rate of 59.4%.
Comparative example 4
The difference from example 1 is that the secondary loading process of step (6) is not performed, and the rest is the same as example 1.
The detoxified plant of the Huaiyshan mountain potatoes cultured has small leaves, yellow leaves and a seedling rate of 59.1 percent.
Comparative example 5
The difference from example 1 is that the dark culture process of step (8) is not performed, and the rest is the same as example 1.
The detoxified plant of the Huaiyshan mountain high-mountain potato obtained by cultivation has few new leaves, smaller leaves, yellow leaves and the seedling rate of 52.4 percent.
The DAS-ELISA method is used for detecting the detoxification rate of the common potato viruses of the examples 1 to 4 and the comparative examples 1 to 5, and the results are shown in the table 1.
TABLE 1
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. A method for ultra-low temperature detoxification of Huaiyoshan high mountain potatoes by an embedding vitrification method is characterized by comprising the following steps:
(1) under the aseptic condition, uniformly mixing the stem tip of the Huaiyoshan high-mountain potato with a calcium-ion-free modified MS culture solution containing sodium alginate and sucrose to obtain a mixed solution;
(2) embedding: under the aseptic condition, the mixed solution obtained in the step (1) is dripped into a solution containing chitosan oligosaccharide, graphene quantum dots, carbon nano tubes and CaCl2Embedding the mixture and a sucrose MS culture solution to obtain a Huaiyoshan high-mountain potato stem tip embedded bead;
(3) primary pre-culture: under the aseptic condition, carrying out primary pre-culture on the stem tip embedded beads of the Huaiyoshan high-mountain potatoes by using MS culture solution containing chitosan oligosaccharide and graphene quantum dots;
(4) secondary pre-culture: under the aseptic condition, carrying out secondary pre-culture on the stem tip embedded beads of the Huaiyoshan high-mountain potatoes subjected to primary pre-culture treatment by using MS culture solution containing chitosan oligosaccharide and carbon nano tubes;
(5) primary loading: under the aseptic condition, loading the stem tip embedded beads of the Huaiyoshan high-mountain potatoes pre-cultured twice with MS culture solution containing chitosan oligosaccharide and graphene quantum dots;
(6) secondary loading: under the aseptic condition, carrying out secondary loading on the stem tip embedded beads of the Huaiyoshan high-mountain potatoes obtained in the step (5) by using MS culture solution containing chitosan oligosaccharide and carbon nano tubes;
(7) and (3) dehydration treatment: under the aseptic condition, dehydrating the stem tip embedded beads of the Huaiyoshan high-mountain potatoes obtained in the step (6) by using an MS culture solution containing 25% of glycerol, 20% of ethylene glycol, 15% of chitosan oligosaccharide, 0.4M of graphene quantum dots and 0.4M of carbon nano tubes;
(8) and (3) under the aseptic condition, placing the stem tip embedded beads of the Huaiyoshan mountain high-mountain potatoes subjected to dehydration treatment in the step (7) in liquid nitrogen for preservation, then thawing, taking out the stem tips from the thawed stem tip embedded beads of the Huaiyoshan mountain high-mountain potatoes, washing in an MS liquid culture medium containing 1.2M sucrose, MS, 0.05g/L chitosan oligosaccharide, 0.03-0.05g/L graphene quantum dots and 0.03-0.05g/L carbon nano tubes, firstly carrying out dark culture on the washed stem tips, and then carrying out culture under the illumination culture condition to obtain the detoxified plants of the Huaiyoshan mountain high-mountain potatoes.
2. The embedding and vitrifying ultra-low temperature detoxification method for high mountain potatoes in Huaiyoshan as claimed in claim 1, wherein the mass volume ratio of the sodium alginate to the modified MS culture solution without calcium ions in step (1) is 3%, and the sucrose concentration is 0.2 mol/L; the length of the potato stem tip is 0.5-1 cm.
3. The method for ultra-low temperature detoxification of Huaiyanshan high mountain potatoes through embedding vitrification, as claimed in claim 1, wherein in the step (2), the concentration of chitosan oligosaccharide is 0.05g/L, the concentration of graphene quantum dots is 0.03-0.05g/L, and the concentration of carbon nanotubes is 0.03-0.05g/L, CaCl2The concentration is 0.1mol/L, and the sucrose concentration is 0.2 mol/L.
4. The embedding vitrification ultra-low temperature detoxification method for Huaiyishan high-mountain potatoes as claimed in claim 1, wherein the embedding time in step (2) is 38-42min, and the particle size of the embedding beads of the stem tips of the Huaiyishan high-mountain potatoes is 1.8-2 cm.
5. The embedding and vitrification ultra-low temperature detoxification method of Huaiyoshan high mountain potatoes as claimed in claim 1, wherein in the step (3), the concentration of chitosan oligosaccharide is 0.05g/L, the concentration of graphene quantum dots is 0.3-0.5g/L, and the pre-culture time is 24 h.
6. The embedding and vitrification ultra-low temperature detoxification method of Huaiyanshan high mountain potatoes as claimed in claim 1, wherein the concentration of chitosan oligosaccharide in step (4) is 0.05g/L, the concentration of carbon nanotubes is 0.3-0.5g/L, and the pre-culture time is 24 h.
7. The embedding vitrification ultra-low temperature detoxification method of Huaiyoshan high mountain potatoes as claimed in claim 1, wherein in the step (5), the concentration of chitosan oligosaccharide is 2M, the concentration of graphene quantum dots is 0.4M, and the loading time is 1 h; in the step (6), the concentration of the chitosan oligosaccharide is 2M, the concentration of the carbon nano tube is 0.4M, and the loading time is 1 h; the dehydration time in the step (7) is 1 h.
8. The embedding vitrification ultra-low temperature detoxification method of Huaiyoshan high mountain potatoes as claimed in claim 1, wherein the temperature of thawing in step (8) is 37 ± 1 ℃, and the temperature of dark culture is 25 ± 2 ℃.
9. The method as claimed in claim 1, wherein the conditions of the step (8) are 14h/d light time, 1500-2000lx light intensity and 25 ± 2 ℃ light temperature.
10. The embedding vitrification ultra-low temperature detoxification method of Huaiyoshan high mountain potatoes as claimed in claim 1, wherein the thickness of the graphene quantum is 1-2nm, and the particle size is 6-7 nm.
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