CN112106635A - Method for rapidly screening label-free gene editing tobacco material seeds by using kanamycin - Google Patents
Method for rapidly screening label-free gene editing tobacco material seeds by using kanamycin Download PDFInfo
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- CN112106635A CN112106635A CN202011003737.0A CN202011003737A CN112106635A CN 112106635 A CN112106635 A CN 112106635A CN 202011003737 A CN202011003737 A CN 202011003737A CN 112106635 A CN112106635 A CN 112106635A
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
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Abstract
The invention relates to a method for rapidly screening label-free gene editing tobacco material seeds by using kanamycin, belonging to the technical field of plant cell engineering. The method comprises the steps of preparation of a screening culture solution, disinfection of gene editing tobacco seeds, seed screening culture and judgment of whether the kanamycin resistance label is carried. The method can conveniently and effectively screen out the materials without the transgenic tags in the gene editing materials. The invention also verifies the reliability of the screening result of kanamycin soaked seeds by utilizing a PCR amplification carrier fragment method, provides an economical, convenient and reliable method for screening label-free gene editing tobacco materials for heavy screening work in the breeding process of gene editing tobacco, and can reduce the screening cost and improve the production efficiency.
Description
Technical Field
The invention belongs to the technical field of plant cell engineering, and particularly relates to a method for rapidly screening tag-free gene editing tobacco material seeds by using kanamycin.
Background
The gene editing technology is not only an important tool for analyzing and clarifying gene functions, but also an epoch-making and important breakthrough in plant breeding. CRISPR/Cas (Clustered regulated interstitial short palindromic repeats) is a gene site-directed editing technology newly discovered in recent years, CRISPR/Cas9 is the most applied at present, a specific exogenous DNA sequence can be cut only by a nuclease Cas9 and a mature crRNA (CRISPR-derived RNA: trans-activating RNA) complex, and the genome editing technology based on the CRISPR/Cas system has the characteristics of high efficiency, convenience and the like. The technology has been successfully edited in various plants such as arabidopsis, tobacco, rice and the like, proves the feasibility of the technology, and realizes the effects of stress resistance of the plants, fruit maturity delay, herbicide tolerance increase, disease resistance and the like.
To facilitate the pre-screening of gene editing materials, kanamycin resistance genes have been integrated into the construction of vectors currently used for plant gene editing, as shown in FIG. 1. The agrobacterium is utilized to transform the leaf disc, usually kanamycin with a certain concentration is added into a culture medium, so that the transformed seedling differentiated in a selective culture medium can be effectively obtained, and the workload of later detection and identification can be reduced. In the process of tobacco gene editing breeding, a plant with kanamycin resistance needs to be screened from T0 generation differentiated seedlings obtained by tissue culture so as to ensure that a gene editing vector enters the plant body; then, after each generation of continuous selfing homozygosis, the target plants needing to be screened out in the T1 generation and later are homozygous plants with successfully edited gene targets and without carrier labels; the screening workload of the material after the inbreeding and the homozygosis is multiplied, and the cost of molecular detection is high. Therefore, how to overcome the defects of the prior art and provide a quick, convenient, strong-visualization, economical and practical method for screening the label-free materials for gene editing is a problem which needs to be solved urgently in the technical field of plant cell engineering at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for rapidly screening non-label gene editing tobacco material seeds by using kanamycin.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for rapidly screening non-label gene editing tobacco material seeds by using kanamycin comprises the following steps:
preparing a screening culture solution, wherein the screening culture solution is 1/2MS minimal medium, 15g/L sucrose and 100-400 mg/L kanamycin;
step (2), sterilizing the gene editing tobacco seeds;
step (3), laying sterile filter paper at the bottom of a culture dish, transferring the sterilized seeds in the step (2) to the filter paper to be spread, adding the screening culture solution in the step (1) to culture, supplementing the culture solution in time during the culture period, and keeping the filter paper in a wet state;
step (4), when the seeds are observed to germinate and grow cotyledons, if the whole seedlings are yellow or white, the seeds are not labeled; if green, the seeds carry a kanamycin resistance tag.
Further, preferably, in the step (1), the screening culture solution is 1/2MS minimal medium +15g/L sucrose +200mg/L kanamycin;
further, preferably, in the step (1), the specific steps of preparing the screening culture solution are as follows: adding sucrose into 1/2MS minimal medium to reach concentration of 15g/L and pH value of 5.5-5.9, sterilizing at high temperature and high pressure, cooling, adding kanamycin after filtering sterilization to reach concentration of 100-400 mg/L, and storing at 4 ℃ for later use.
Further, it is preferable that sucrose is added to 1/2MS minimal medium to a concentration of 15g/L and a pH of 5.7; the temperature of high temperature and high pressure sterilization is 121 deg.C, the pressure is 103.4kPa, and the time is 15-20 min.
Further, it is preferable that, in the step (2), the specific method for disinfecting the seeds is as follows: placing gene-edited tobacco seeds on a superclean workbench, soaking the seeds in 75% alcohol for 30s, washing with sterile water, and then washing with 1% AgNO3Sterilizing the solution for 7-10min, soaking in sterile water, cleaning, and drying with sterile filter paper.
Further, it is preferable that the number of times of washing with sterile water is at least 2; the number of the soaking and cleaning in the sterile water is at least 5.
Further, it is preferable that in the step (3), a sterile filter paper is laid on the bottom of the culture dish, the seeds sterilized in the step (2) are transferred to the filter paper and spread, and the screening culture solution in the step (1) is added until the filter paper is soaked and half of the height of the seeds is reached.
Further, it is preferable that, in the step (3), the seed selection culture conditions are: the temperature is 25 +/-1 ℃, and the illumination intensity is 30-50 mu mol/(m)2S), the illumination time is 16h/d, and the culture lasts for 14-21 d.
Further, preferably, the seedlings germinated from the seeds in the step (4) are collected, DNA is extracted, primers are designed aiming at specific gene sequences or national standard universal primers are adopted to carry out PCR and gel electrophoresis detection on label bands, so as to confirm the accuracy of the result in the step (4).
Further, it is preferable that the DNA is extracted using a plant genomic DNA extraction kit according to the instructions; the PCR detection method uses the national standard of tobacco and tobacco product transgenic detection method GBT 24310-2009: 35S promoter sequence amplification, wherein an amplification primer pair is 35S-1 and 35S-2, and the length of an amplification fragment of the primer pair is 205 bp; amplifying NOS terminator sequences, wherein the amplification primer pair is NOS-1 and NOS-2, and the length of the amplification fragment of the primer pair is 213 bp; performing 1.5% agarose gel electrophoresis at 145V for 45min, and detecting a label band;
wherein, 35S-1: 5'-ctacaaatgccatcattgcg-3', respectively;
35S-2：5’-gggtcttgcgaaggatagtg-3’；
NOS-1：5’-gattgaatcctgyygccggt-3’；
NOS-2：5’-gtaacatagatgacaccgcg-3’。
in the invention, when the seed germination and cotyledon growth are observed, if the whole seedling is yellow or white, the seed is not provided with a label and is usually accompanied with the phenomenon of slow plant growth; when seed germination is observed to give rise to cotyledons, if the entire plantlet is green, the seed carries a kanamycin resistance tag, usually accompanied by a phenomenon of normal plant growth.
The filtration of the kanamycin added in the present invention after the filtration sterilization is usually performed by a 0.22 μm membrane filtration.
Compared with the prior art, the invention has the beneficial effects that:
aiming at the purpose that gene editing breeding is to obtain plants which are successfully edited, homozygous and have no transgenic label, the invention can conveniently and visually screen whether the transgenic label exists in seeds of a certain generation of the edited tobacco materials by utilizing kanamycin. The general breeding work has the problem of large screening material quantity, the method for screening label-free plants in large batch by methods of DNA extraction, PCR amplification or molecular detection usually consumes manpower and high cost, the large-batch molecular detection method needs to consume 2-4 weeks for detecting and comparing sequences, and the cost is 20 times of that of the invention; the method provided by the invention is convenient, visual, high in accuracy and more economical and practical.
Drawings
FIG. 1 is a gene editing plasmid construction map;
FIG. 2 is a diagram of seed germination of a kanamycin-screened gene-edited tobacco material at different concentrations; wherein, the concentrations of A to D are respectively 0, 200, 400 and 600mg/L, and the four areas a to D are respectively seeds of wild WT (negative control), GE1, GE2 and GE 3.
FIG. 3 is a gel electrophoresis image of the PCR amplified carrier fragment; wherein, the A-C primers are 35S, NOS and CSN respectively.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.
Example 1
A method for rapidly screening non-label gene editing tobacco material seeds by using kanamycin comprises the following steps:
preparing a screening culture solution, wherein the screening culture solution is 1/2MS minimal medium, 15g/L sucrose and 100mg/L kanamycin;
step (2), sterilizing the gene editing tobacco seeds;
step (3), laying sterile filter paper at the bottom of a culture dish, transferring the sterilized seeds in the step (2) to the filter paper to be spread, adding the screening culture solution in the step (1) to culture, supplementing the culture solution in time during the culture period, and keeping the filter paper in a wet state;
step (4), when the seeds are observed to germinate and grow cotyledons, if the whole seedlings are yellow or white, the seeds are not labeled; if green, the seeds carry a kanamycin resistance tag.
Example 2
A method for rapidly screening non-label gene editing tobacco material seeds by using kanamycin comprises the following steps:
preparing a screening culture solution, wherein the screening culture solution is 1/2MS minimal medium, 15g/L sucrose and 400mg/L kanamycin;
step (2), sterilizing the gene editing tobacco seeds;
step (3), laying sterile filter paper at the bottom of a culture dish, transferring the sterilized seeds in the step (2) to the filter paper to be spread, adding the screening culture solution in the step (1) to culture, supplementing the culture solution in time during the culture period, and keeping the filter paper in a wet state;
step (4), when the seeds are observed to germinate and grow cotyledons, if the whole seedlings are yellow or white, the seeds are not labeled; if green, the seeds carry a kanamycin resistance tag.
In the step (1), the specific steps of preparing the screening culture solution are as follows: adding sucrose into 1/2MS minimal medium to reach concentration of 15g/L and pH of 5.5, sterilizing at high temperature under high pressure, cooling, adding kanamycin after filtering sterilization to reach concentration of 400mg/L, and storing at 4 deg.C for use. The temperature of high temperature and high pressure sterilization is 121 deg.C, the pressure is 103.4kPa, and the time is 15 min.
In the step (2), the specific method for disinfecting the seeds comprises the following steps: placing gene-edited tobacco seeds on a superclean workbench, soaking the seeds in 75% alcohol for 30s, washing with sterile water, and then washing with 1% AgNO3Sterilizing the solution for 7min, soaking in sterile water, cleaning, and drying with sterile filter paper. The times of the sterile water washing are 2 times; the number of the soaking and cleaning with sterile water is 5.
In step (3), a piece of sterile filter paper is laid at the bottom of the culture dish, the seeds sterilized in step (2) are transferred to the filter paper and spread, and the screening culture solution in step (1) is added until the filter paper is soaked and is half of the height of the seeds. Seed screening culture conditions: the temperature is 25 +/-1 ℃, and the illumination intensity is 30-50 mu mol/(m)2S), the illumination time was 16h/d, and the culture was carried out for 14 d.
Example 3
A method for rapidly screening non-label gene editing tobacco material seeds by using kanamycin comprises the following steps:
preparing a screening culture solution, wherein the screening culture solution is 1/2MS minimal medium, 15g/L sucrose and 200mg/L kanamycin;
step (2), sterilizing the gene editing tobacco seeds;
step (3), laying sterile filter paper at the bottom of a culture dish, transferring the sterilized seeds in the step (2) to the filter paper to be spread, adding the screening culture solution in the step (1) to culture, supplementing the culture solution in time during the culture period, and keeping the filter paper in a wet state;
step (4), when the seeds are observed to germinate and grow cotyledons, if the whole seedlings are yellow or white, the seeds are not labeled; if green, the seeds carry a kanamycin resistance tag.
In the step (1), the specific steps of preparing the screening culture solution are as follows: adding sucrose into 1/2MS minimal medium to reach concentration of 15g/L and pH of 5.9, sterilizing at high temperature under high pressure, cooling, adding kanamycin after filtering sterilization to reach concentration of 200mg/L, and storing at 4 deg.C for use.
The temperature of high temperature and high pressure sterilization is 121 deg.C, the pressure is 103.4kPa, and the time is 20 min.
In the step (2), the specific method for disinfecting the seeds comprises the following steps: placing gene-edited tobacco seeds on a superclean workbench, soaking the seeds in 75% alcohol for 30s, washing with sterile water, and then washing with 1% AgNO3Sterilizing the solution for 10min, soaking in sterile water, cleaning, and drying with sterile filter paper. The number of times of washing with sterile water is 3; the number of times of soaking and cleaning in sterile water is 6.
In step (3), a piece of sterile filter paper is laid at the bottom of the culture dish, the seeds sterilized in step (2) are transferred to the filter paper and spread, and the screening culture solution in step (1) is added until the filter paper is soaked and is half of the height of the seeds. Seed screening culture conditions: the temperature is 25 +/-1 ℃, and the illumination intensity is 30-50 mu mol/(m)2S) light exposure time of 16h/d, culture 21d。
Example 4
A method for rapidly screening non-label gene editing tobacco material seeds by using kanamycin comprises the following steps:
preparing a screening culture solution, wherein the screening culture solution is 1/2MS minimal medium, 15g/L sucrose and 200mg/L kanamycin;
step (2), sterilizing the gene editing tobacco seeds;
step (3), laying sterile filter paper at the bottom of a culture dish, transferring the sterilized seeds in the step (2) to the filter paper to be spread, adding the screening culture solution in the step (1) to culture, supplementing the culture solution in time during the culture period, and keeping the filter paper in a wet state;
step (4), when the seeds are observed to germinate and grow cotyledons, if the whole seedlings are yellow or white, the seeds are not labeled; if green, the seeds carry a kanamycin resistance tag.
In the step (1), the specific steps of preparing the screening culture solution are as follows: adding sucrose into 1/2MS minimal medium to reach concentration of 15g/L and pH of 5.7, sterilizing at high temperature under high pressure, cooling, adding kanamycin after filtering sterilization to reach concentration of 200mg/L, and storing at 4 deg.C for use. The temperature of high temperature and high pressure sterilization is 121 deg.C, the pressure is 103.4kPa, and the time is 18 min.
In the step (2), the specific method for disinfecting the seeds comprises the following steps: placing gene-edited tobacco seeds on a superclean workbench, soaking the seeds in 75% alcohol for 30s, washing with sterile water, and then washing with 1% AgNO3Sterilizing the solution for 7-10min, soaking in sterile water, cleaning, and drying with sterile filter paper. The times of the sterile water washing are 4 times; the number of the soaking and cleaning with sterile water is 7.
In step (3), a piece of sterile filter paper is laid at the bottom of the culture dish, the seeds sterilized in step (2) are transferred to the filter paper and spread, and the screening culture solution in step (1) is added until the filter paper is soaked and is half of the height of the seeds. Seed screening culture conditions: the temperature is 25 +/-1 ℃, and the illumination intensity is 30-50 mu mol/(m)2S) in the lightThe time is 16h/d, and the culture lasts 18 d.
And (3) collecting seedlings germinated from the seeds in the step (4), extracting DNA, designing a primer aiming at a specific gene sequence or detecting a label strip by using a national standard universal primer to carry out PCR and gel electrophoresis so as to confirm the accuracy of the result in the step (4).
Extracting DNA by using a plant genome DNA extraction kit according to the operation of an instruction; the PCR detection method uses the national standard of tobacco and tobacco product transgenic detection method GBT 24310-2009: 35S promoter sequence amplification, wherein an amplification primer pair is 35S-1 and 35S-2, and the length of an amplification fragment of the primer pair is 205 bp; amplifying NOS terminator sequences, wherein the amplification primer pair is NOS-1 and NOS-2, and the length of the amplification fragment of the primer pair is 213 bp; performing 1.5% agarose gel electrophoresis at 145V for 45min, and detecting a label band;
wherein, 35S-1: 5'-ctacaaatgccatcattgcg-3', respectively;
35S-2：5’-gggtcttgcgaaggatagtg-3’；
NOS-1：5’-gattgaatcctgyygccggt-3’；
NOS-2：5’-gtaacatagatgacaccgcg-3’。
in the invention, when the seed germination and cotyledon growth are observed, if the whole seedling is yellow or white, the seed is not provided with a label and is usually accompanied with the phenomenon of slow plant growth; when seed germination is observed to give rise to cotyledons, if the entire plantlet is green, the seed carries a kanamycin resistance tag, usually accompanied by a phenomenon of normal plant growth.
Application example 1
A method for rapidly screening non-label gene editing tobacco material seeds by using kanamycin comprises the following steps:
step (1), screening culture solution preparation: 1/2 adding sucrose 15g/L into the basic culture medium, adjusting pH to 5.7, sterilizing at 121 deg.C for 15min under high temperature and high pressure. After cooling, separately adding kanamycin to 1/2MS liquid medium (filtration sterilization), preparing four culture solutions with screening concentration (containing kanamycin 0, 200, 400, 600 mg/L), and storing at 4 ℃ for later use;
step (2), Gene editing tobaccoSeed disinfection: collecting 3 parts of gene-edited tobacco seeds (GE 1, GE2 and GE 3) and 1 part of control tobacco seeds (wild type WT), soaking the seeds with 75% alcohol for 30s on an ultraclean bench, washing with sterile water for 2 times, and adding AgNO with a mass concentration of 1%3Sterilizing the solution for 10min, soaking and cleaning with sterile water for 5 times, and inoculating after absorbing water on the surface of the seeds with sterile filter paper, wherein the sterile water is ultrapure water sterilized under high pressure;
and (3) screening and culturing seeds: taking four culture dishes, respectively putting a piece of sterile filter paper, uniformly transferring four seeds sterilized in the step (2) to four areas of the filter paper, spreading, adding the screening culture solution in the step (1) until the filter paper is soaked and is half of the height of the seeds, timely supplementing the culture solution during culture, and keeping the filter paper in a wet state; seed screening culture conditions: the temperature is 25 +/-1 ℃, the illumination intensity is 30-50 mu mol/(m2 & s), and the illumination time is 16 h/d;
step (4), when the seeds are cultured for 14-21d, the seeds can be observed to germinate and grow cotyledons, and if the whole seedlings are yellow or white and grow slowly, the seeds are not labeled; if the seeds are green and grow normally, the seeds carry a tag containing kanamycin resistance;
and (5) collecting seedlings germinated from the seeds in the step (4), extracting DNA, designing a primer aiming at a specific gene sequence or detecting a label strip by using a national standard universal primer to carry out PCR and gel electrophoresis so as to confirm the accuracy of the result in the step (4).
Wherein, the DNA is extracted by using a plant genome DNA extraction kit (Beijing Tiangen) according to the instruction; the PCR detection method uses the national standard of tobacco and tobacco product transgenic detection method GBT 24310-2009: 35S promoter sequence amplification, wherein an amplification primer pair is 35S-1 and 35S-2, and the length of an amplification fragment of the primer pair is 205 bp; amplifying NOS terminator sequences, wherein the amplification primer pair is NOS-1 and NOS-2, and the length of the amplification fragment of the primer pair is 213 bp; amplification was performed using a Cas9(CSN) primer pair [ Cas9(CSN) -1 and Cas9(CSN) -2], which amplifies a fragment 358bp in length.
Performing 1.5% agarose gel electrophoresis at 145V for 45min, and detecting a label band;
wherein, the 35S primer pair:
35S-1：5’-ctacaaatgccatcattgcg-3’；（SEQ ID NO.1）
35S-2：5’-gggtcttgcgaaggatagtg-3’；（SEQ ID NO.2）
NOS primer pairs:
NOS-1：5’-gattgaatcctgyygccggt-3’；（SEQ ID NO.3）
NOS-2：5’-gtaacatagatgacaccgcg-3’。（SEQ ID NO.4）
cas9(CSN) primer pair:
Cas9(CSN)-1：5'-cattgcgttgtcactcgg-3’；（SEQ ID NO.5）
Cas9(CSN)-2：5'-tgcgtagccattcttgga-3’；（SEQ ID NO.6）
the length of the amplified fragment is 358 bp.
The PCR amplification system can be obtained according to the national standard of tobacco and tobacco product transgenic detection method GBT 24310-.
PCR amplification procedure: for primers NOS and 35S, Cas9(CSN) 3, the annealing temperatures are 63 ℃, 64 ℃ and 56 ℃, respectively, the reaction system is 50 mu L, and the amplification program is 95 ℃ for 3 min; 95 ℃ for 15s, annealing temperature for 15s, 72 ℃ for 10s, 35 cycles; 5min at 72 ℃; infinity at 12 ℃.
In this example, all seeds were green and growing normally in the 0mg/L kanamycin control group, only GE3 was green and growing normally in the 200, 400, 600mg/L kanamycin experimental group, and WT, GE1 and GE2 were yellow and growing slowly as shown in FIG. 2. The gel was positive for the GE3 tag and negative for the WT, GE1 and GE2 tags as determined by PCR amplification gel electrophoresis, consistent with the kanamycin screening assay, as shown in FIG. 3.
The step (5) is to further confirm the result, and the accuracy is verified by the test and the molecular detection result, and the step can be omitted in the actual operation process. Kanamycin is found to be lower than the protection range of the invention in research, for example, the MS liquid culture medium is 1/2MS minimal medium +15g/L sucrose +50mg/L kanamycin, and after treatment, when all seeds germinate and grow cotyledons, no obvious color distinction is shown; kanamycin is higher than the protection range of the invention, the screening pressure is high due to too high kanamycin concentration, and plants are easy to die after seeds germinate.
The method is adopted to simultaneously detect the tobacco varieties of K326, NC102, KRK26, Yunyan 85, Yunyan 87, Yunyan 97 and Longjiang 911, 20 seeds are randomly extracted from each variety, and the result of detecting whether the material has the label or not obtained by screening through the method is basically consistent with the PCR detection result of the currently accepted label detection method, and the accuracy of the method is more than 99%.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
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Claims (10)
1. A method for rapidly screening label-free gene editing tobacco material seeds by using kanamycin is characterized by comprising the following steps:
preparing a screening culture solution, wherein the screening culture solution is 1/2MS minimal medium, 15g/L sucrose and 100-400 mg/L kanamycin;
step (2), sterilizing the gene editing tobacco seeds;
step (3), laying sterile filter paper at the bottom of a culture dish, transferring the sterilized seeds in the step (2) to the filter paper to be spread, adding the screening culture solution in the step (1) to culture, supplementing the culture solution in time during the culture period, and keeping the filter paper in a wet state;
step (4), when the seeds are observed to germinate and grow cotyledons, if the whole seedlings are yellow or white, the seeds are not labeled; if green, the seeds carry a kanamycin resistance tag.
2. The method for rapidly screening unlabeled gene editing tobacco material seeds using kanamycin according to claim 1, wherein in the step (1), the screening culture solution is 1/2MS minimal medium +15g/L sucrose +200mg/L kanamycin.
3. The method for rapidly screening tagless gene editing tobacco material seeds by using kanamycin as claimed in claim 1, wherein in the step (1), the specific steps of preparing the screening culture solution are as follows: adding sucrose into 1/2MS minimal medium to reach concentration of 15g/L and pH value of 5.5-5.9, sterilizing at high temperature and high pressure, cooling, adding kanamycin after filtering sterilization to reach concentration of 100-400 mg/L, and storing at 4 ℃ for later use.
4. The method for rapidly screening unlabeled gene editing tobacco material seeds using kanamycin as claimed in claim 2, wherein sucrose is added to 1/2MS minimal medium to a concentration of 15g/L and pH value of 5.7; the temperature of high temperature and high pressure sterilization is 121 deg.C, the pressure is 103.4kPa, and the time is 15-20 min.
5. The method for rapidly screening the label-free gene editing tobacco material seeds by using kanamycin according to claim 1, wherein in the step (2), the specific method for sterilizing the seeds is as follows: placing gene-edited tobacco seeds on a superclean workbench, soaking the seeds in 75% alcohol for 30s, washing with sterile water, and then washing with 1% AgNO3Sterilizing the solution for 7-10min, soaking in sterile water, cleaning, and drying with sterile filter paper.
6. The method for rapidly screening the label-free gene editing tobacco material seeds by using kanamycin as claimed in claim 5, wherein the number of times of washing with sterile water is at least 2; the number of the soaking and cleaning in the sterile water is at least 5.
7. The method for rapidly screening tabellated gene editing tobacco material seeds using kanamycin as claimed in claim 1, wherein in step (3), a sterile filter paper is laid on the bottom of the culture dish, the seeds sterilized in step (2) are transferred to the filter paper and spread, and the screening culture solution in step (1) is added until the filter paper is soaked and half of the height of the seeds is exceeded.
8. The method for rapidly screening the seeds of the tobacco material edited by the tag-free gene by using kanamycin as claimed in claim 1, wherein in the step (3), the seed screening culture conditions are as follows: the temperature is 25 +/-1 ℃, and the illumination intensity is 30-50 mu mol/(m)2S), the illumination time is 16h/d, and the culture lasts for 14-21 d.
9. The method for rapidly screening the label-free gene editing tobacco material seeds by using kanamycin according to claim 1, wherein seedlings germinated from the seeds in the step (4) are collected, DNA is extracted, primers are designed aiming at specific gene sequences or national standard universal primers are adopted to carry out PCR and gel electrophoresis detection on label strips so as to confirm the accuracy of the result in the step (4).
10. The method for rapidly screening the label-free gene editing tobacco material seeds by using kanamycin according to claim 9, wherein the DNA extraction is performed by using a plant genome DNA extraction kit according to instructions; the PCR detection method uses the national standard of tobacco and tobacco product transgenic detection method GBT 24310-2009: 35S promoter sequence amplification, wherein an amplification primer pair is 35S-1 and 35S-2, and the length of an amplification fragment of the primer pair is 205 bp; amplifying NOS terminator sequences, wherein the amplification primer pair is NOS-1 and NOS-2, and the length of the amplification fragment of the primer pair is 213 bp; performing 1.5% agarose gel electrophoresis at 145V for 45min, and detecting a label band;
wherein, 35S-1: 5'-ctacaaatgccatcattgcg-3', respectively;
35S-2：5’-gggtcttgcgaaggatagtg-3’；
NOS-1：5’-gattgaatcctgyygccggt-3’；
NOS-2：5’-gtaacatagatgacaccgcg-3’。
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