CN103004585A - Method for quickly screening transgene chrysanthemum plant by kanamycin - Google Patents
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Abstract
The invention discloses a method for quickly screening transgene chrysanthemum plant by kanamycin. The method comprises the following steps of: (1) determining chrysanthemum leave tray adventitious bud differentiation Kan screening concentration, chrysanthemum stem root generation Kan screening concentration, Kan solution immersion screening concentration and observation time; (2) performing differentiation regeneration and root generationon chrysanthemum conversion plants, selecting to obtain root generation plant systems, and soaking the root generation plant systems in a Kan solution, and selecting to obtain a transgene Kan resistant chrysanthemum plant system; and (3) performing PCR (polymerase chain reaction) verification on the transgene chrysanthemum, namely, performing PCR expansion on the selected transgene Kan resistant chrysanthemum plant system according to a 35S starter sequence design primer in a transgene carrier, and verifying whether a carrier section is inserted into a chrysanthemum gene group. The method is simple and accurate; a large number of false positive plants can be effectively eliminated; and the method has great significance on large-scale quick screening and acceleration of breeding progress of the transgene chrysa nthemum plants.
Description
Technical field
The invention belongs to the plant biological engineering technical field, relate to a kind of method of utilizing kanamycin rapid screening chrysanthemum transfer-gen plant.
Background technology
Transgenic technology is the effective ways of improvement chrysanthemum (Chrysanthemum morifolium) genetic character, over nearly 20 years, people are obtaining certain progress aspect the agronomic trait gene engineering germplasm improvement such as chrysanthemum pattern, the flower fancy points such as type and disease resistance, abiotic stress patience, but transformation efficiency is low to be a difficult problem that exists in the chrysanthemum genetic engineering breeding always, in recent years, though oneself has obtained some transgenosis chrysanthemums, these transfer-gen plants mostly are just to obtain after having carried out a large amount of repeated experiments.Therefore, set up a kind of fast and effectively transgenosis screening technique most important for the genetic transformation of realizing chrysanthemum.For the ease of selection, great majority research utilizes the linkage relationships of marker gene and genes of interest usually, by the existence of certification mark gene, and the existence of indirect proof genes of interest.The multiple genetic selection marker gene that is used for the transfer-gen plant screening, still be most widely used with npt-II gene, its coded product-aminoglycoside phosphotransferase (npt-II), can make aminoglycoside antibiotics (such as kanamycin, neomycin etc.) phosphorylation occur and inactivation, thereby make plant produce corresponding antibiotic resistance, therefore, can utilize kanamycin (Kanamycin writes a Chinese character in simplified form Kan) that transformed plant is screened.
At present, the chrysanthemum breeding worker is many to comprise the screening in the screening in differentiation and regeneration stage and the stage of taking root organizing cultivation stage to utilize Kan that the chrysanthemum transformed plant is screened, and determines transgenic line by Molecular Detection at last.Kan not only can play the screening effect in the Plant Transformation process, and can carry out genetic analysis and measure seed purity the offspring by it in transformation generation, also can be used for simultaneously the screening of offspring field strain.But utilizing at present Kan that transgenosis chrysanthemum regeneration plant is carried out Field Screening not yet has report.
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Summary of the invention
Low and transgenosis triage techniques is that all right ripe this present situation the object of the present invention is to provide a kind of method of utilizing kanamycin (Kan) rapid screening chrysanthemum transfer-gen plant for present chrysanthemum transgene efficiency.The present invention has determined a series of Kan screening concentrations in the chrysanthemum transgenosis process, has realized the multi-level screening to the transgenosis chrysanthemum.The present invention proposes the method for utilizing Kan infusion method screening transgenic chrysanthemum, phenomenon is obvious, screening time is short.The method is simply accurate, can effectively reject a large amount of false positive plant, and is significant for scale rapid screening, the quickening breeding process of realization chrysanthemum transfer-gen plant.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of method of utilizing kanamycin rapid screening chrysanthemum transfer-gen plant may further comprise the steps:
(1) determine that florists chrysanthemum leaf dish differentiation adventitious buds kanamycin (Kan) screening concentration, chrysanthemum stem section take root Kan screening concentration and Kan solution soaking and screening concentration and observing time:
Determining of florists chrysanthemum leaf dish differentiation adventitious buds Kan screening concentration: non-transgenic florists chrysanthemum leaf dish is inoculated on the regeneration culture medium of the Kan that contains variable concentrations, find a Kan critical concentration that can just 100% suppresses florists chrysanthemum leaf dish differentiation adventitious buds, be florists chrysanthemum leaf dish differentiation adventitious buds Kan screening concentration;
Chrysanthemum stem section the determining of Kan screening concentration of taking root: non-transgenic chrysanthemum band top leaf stem section is inoculated on the root media of the Kan that contains variable concentrations, find one can just 100% to suppress the Kan critical concentration that chrysanthemum stem section is taken root, be the chrysanthemum stem section Kan screening concentration of taking root;
Determining of Kan solution soaking and screening concentration and observing time: the 3rd full exhibition leaf getting the consistent non-transgenic chrysanthemum of growing way, clean and be placed in the culture dish that fills a small amount of variable concentrations Kan solution, add solution amount and be as the criterion with submergence blade just, determine Kan screening concentration and observing time according to the blade victimization state;
(2) adopt the determined florists chrysanthemum leaf dish of step (1) differentiation adventitious buds Kan screening concentration, the chrysanthemum stem section Kan screening concentration of taking root that the screening that the chrysanthemum transformed plant carries out differentiation and regeneration stage and the stage of taking root is obtained the strain of taking root; And then Kan solution soaking and screening concentration and the observing time of adopting step (1) to determine obtain transgenosis chrysanthemum Kan resistance strain with the described strain of taking root behind Kan solution soaking and screening;
(3) PCR of transgenosis chrysanthemum checking: according to the 35S promoter primers in the transgene carrier transgenosis chrysanthemum Kan resistance strain of step (2) screening is carried out the PCR amplification, whether the checking carrier fragment inserts the chrysanthemum genome.
The above-mentioned method of utilizing kanamycin screening chrysanthemum transfer-gen plant, the regeneration culture medium described in the step (1) is: MS medium+6-BA 1.0 mgL
-1+ NAA 0.5 mgL
-1, described root media is: 1/2 MS medium, namely the macroelement in the MS medium reduces by half, other components unchanged.
According to the extent of injury of chrysanthemum leaf, the end reaction degree after can processing chrysanthemum leaf to Kan is divided into 4 grades: the I level: the leaf look normal, and the scab of being injured does not occur in the step (1); The II level: the flavescence of leaf look slight, the scab of being injured accounts for leaf area 0～5%; The III level: the flavescence of leaf look, be injured obviously, obvious with normal blade difference, the scab of being injured accounts for leaf area 5%～10%; The IV level: leaf chlorosis, brown scab large tracts of land occurs, and the scab of being injured accounts for leaf area more than 10%; Chrysanthemum leaf just occur obviously the being injured concentration for the treatment of of symptom (III order reaction) is critical screening concentration, and blade substantially constant processing time of progression of being injured is observing time.
Sequence according to the 35S promoter primers in the transgene carrier described in the step (3) is:
35S-F：5’-?AGATACAGTCTCAGAAGACCAAAGG-3’；
35S-R：5’-?TTGATATTCTTGGAGTAGACGAGAG-3’。
The screening principle:
Kanamycin (Kan) is the selected marker that is widely used in the present plant genetic engineering.The function of selected marker is under selection pressure transformant to be chosen.For reaching this purpose, to produce a kind of selection pressure selecting medium or select to add selective agent in the solution first, no transformed cells can not be grown; Next is that the product of selectable marker gene produces resistance to selective agent and makes transformant not be subjected to the impact of selective agent, normal growth, growth, differentiation, and transformant is screened.
Kan can screen in fact genetically modified plants and work by the Kan resistant gene.Kan resistance (Kan
r) gene is neomycin phosphotransferase gene (npt-II), also can be described as aminoglycoside phosphotransferase II gene (aph-II), it is from the aphA2 gene of Escherichia coli (E. coli), is present most widely used selectable marker gene in plant gene transforms.The product aminoglycoside phosphotransferase (APH (3 ') II-enzyme) of its coding has resistance to glucosides class antibiotic-kanamycin.This enzyme separates from bacterial transposon Tn5 the earliest and obtains, and its action principle is: npt-II gene outcome lost efficacy aminoglycoside antibiotics by Enzymatic Phosphorylation, and removed toxicity.Because Kan can disturb general plant cell chloroplast and mitochondrial protein to synthesize, cause the yellow of green plant organ, finally cause the death of plant cell.And genetically modified plants are owing to containing Kan resistance (npt-II), thereby have suppressed the effect of Kan, so the Kan transformant just is easy to screen from non-transformed body.
The detailed process of technical solution of the present invention is:
Determining of a, florists chrysanthemum leaf dish differentiation adventitious buds stage Kan screening concentration
Get the non-transgenic chrysanthemum aseptic seedling tender leaf of 25-30 d seedling age, be cut into 5 mm
2Square fritter (leaf dish) is inoculated into respectively that to contain Kan concentration be 0,5,7.5,10,15,20 mgL
-1Regeneration culture medium (MS medium+6-BA 1.0 mgL
-1+ NAA 0.5 mgL
-1) on.50 leaf dishes of every processing are added up leaf dish regeneration situation behind 15 d, determine the Kan screening concentration of leaf regeneration.
B, chrysanthemum stem section the determining of stage Kan screening concentration of taking root
Non-transgenic chrysanthemum aseptic seedling band top leaf stem section is inoculated into to contain Kan concentration be 0,5,7.5,10,15,20 mgL
-1Root media (1/2 MS medium) on, each processes 30 stem sections, adds up rooting rate behind 20 d, the stage Kan concentration of determining to take root.
Determining of c, Kan solution soaking and screening concentration
Get the 3rd of the consistent non-transgenic chrysanthemum of growing way full exhibition leaf, clean and be placed in the culture dish that fills a small amount of variable concentrations Kan solution, add solution amount and be as the criterion with submergence blade just, 0,50,100,200,300,500,700,1000 mgL are set
-1Totally 8 concentration gradients, each processes 30 blades.According to the extent of injury of chrysanthemum leaf, the end reaction degree after can processing chrysanthemum leaf to Kan is divided into 4 grades.The I level: the leaf look normal, and the scab of being injured does not occur; The II level: the flavescence of leaf look slight, the scab of being injured accounts for leaf area 0～5%; The III level: the flavescence of leaf look, be injured obviously, obvious with normal blade difference, the scab of being injured accounts for leaf area 5～10%; The IV level: leaf chlorosis, brown scab large tracts of land occurs, and the scab of being injured accounts for leaf area more than 10%.Observe the response situation of blade every day, to determine Kan solution soaking and screening concentration and observing time.
The acquisition of d, transgenosis chrysanthemum regeneration plant
The method of chrysanthemum genetic transformation is with reference to the method for He Junping (evaluation of Cut Flower Chrysanthemum Morifolium aphis resistance and Exploration of Mechanism and LLA transgenic research), the genetic transformation carrier that the transgenosis process is used is pBIG-CmPT1(Fig. 1), be built with npt-II plant Select gene in the carrier.Its middle period dish differentiation and regeneration stage Kan screening concentration and the stage Kan screening concentration of taking root are the concentration of determining among a, the b, after offspring to be generated grows to neat tissue culture bottle mouth, wait for next step detection to the land for growing field crops through acclimatization and transplants.
E, transgenosis chrysanthemum Kan solution soaking and screening and PCR checking
Soak transgenosis chrysanthemum leaf, statistics behind definite observation fate according to the Kan solution of determining concentration among the step c.The DNA of the transgenosis chrysanthemum plant that employing CTAB method extraction Kan solution soaking and screening goes out, the 35S promoter primers among the usefulness carrier pBIG-CmPT1 (35S-F:5 '-AGATACAGTCTCAGAAGACCAAAGG-3 '; 35S-R:5 '-TTGATATTCTTGGAGTAGACGAGAG-3 ') carry out the PCR amplification, amplification purpose fragment length is 325 bp, and whether the checking carrier fragment inserts the chrysanthemum genome.
Beneficial effect of the present invention:
1. the present invention is the cover research method for the screening of chrysanthemum transgenosis of present comparatively system, i.e. screening by four levels: the screening of leaf dish differentiation adventitious buds, the screening of taking root, Kan solution soaking and screening and PCR identify, and the method is simply effective, can effectively reject a large amount of false positive strains, filter out transgenic line.
2. propose first to utilize Kan solution to soak chrysanthemum leaf screening chrysanthemum transgenic line, the method is intuitive and reliable, and does not affect plant strain growth, and is significant for scale rapid screening, the quickening breeding process of realization chrysanthemum transfer-gen plant.
Description of drawings
The T-DNA plot structure schematic diagram of Fig. 1 chrysanthemum genetic transformation carrier pBIG-CmPT1
The PCR of Fig. 2 Kan resistance strain identifies
Note:M:DL2000; 1:H
2O is the blank of template; 2～7:Kan resistance strain; 8:Kan perception strain; 9: unconverted plant; 10: the positive control take plasmid pBIG-CmPT1 as template.
Embodiment
1, florists chrysanthemum leaf dish differentiation adventitious buds stage Kan screening concentration determines
Get non-transgenic Dendranthema morifolium Varieties ' refreshing horse ' the aseptic seedling tender leaf of 25-30 d seedling age, be cut into 5 mm
2Square fritter (leaf dish) is inoculated into respectively that to contain Kan concentration be 0,5,7.5,10,15,20 mgL
-1Regeneration culture medium (MS medium+6-BA 1.0 mgL
-1+ NAA 0.5 mgL
-1) on.50 leaf dishes of every processing are added up leaf dish regeneration situation behind 15 d, determine the Kan screening concentration of leaf regeneration.
The differentiation of ' refreshing horse ' leaf dish is relatively more responsive to Kan, and along with increasing progressively of Kan concentration, the regeneration capacity of ' refreshing horse ' leaf dish reduces rapidly, and on average each leaf disk regenerated adventitious bud digital display work reduces, and leaf dish extent of injury is constantly deepened.When the concentration of Kan is 10 mgL
-1, the regeneration capacity of ' refreshing horse ' leaf dish indefinite bud reduces to 0 from 100% of contrast, and the albefaction of leaf dish is also dead gradually, so 10 mgL
-1Can be used as the critical tolerance concentration (table 1) of ' refreshing horse ' genetic transformation screening resistance indefinite bud.
Table 1 Kan is on the impact of ' refreshing horse ' leaf dish differentiation adventitious buds
2, chrysanthemum stem section the determining of stage Kan screening concentration of taking root
Non-transgenic ' refreshing horse ' aseptic seedling band top leaf stem section is inoculated into to contain Kan concentration be 0,5,7.5,10,15,20 mgL
-1Root media (1/2 MS medium) on, each processes 30 stem sections, adds up rooting rate behind 20 d, the stage Kan concentration of determining to take root.
' refreshing horse ' stem section is along with the gradually rising of Kan concentration in the medium, and rooting rate and the quantity of on average taking root significantly descend, and are 7.5 mgL in Kan concentration
-1The time, the rooting rate of ' refreshing horse ' stem section reduces to 0 from 100% of contrast, and plant can not grow, and albefaction is dead gradually.Therefore, 7.5 mgL
-1Kan concentration can be used as the antibiotic critical concentration (table 2) that resistant buds uses when taking root screening and culturing.
The impact that table 2 Kan is taken root on ' refreshing horse ' stem section
3, Kan solution soaking and screening concentration determines
Get the 3rd full exhibition leaf of the consistent non-transgenic of growing way ' refreshing horse ' seedling, clean and be placed in the culture dish that fills a small amount of variable concentrations Kan solution, add solution amount and be as the criterion with submergence blade just, 0,50,100,200,300,500,700,1000 mgL are set
-1Totally 8 concentration gradients, each processes 30 blades.According to the extent of injury of chrysanthemum leaf, the end reaction degree after can processing chrysanthemum leaf to Kan is divided into 4 grades.The I level: the leaf look normal, and the scab of being injured does not occur; The II level: the flavescence of leaf look slight, the scab of being injured accounts for leaf area 0～5%; The III level: the flavescence of leaf look, be injured obviously, obvious with normal blade difference, the scab of being injured accounts for leaf area 5～10%; The IV level: leaf chlorosis, brown scab large tracts of land occurs, and the scab of being injured accounts for leaf area more than 10%.Observe the response situation of blade every day, to determine Kan solution soaking and screening concentration and observing time.
' refreshing horse ' blade table reveals certain Kan resistance, as screening concentration≤100 mgL
-1The time, along with the prolongation leaf color in reaction time is almost unchanged, mainly present the I order reaction; When screening concentration is 200,300 mgL
-1The time, although the scab of being injured appears in most of blade, little, the difficult identification of scab, partial blade there is no significant change, illustrates that these two concentration do not eliminate the individual difference of chrysanthemum leaf, if screen with these concentration, can increase the quantity of false positive individual plant, the effect that impact is selected; As kanamycin concentration 〉=700 mgL
-1The time, blade moves back green, and the scab large tracts of land of being injured occurs.And 500 mgL
-1Comparatively desirable as critical screening concentration, under this concentration, all the scab of being injured appears in blade, easily identification (table 3).Respectively organize as can be seen from Table 4 blade and substantially tend towards stability at processing 3d afterreaction progression, therefore be advisable with 3 d observing time.
Table 3 variable concentrations Kan is on the impact of ' refreshing horse ' blade
The order of reaction of ' refreshing horse ' blade after table 4 variable concentrations Kan processes
4, the acquisition of transgenosis chrysanthemum regeneration plant
The method of chrysanthemum genetic transformation is with reference to the method described in the He Junping (evaluation of Cut Flower Chrysanthemum Morifolium aphis resistance and Exploration of Mechanism and LLA transgenic research), the genetic transformation carrier that the transgenosis process is used is pBIG-CmPT1(Fig. 1), be built with npt-II plant Select gene in the carrier.Its middle period dish differentiation and regeneration stage Kan screening concentration is 10 mgL
-1, the stage Kan screening concentration of taking root is 7.5 mgL
-1, after the seedling of taking root that obtains grows to neat tissue culture bottle mouth, wait for next step detection to the land for growing field crops through acclimatization and transplants after screening with the stage Kan of taking root through the leaf dish differentiation and regeneration stage successively.
5, transgenosis chrysanthemum Kan solution soaking and screening and PCR the result
With successively through 65 strains of taking root obtaining after leaf dish differentiation and regeneration stage and the stage Kan screening of taking root through concentration 500 mgL
-1Kan solution soaking and screening after, it is Kan resistance strain that 6 strains are the I order reaction, 59 strains mainly are III order reaction (small part is the II order reaction) and are Kan perception strain.Adopt the CTAB method to extract the DNA of 6 Kan resistance strains, the 35S promoter primers among the usefulness carrier pBIG-CmPT1 (35S-F:5 '-AGATACAGTCTCAGAAGACCAAAGG-3 '; 35S-R:5 '-TTGATATTCTTGGAGTAGACGAG
AG-3 ') carry out the PCR amplification, amplification purpose fragment length is 325 bp, and whether the checking carrier fragment inserts the chrysanthemum genome.6 resistance strains detect through PCR and all are positive (Fig. 2), illustrate that target gene successfully is incorporated in the chrysanthemum genome.
6, the screening effect analysis that leaf dish differentiation adventitious buds, stem section are taken root and blade soaks
This embodiment transforms 2400 leaf dishes, obtains 358 of resistance indefinite buds after the Kan screening.358 resistance indefinite buds obtain 65 strains of taking root after Kan is taken root screening, eliminate 81.84% false positive plant.65 strains of taking root obtain 6 Kan resistance strains behind the Kan soaking and screening, eliminate 90.77% false positive plant.6 Kan resistance strain systems detect through PCR and all are positive, and degree of conformity is 100%.By screening and the evaluation of four-wheel, success filter out the chrysanthemum transgenic line, illustrate that the method can be applied to the chrysanthemum transgenosis and screen.
Claims (4)
1. method of utilizing kanamycin rapid screening chrysanthemum transfer-gen plant is characterized in that may further comprise the steps:
(1) determine that florists chrysanthemum leaf dish differentiation adventitious buds Kan screening concentration, chrysanthemum stem section take root Kan screening concentration and Kan solution soaking and screening concentration and observing time:
Determining of florists chrysanthemum leaf dish differentiation adventitious buds Kan screening concentration: non-transgenic florists chrysanthemum leaf dish is inoculated on the regeneration culture medium of the Kan that contains variable concentrations, find a Kan critical concentration that can just 100% suppresses florists chrysanthemum leaf dish differentiation adventitious buds, be florists chrysanthemum leaf dish differentiation adventitious buds Kan screening concentration;
Chrysanthemum stem section the determining of Kan screening concentration of taking root: non-transgenic chrysanthemum band top leaf stem section is inoculated on the root media of the Kan that contains variable concentrations, find one can just 100% to suppress the Kan critical concentration that chrysanthemum stem section is taken root, be the chrysanthemum stem section Kan screening concentration of taking root;
Determining of Kan solution soaking and screening concentration and observing time: the 3rd full exhibition leaf getting the consistent non-transgenic chrysanthemum of growing way, clean and be placed in the culture dish that fills a small amount of variable concentrations Kan solution, add solution amount and be as the criterion with submergence blade just, determine Kan screening concentration and observing time according to the blade victimization state;
(2) adopt the determined florists chrysanthemum leaf dish of step (1) differentiation adventitious buds Kan screening concentration, the chrysanthemum stem section Kan screening concentration of taking root that the screening that the chrysanthemum transformed plant carries out differentiation and regeneration stage and the stage of taking root is obtained the strain of taking root; And then Kan solution soaking and screening concentration and the observing time of adopting step (1) to determine obtain transgenosis chrysanthemum Kan resistance strain with the described strain of taking root behind Kan solution soaking and screening;
(3) PCR of transgenosis chrysanthemum checking: according to the 35S promoter primers in the transgene carrier transgenosis chrysanthemum Kan resistance strain of step (2) screening is carried out the PCR amplification, whether the checking carrier fragment inserts the chrysanthemum genome.
2. the method for utilizing kanamycin screening chrysanthemum transfer-gen plant according to claim 1 is characterized in that the regeneration culture medium described in the step (1) is: MS medium+6-BA 1.0 mgL
-1+ NAA 0.5 mgL
-1, described root media is: 1/2 MS medium, namely the macroelement in the MS medium reduces by half, other components unchanged.
3. the method for utilizing kanamycin screening chrysanthemum transfer-gen plant according to claim 1, it is characterized in that determining described in the step (1) that Kan solution soaking and screening concentration and the method for observing time are the extent of injury according to chrysanthemum leaf, end reaction degree after chrysanthemum leaf processed Kan is divided into 4 grades: the I level: the leaf look normal, and the scab of being injured does not occur; The II level: the flavescence of leaf look slight, the scab of being injured accounts for leaf area 0～5%; The III level: the flavescence of leaf look, be injured obviously, obvious with normal blade difference, the scab of being injured accounts for leaf area 5%～10%; The IV level: leaf chlorosis, brown scab large tracts of land occurs, and the scab of being injured accounts for leaf area more than 10%; The chrysanthemum leaf symptom that just occurs obviously being injured is that the concentration for the treatment of of III order reaction is critical screening concentration, and blade substantially constant processing time of progression of being injured is observing time.
4. the method for utilizing kanamycin screening chrysanthemum transfer-gen plant according to claim 1 is characterized in that the sequence according to the 35S promoter primers in the transgene carrier is described in the step (3):
35S-F：5’-?AGATACAGTCTCAGAAGACCAAAGG-3’；
35S-R：5’-?TTGATATTCTTGGAGTAGACGAGAG-3’。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104017823A (en) * | 2014-06-16 | 2014-09-03 | 山东省农业科学院生物技术研究中心 | Screening method for fast and effectively reducing false positive rate of peanut genetic transformation plant |
| CN105830763A (en) * | 2016-04-12 | 2016-08-10 | 扬州大学 | Application of hygromycin serving as selective agent in wheat transformation |
| CN108739348A (en) * | 2018-04-19 | 2018-11-06 | 贵州大学 | A kind of method of quick discriminating transgenic positive strain |
| CN112075343A (en) * | 2020-09-22 | 2020-12-15 | 云南中烟工业有限责任公司 | Method for simply, conveniently and effectively detecting existence of label in tobacco gene editing material |
| CN112106635A (en) * | 2020-09-22 | 2020-12-22 | 云南中烟工业有限责任公司 | Method for rapidly screening label-free gene editing tobacco material seeds by using kanamycin |
| CN112075343B (en) * | 2020-09-22 | 2022-07-01 | 云南中烟工业有限责任公司 | Method for simply, conveniently and effectively detecting existence of label in tobacco gene editing material |
| CN116024255A (en) * | 2022-07-11 | 2023-04-28 | 南京农业大学 | Method for improving chrysanthemum plant gene editing efficiency by utilizing adventitious bud in-vitro regeneration |
| CN116024255B (en) * | 2022-07-11 | 2024-03-19 | 南京农业大学 | Method for improving chrysanthemum plant gene editing efficiency by utilizing adventitious bud in-vitro regeneration |
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