CN105087641A - Novel agrobacterium mediated brassica pekinensis in-situ transgenosis method - Google Patents
Novel agrobacterium mediated brassica pekinensis in-situ transgenosis method Download PDFInfo
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Abstract
The invention discloses a novel agrobacterium mediated brassica pekinensis in-situ transgenosis method, wherein the method comprises the following steps: in the initial blooming period of brassica pekinensis, removing bloomed flowers and reserving flower buds; lightly opening the flower buds with a pair of tweezers by a small opening; completely soaking inflorescences in an agrobacterium dyeing liquid containing a gene to be transgenic for 2-4min; managing normally and collecting seeds; and conducting resistance identification and molecular detection on a transformation generation so as to obtain a transgenic positive plant. In accordance with the characteristic of the brassica pekinensis which is difficult in differentiation, the method disclosed by the invention can avoid an explant regeneration system, so as to simplify and shorten a transgenosis process, and the method can be used for directly obtaining transformed seeds without complex tissue culture, so as to omit a long process of artificial culture from an isolated protoplast to a regenerated plant; and the method, which is convenient, rapid and efficient in operation, low in cost and easy to master, has relatively high application value to the transgenosis research of brassica pekinensis genotype which is difficult to regenerate in tissue culture, and the method also has good application prospect in other crops which are difficult in plant regeneration transgenosis methods.
Description
Technical field
The present invention relates to genetically engineered field, particularly relate to agriculture bacillus mediated Chinese cabbage in-situ transesterification genetic method.
Background technology
1, Chinese cabbage (
brassicarapassp.pekinensis) be Cruciferae Brassica genus (
brassica) in one of most important vegetable crop, be the vegetables that China's cultivated area is maximum, distribution is the widest, people generally like, be called as " staple vegetable ".For a long time, China Chinese cabbage mainly carries out breed improvement by cross-breeding.But traditional cross breeding method, there is many drawbacks such as breeding year limit for length, cost is high, parental autocopulation vitality of many generations is easily degenerated, along with the foundation of tissue culture and DNA recombinant technology and constantly perfect, transgenic technology becomes improved agronomic traits, solves the important means of an agriculture production difficult problem.The In vitro transformation mainly contained by cell and tissue structrue technology with Agrobacterium-medialed transformation method at present infiltrates conversion, such as pollen tube passage method, vacuum-infiltration etc. with the original position of directly carrying out on plant.
At present, the method that Chinese cabbage genetic transformation the most often uses is still the agrobacterium-mediated transformation utilizing common tissue culture mode to carry out, the explants such as the cotyledon of the plant that namely will transform are as transformation receptor incubation growth on pre-culture medium, then immerse 3 ~ 5min in the Agrobacterium bacterium liquid of foreign gene-carrying, aseptic filter paper blots bacterium liquid.First, on Dual culture base, cultivated the regular hour by the explant contaminated, to guarantee that foreign gene is fully incorporated on karyomit(e); Secondly, be transferred to recovery media by the explant contaminated, the microbiotic (having penbritin, Pyocianil, Rifampin etc.) containing certain density bacteria growing inhibiting in recovery media; Finally, explant is transferred to containing the antibiotic Selective agar medium of finite concentration selectivity, screening resistant buds, root induction, and regeneration plant obtains transfer-gen plant through resistance molecule qualification.In recent years, Chinese cabbage transgenic research makes remarkable progress along with the foundation and constantly improving of its high frequency plant regeneration.Turnip mosaic virus resistance gene, modification cowpea trypsin inhibitor gene, antibacterial peptide gene, interleukin-1 gene, nitrate reductase gene, Galanthus nivalis agglutinin GNA etc. are led people Chinese cabbage genome with agrobacterium-mediated transformation by such as Zhu Chang Xiang etc., Yang Guangdong etc., Wang Guanlin etc., Wang Yang etc., Zhang Yanping etc., Yang Guangdong etc. respectively, obtain transfer-gen plant.
According to studying in recent years, the acceptor used with agriculture bacillus mediated Chinese cabbage genetic transformation is the cotyledon of aseptic seedling or hypocotyl mainly, Dou Shi somatic tissue, there are two cover karyomit(e)s, the possibility that foreign gene inserts homologous chromosomes same seat is simultaneously minimum, thus transformed plant is usually heterozygosis, and offspring can be separated.Cao Mingqing etc., using sporule as genetic transformation acceptor, obtain antiweed transfer-gen plant.Because sporule has possessed monoploid and unicellular two characteristics simultaneously, transformant can become the diploid of isozygotying after doubling.
But, because Chinese cabbage tissue culture difficulty is larger, the more difficult foundation of regeneration system, constrain the application of transgenic technology in Chinese cabbage breeding to a certain extent, therefore, seeking the method for transformation not relying on tissue culture approach, is the thing that many investigators being engaged in genetically engineered research pay special attention to.Pollen tube passage method is a kind of recipient cell using sexual cell as foreign DNA, by the sexual reproduction of flowering plant complexity, obtains the method for transfer-gen plant.Its principle makes foreign DNA can infiltrate along pollen tube after mainly pollinating, enter blastular through megarchidium passage, transforms the ovum, zygote or the body early embryo cell that still do not possess normal cell wall.According to statistics, Direct introduction exogenous DNA plant technology succeeds in succession on nearly 30 kinds of crops, example application on the cash crop such as paddy rice, wheat is many, and the application on vegetable breeding is lacked relatively, especially few to some vegetables little kind report.The people such as such as Wang Lei are by pollen tube passage method mediation il-4 gene transformation Chinese cabbage, and " breathing out white No. two " Chinese cabbage about 24h after florescence pollination transforms, and transformation efficiency reaches 1.15%.
It is the seed that Feldmann and Marks utilizes Agrobacterium to infect just to have sprouted that original position infiltrates that conversion method succeeds the earliest, has filtered out resistant strain, and verified by molecular hybridization from its progeny seed.And vacuum infiltration transformation method infiltrates the one transformed as original position, it enters the method for plant by Bechtold etc. by being used for auxiliary plant virus in plant pathology, be applied in the Agrobacterium gene transformation of Arabidopsis plant, its transformation frequency comparatively seed conversion improves 3 ~ 4 times.The method avoids the process of tissue culture, using the plant in growing as transformation receptor, directly screens resistant strain, for the transgenosis work of plant opens new approach from progeny seed.But regrettably current this method except being widely used in Arabidopis thaliana, only has the report of successful conversion in other plant in Chinese cabbage, clover.Chinese cabbage utilizes vacuum-infiltration to transform and obtains transgenosis Chinese cabbage by Cao Mingqing etc.Liu Fan etc. utilize agriculture bacillus mediated vacuum infiltration transformation method by potato proteinase inhibitor gene (
pinII) lead in people's Chinese cabbage " 49 cabbage heart ", obtain insect-resistance material.
Although Chinese cabbage transgenic technology obtains some progress, but because In vitro transformation is more difficult, the agriculture bacillus mediated in vitro transgenic method of Chinese cabbage is restricted, pollen tube passage method and vacuum-infiltration only test successfully on Chinese cabbage, have no report receiving on Chinese cabbage, and these methods also exist some drawbacks, in the urgent need to developing some more efficient, simple and easy, quick, reproducible technology in practice.
Plant in-situ transesterification genetic method (inplantatransformation) of the present invention is that one is easier, quick, reliable, and without the need to can obtain the new gene transformation method of a large amount of transformed plant through the tissue culture stage, its essential characteristics is just that it does not need tissue or cell cultures means, also reaches the conversion of plant at live body (invivo) under Non in vitro (vitro) state without the need to vacuumizing.At present, the application of this method is also only confined to Arabidopis thaliana, and present method is that the non-tissue culture method finding Brassica Crops and even crop in cruciferae High-efficient Genetic Transformation is offered reference, significant.
Summary of the invention
The object of this invention is to provide a kind of new Chinese cabbage in-situ transesterification gene approach, do not need tissue culture and complicated operation and directly obtain the method for transfer-gen plant, to overcome the deficiencies in the prior art.
The present invention is achieved in that
(1) preparing During Agrobacterium liquid: picking is containing needing genetically modified single bacterium colony from flat board, being inoculated into containing in the antibiotic YEB liquid nutrient medium (pH7.0) of screening, 28 DEG C, be cultured to OD under the condition of 225r/min
600=0.6 ~ 0.8.Getting 800 μ L bacterium liquid is diluted in the YEB liquid nutrient medium of 50mL antibiotic-free, is cultured to OD under the same terms
600when=0.6 ~ 0.8, the centrifugal 5min of 4000r/min, throw out 50mL liquid MS medium is resuspended by thalline.
(2) Chinese cabbage in-situ transesterification genetic method: Chinese cabbage, in field normal growth to blooming phase beginning, removes the flower opened, and retains bud, with tweezers gently by bud strip off osculum, inflorescence is all immersed in 2 ~ 4min in During Agrobacterium liquid.Normal management, seed of gathering.
(3) Resistance Identification of Chinese cabbage transformation generation: the Chinese cabbage seeds of results be seeded in containing on antibiotic substratum MS+1mg/LBA, selects the Molecular Detection that the seedling with resistance carries out transformed plant.
(4) Molecular Detection of Chinese cabbage transformed plant: gather the Chinese cabbage young leaflet tablet with resistance, extracts leaf DNA by the CTAB method of routine and carries out PCR detection.Get PCR reaction product 10ul, the sepharose with 1% carries out electrophoresis, observes as needed genetically modified band, then illustrates it is transgenic positive plant, transform successfully.
Owing to have employed technique scheme, compared with prior art, the present invention has 7 large advantages:
(1) for carrying the AA genome type not easily regenerated due to Chinese cabbage, this feature of Chinese cabbage difficulty differentiation, present method does not need the regeneration system rapidly of explant, this has larger using value to the genotypic transgenic research of Chinese cabbage that difficulty occurs indefinite bud during tissue culture, when the transgenic method that other crops relate to plant regeneration is met difficulty, also there is good application prospect.
(2) simplify and shorten transgenic breeding process, directly obtain transformed the seed, need not loaded down with trivial details tissue culture be carried out, avoid easy pollution in group training process and cause transforming unsuccessfully, also eliminate from vitro protoplasma to the very long artificial culture process of regeneration plant, also reduce genotypic impact.
(3) easy and simple to handle, quick, efficient, cost is low, be easy to grasp, without the need to plant and instrument and the pharmaceutical chemicals of costliness, researchist can directly operate in field.
(4) possess engineered advance, not only can import the STb gene of donor, may also be part DNA fragmentation or goal gene, the DNA of importing is easy to integrate, the proterties that controls by transgenosis in recipient plant, be easy to stable.
(5) in transgene expression vector builds, sometimes can omit antibiotic marker genes, thus in transgenic product, may not express containing microbiotic, security can be better.
(6) basic characteristics (can directly require to select transformation generation according to breeding) of conventional breeding are remained.
(7) in the prior art, never From Flower Bud of Chinese Cabbage is directly immersed in containing treating in genetically modified During Agrobacterium liquid both at home and abroad, converted in-situ under condition of living organism and directly obtain Chinese cabbage transformed the seed.
The present invention, compared with the agriculture bacillus mediated In vitro transformation method of prior art, pollen tube passage method and vacuum-infiltration, has following 3 different points:
(1) conventional need through following steps by agriculture bacillus mediated In vitro transformation method: one, select vegetable material to carry out sterile culture, cultivate the tissues such as cotyledon, hypocotyl, sporule or organ as transformation receptor (external piles is carried); Two, high frequency regenerating system is set up; Three, the determination of Material selec-tion pressure; Four, the activation of Agrobacterium; Five, the preculture of acceptor material; Six, infected material and Dual culture; Seven, micro-organisms; Eight, select to cultivate; Nine, transfer-gen plant qualification; Ten, the genetic stability analysis of transgenic progeny.The shortcoming of the method is:
chinese cabbage is a class crop of the most difficult life in Brassica genus, and because Chinese cabbage has AA genome, other have the genomic plant of BB with CC and compare with Brassica genus, its indefinite bud and plant regeneration more difficult;
chinese cabbage tissue culture and transformation tissue culture ability stronger to genotype-independent, its regeneration rate of different genotype and to transform complexity widely different, so for specific Chinese cabbage material, needs to carry out regenerating and the optimization of transformation system;
the differentiation of agrobacterium strains to explant of different antibacterial microbiotic and conversion has a certain impact;
some kind also exists regenerable cell and the inconsistent problem of transformed competence colibacillus cell area, causes escape body, mosaic, low conversion rate inferior;
easily produce anaphylaxis between some kind Agrobacteriums and infected cell, cause brownization and the necrosis of infection site;
the fertility likely producing somatic variation and transfer-gen plant in cell dedifferentiation and again differentiation reduces or loses, the analysis and application of this likely interference of transgene plant.
Transgenic protocol of the present invention is different from above-mentioned, directly From Flower Bud of Chinese Cabbage is immersed in containing treating in genetically modified During Agrobacterium liquid, directly obtain Chinese cabbage transformed the seed, do not need tissue culture procedures, simple, greatly save the time, shortened conversion process, and avoid the above-mentioned 6 large problems such as issuable somatic variation and genotype-independent in plant tissue culture course.
(2) pollen tube passage method, carry out artificial pollination flowering period in Chinese cabbage, after pollination, 24h transforms, namely column cap is cut away with double-edged razor blade, then drip containing needing genetically modified plasmid 5u1 by the incision of micropipet at style, make it form the drop of a parcel otch, after seed maturity, obtain transformed the seed.Pollen tube channel working method has: microinjection, column cap instillation, Ovary injection and pollen granule carry.The method shortcoming:
existing result of study shows, the transfer-gen plant foreign gene obtained by pollen tube passage method is inserted mainly with multiple copied, and the position of inserting also has randomness, after plasmid vector enters recipient cell, not necessarily be incorporated in the genome of Chinese cabbage, in exogenous origin gene integrator process, karyomit(e) there occurs the sudden change such as transposition, disappearance, causes transgenic progeny to show variation in phenotype, presents foreign DNA genetic instability.
the importing of foreign gene affects the survival ability of gamete sometimes, and gene may be inserted into the gene locus affecting Pollen Activity, the germinating power of transfer-gen plant pollen, pollen tube growth ability and fertility is reduced, causes transformation efficiency on the low side;
repeatability is poor, although the such as existing report utilizing pollen tube passage method successful conversion soybean, Shou reports, utilizes pollen tube passage method can not successful conversion soybean;
at present, this technology also exists the problems such as molecular biology evidence is abundant not, transformation frequency is low, the poor repeatability of transformation, particularly how naked DNA crosses cell-wall barriers, cytolemma selects permeability, resist the Degradation of the enzyme such as nuclease in histocyte, enter sexual cell, and be finally incorporated on karyomit(e), the mechanism of this serial procedures is not also elucidated.
The present invention transforms in Chinese cabbage flowering period, but method is completely different, do not need artificial pollination, also without the need to cutting away column cap, do not need the processes such as injection, instillation and pollen granule carry, but directly containing needing genetically modified Agrobacterium solution dip-dye inflorescence, damage will not caused to plant, avoid the above-mentioned drawback of pollen tube passage method, and simply, fast, efficiently.
(3) vacuum-infiltration, its method is by containing treating that the inorganic+B5 organism of genetically modified agrobacterium tumefaciens I/2MS infiltrates substratum dilution, bacterium liquid OD
600=O.8 time put into the container vacuumized, be inverted by plant, bud position is dipped in be infiltrated in substratum, vacuumizes, and the vacuum time of infiltrating is 2min/ interval 30s/30s, then recovers normal pressure.The shortcoming of the method is:
plant after vacuum-treat is in a kind of state of extremely feeble, needs first to be positioned in high humidity environment to carry out restoration ecosystem, under being then just transplanted to normal growing conditions, and results seed;
vacuum-infiltration needs vacuum unit, after vacuum-treat plant choose and again cultivation add workload;
changing effect oozes the impact of the many factors such as people's time, inoculum optical density(OD) by plant development period, vacuum.
The present invention is the flower-dipping method directly contaminating bud with the Agrobacterium bacterium liquid containing tensio-active agent and sucrose, eliminates vacuum treated equipment and step, also therefore avoids the drawback of vacuum-infiltration; With the tensio-active agent Silwet of 5% sucrose, 500 μ L/L, OD
600the dip-dye immersion libation at an ancient wedding ceremony Chinese cabbage inflorescence 2 ~ 4min of=0.6 ~ 0.8, normal growth, obtains transgenic seed.This method for transformation is more convenient, quick, efficient, reliable, does not need group training and directly obtains transfer-gen plant, being with a wide range of applications in the genetic transformation of cress.
Accompanying drawing explanation
Fig. 1: anti-Kan gene in Chinese cabbage transfer-gen plant
nptIIpCR detected result: detect anti-Kan gene with primer P144/P145
nptII(1118bp).M:MakerDL2000; 1: positive control (plasmid); 2: negative control (water); 3 ~ 7: transgenosis breeds of Chinese cabbage zui4-4 '; 8 ~ 9: transgenosis breeds of Chinese cabbage zui4-2 '; 10: transgenosis breeds of Chinese cabbage zui4-3 '; 11 ~ 12: transgenosis breeds of Chinese cabbage zui5-2 '; 13 ~ 14: transgenosis breeds of Chinese cabbage zui4-3 '.
Fig. 2: in Chinese cabbage transfer-gen plant
cBF3the PCR detected result of gene: detect with primer P205/P264
cBF3gene (967bp).1 ~ 11: transgenosis Chinese cabbage cultivar zui4-4 '; 2 CK-: negative control (being followed successively by water and WT lines); CK+: positive control, containing the Agrobacterium plasmid of pCBF3.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Embodiment 1:
1. materials and methods
1.1 experiment materials:
Vegetable material: the Chinese cabbage cultivar for examination provides for " Guizhou Province is white " serial Chinese cabbage zui4, zui5, b26, gh2, sd4(Guizhou Horticultural Research Institute).
Bacterial classification: containing Cold resistant genes
cBF3expression vector pCBF3 plasmid and Agrobacterium tumefaciens strain LBA4404, be so kind as to give by institute vegetables laboratory, Southwestern University's gardening gardens.
Main agents and primer: pcr amplification
cBF3gene the primer is: 5 ' primer p50 in P-RD29A promotor (5 '-CTGCAAGAATCTCAAACACG-3 ') and be positioned at
cBF33 ' end primer p49 on gene (5 '-GTACTAAAAATGGAAATAATAATCTGAG-3 ').Increase anti-Kan gene
npt IIthe primer is P144B (GAATTCAGGGAGTCACGTTATGACC) and P145(CGCTCGAGTCCCGCTCAGAAGAAC).
1.2 experimental techniques:
The cultivation of Agrobacterium: picking is containing needing single bacterium colony of expression vector pCBF3 of transgenosis CBF3 from flat board, be inoculated in the YEB liquid nutrient medium (pH7.0) containing Kan50mg/L, Streptomycin sulphate 100mg/L, Rifampin 50mg/L, 28 DEG C, be cultured to OD under the condition of 225r/min
600=0.6 ~ 0.8.Getting 800 μ L bacterium liquid is diluted in the YEB liquid nutrient medium of 50mL antibiotic-free, is cultured to OD under the same terms
600when=0.6 ~ 0.8, the centrifugal 5min of 4000r/min, throw out 50mL liquid MS medium is resuspended by thalline.
Chinese cabbage in-situ transesterification genetic method: " Guizhou Province is white " serial Chinese cabbage, in field normal growth to blooming phase beginning, removes the flower opened, and retains bud, with tweezers gently by bud strip off osculum, inflorescence is all immersed in 2 ~ 4min in During Agrobacterium liquid.Normal management, seed of gathering.
The Kan Resistance Identification of Chinese cabbage transformation generation: by the planting seed of the transformed plant of results on substratum MS+1mg/LBA+5mg/LKan, select the Molecular Detection that the seedling with Kan resistance carries out transformed plant.
The Molecular Detection of Chinese cabbage transformed plant: gather the Chinese cabbage young leaflet tablet with Kan resistance, extracts leaf DNA by the CTAB method of routine and carries out PCR detection.Reaction system is: each 2ul of 10xBuffer5ul, MgCL23ul, dNTP4ul, primer, DNA1.5ul, Taq enzyme 2.8ul, add sterilized water to 50ul.Negative control is water, and positive control is the plasmid containing treating transgene expression vector.Amplification program is 94 DEG C of sex change 3min, then successively 94 DEG C of sex change 30s, 54 DEG C of renaturation 30s, 72 DEG C extend 1.5min, circulate 3 times; 94 DEG C of 30s, 59 DEG C of 30s, 72 DEG C of 1.5min successively again, circulate 32 times, and 72 DEG C extend 10min, 4 DEG C of preservations.Get PCR reaction product 10ul, the sepharose with 1% carries out electrophoresis, observes and whether contains goal gene band.
2 results
Carry out PCR detection with different primers to Chinese cabbage transformed plant, result has 21 strains to be positive in 88 strain offsprings, and transformation efficiency reaches 26 ~ 57%(in table 1).Part amplification is as Fig. 1 and Fig. 2: be have 10 transformed plants to have an amplified band (Fig. 1) at 1118bp place in the PCR primer of primer with P144/P145, show Kan resistant gene
nptIIbe transferred in Chinese cabbage genome; Be that in the PCR primer of primer, 2 plant have an amplified band (Fig. 2) at 967bp place with P205/P264, show Cold resistant genes
cBF3be transferred in Chinese cabbage genome.More than test proves by in-situ transesterification genetic method, successfully will treat that transgenosis proceeds to in examination Chinese cabbage " Guizhou Province is white " serial Chinese cabbage.
Certainly, the embody rule example of more than just invention, the technical scheme that the present invention also has other embodiment, all employings to be equal to replacement or equivalent transformation to be formed, all drops within protection domain of the presently claimed invention.
sequence table
SEQUENCELISTING
<110> Guizhou Horticultural Research Institute
The Chinese cabbage in-situ transesterification genetic method that <120> is agriculture bacillus mediated
<130>2015
<160>4
<170>PatentInversion3.3
<210>1
<211>20
<212>DNA
<213> artificial sequence
<220>
<222>(1)...(20)
5 ' primer p50 in <223>P-RD29A promotor, for pcr amplification
cBF3gene
<400>1
ctgcaagaatctcaaacacg20
<210>2
<211>20
<212>DNA
<213> artificial sequence
<220>
<222>(1)...(28)
<223> is positioned at
cBF33 ' end primer p49 on gene, for pcr amplification
cBF3gene
<400>2
gtactaaaaatggaaataataatctgag28
<210>3
<211>27
<212>DNA
<213> artificial sequence
<220>
<222>(1)...(25)
<223>P144B, for the anti-Kan gene that increases
npt II
<400>3
gaattcagggagtcacgttatgacc25
<210>4
<211>26
<212>DNA
<213> artificial sequence
<220>
<222>(1)...(24)
<223>P145, for the anti-Kan gene that increases
npt II
<400>4
cgctcgagtcccgctcagaagaac24
Claims (1)
1. a new agriculture bacillus mediated Chinese cabbage in-situ transesterification genetic method, is characterized in that:
(1) During Agrobacterium liquid is prepared: picking, containing needing genetically modified single bacterium colony, is inoculated into containing in the antibiotic YEB liquid nutrient medium of screening, is cultured to OD
600=0.6 ~ 0.8; Get bacterium liquid to be diluted in the YEB liquid nutrient medium of antibiotic-free, be cultured to OD
600when=0.6 ~ 0.8, centrifugal treating, throw out liquid MS medium is resuspended by thalline;
(2) Chinese cabbage in-situ transesterification genetic method: Chinese cabbage, in field normal growth to blooming phase beginning, removes the flower opened, and retains bud, with tweezers gently by bud strip off osculum, inflorescence is all immersed in 2 ~ 4min in above-mentioned During Agrobacterium liquid; Normal management, seed of gathering;
(3) Resistance Identification of Chinese cabbage transformation generation: the Chinese cabbage seeds of results be seeded in containing on antibiotic substratum MS+1mg/LBA, selects the Molecular Detection that the seedling with resistance carries out transformed plant;
(4) Molecular Detection of Chinese cabbage transformed plant: gather the Chinese cabbage young leaflet tablet with resistance, extracts leaf DNA by the CTAB method of routine and carries out PCR detection; Get PCR reaction product, the sepharose with 1% carries out electrophoresis, as needed genetically modified band, then illustrates it is transgenic positive plant, transforms successfully.
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Cited By (9)
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| CN107849582A (en) * | 2015-05-19 | 2018-03-27 | Kws种子欧洲股份公司 | For the In Planta transformation method and manufacturing process of plant and based on it and therefrom obtainable product |
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| CN109566402A (en) * | 2019-01-14 | 2019-04-05 | 安徽农业大学 | A kind of Fast synchronization obtains B. campestris L.ssp. Chinensis male sterility and holding is transgenic plant method |
| CN109566402B (en) * | 2019-01-14 | 2021-09-14 | 安徽农业大学 | Method for rapidly and synchronously obtaining male sterile and maintainer line transgenic plants of black-bone dish |
| CN109652455A (en) * | 2019-02-19 | 2019-04-19 | 南京农业大学 | The Chinese cabbage high-efficiency genetic transforming method and its application that a kind of magnetic nano-carrier mediates |
| CN111647620A (en) * | 2020-06-10 | 2020-09-11 | 北京市农林科学院 | Method for creating non-transgenic mutant strain of flowering Chinese cabbage |
| CN111893133A (en) * | 2020-07-22 | 2020-11-06 | 华南农业大学 | Agrobacterium-mediated cabbage heart genetic transformation method |
| CN115232833A (en) * | 2022-09-01 | 2022-10-25 | 深圳大学 | Agrobacterium-mediated efficient genetic transformation method for brassica crops |
| CN115232833B (en) * | 2022-09-01 | 2023-09-12 | 深圳大学 | Agrobacterium-mediated brassica crop efficient genetic transformation method |
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