CN113462719A - Agrobacterium-mediated cotton genetic transformation method - Google Patents
Agrobacterium-mediated cotton genetic transformation method Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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Abstract
The invention provides a cotton genetic transformation method, which comprises the steps of taking a shoot apical meristem at the lower part of a leaf primordium stripped from soaked cotton mature seeds as a genetic transformation receptor, infecting the genetic transformation receptor by recombinant agrobacterium tumefaciens containing target DNA under the condition of ultrasonic treatment to obtain an infected explant, and culturing the infected explant to obtain a transgenic cotton plant. The invention has the advantages of simple operation, short growth cycle, good repeatability, small restriction of receptor genotype, unified and simple transformation mode, high transformation efficiency, good genetic stability of the obtained transgenic cotton plant and the like.
Description
Technical Field
The invention relates to a cotton genetic transformation method in the field of biotechnology, in particular to an agrobacterium-mediated cotton genetic transformation method.
Background
Cotton is one of the most important natural fiber sources and commercial crops in the world. The completion of cotton whole genome sequencing enables the confirmation and the separation of a large number of genes to be faster, simpler and more convenient, and the emerging genome editing technology provides a simple and accurate gene improvement means and becomes a subversive technical breakthrough in the field of life science. The verification of gene function requires that the genes are transformed into cotton to verify the practicability of candidate genes. At present, the conventional cotton genetic transformation method obtains a transgenic plant through a tissue culture way after gene gun bombardment and agrobacterium infection, and the process usually needs about 10 to 11 months, thereby not only wasting time and labor, but also being only suitable for a few cotton varieties. And gene editing requires a mature, stable and efficient regeneration system. However, the cotton varieties popularized and planted at present are severely limited by genotypes, and basically, agrobacterium-mediated genetic transformation cannot be carried out; even if the cotton varieties can be genetically transformed, the problems of long transformation period, high somatic embryo aberration rate, small number of normal regenerated seedlings and the like exist, most of cotton varieties with important economic values have large genotype difference and poor regeneration performance and are still difficult to transform, and therefore, the establishment of the cotton genetic transformation method with low dependence degree of transformation receptor genotypes has great significance.
Disclosure of Invention
Aiming at the current situation that the dependence degree of the genotype of a transformation receptor is higher in the transgenic process of cotton at present, the invention aims to provide a cotton genetic transformation method with lower dependence degree of the genotype of the transformation receptor.
The method for cotton genetic transformation comprises the steps of taking a shoot apical meristem at the lower part of a leaf primordium stripped from soaked cotton mature seeds as a genetic transformation receptor, infecting the genetic transformation receptor by recombinant agrobacterium tumefaciens containing target DNA under the condition of ultrasonic treatment to obtain an infected explant, and culturing the infected explant to obtain a transgenic cotton plant.
In the above method, culturing the infected explant does not include callus proliferation, embryogenic callus induction, callus subculture, callus screening, shoot induction and/or root induction.
In the method, the ultrasonic treatment is carried out at the frequency of 40-100kHz and the time length of 40-80 s. Preferably, the frequency is 40kHz for a duration of 40 s.
In the method, the soaking is to soak the cotton mature seeds with MSB liquid culture medium for 18-24 hours.
Wherein the MSB liquid culture medium has a pH value of 5.6 and comprises the following components: the mixture concentration of MS salt and B5 vitamin is 4.4g/L, the glucose concentration is 20g/L, the calcium gluconate concentration is 1.29g/L, and the rest is water; the mixture of the MS salt and the vitamin B5 consists of the following raw materials in parts by weight: 1900 parts of potassium nitrate, 1650 parts of ammonium nitrate, 170 parts of monopotassium phosphate, 598 parts of calcium nitrate dihydrate, 181 parts of anhydrous magnesium sulfate, 27.85 parts of ferrous sulfate heptahydrate, 37.3 parts of EDTA-na, 17.1 parts of manganese sulfate monohydrate, 0.83 part of potassium iodide, 6.2 parts of boric acid, 0.25 part of sodium molybdate dihydrate, 0.02 part of modified cobalt, 0.025 part of copper sulfate pentahydrate, 100 parts of inositol, 2 parts of glycine, 10.1 parts of VB, 60.5 parts of VB and 30.5 parts of VB.
In the method, the infection is carried out in an infection culture medium containing acetosyringone.
Wherein the pH value of the infection culture medium is 5.4, and the composition is as follows: the concentration of CA mother liquor is 10ml/L, the concentration of glucose is 30g/L, the concentration of MES is 4.2g/L, the concentration of B5 vitamin is 0.1ml/L, the concentration of 6-BA is 1mg/L, the concentration of NAA is 0.1mg/L, the concentration of acetosyringone is 0.2mM, and the balance is water; the CA mother liquor comprises the following components: 10g/L magnesium sulfate, 5.36g/L ammonium sulfate, 6g/L sodium phosphate monohydrate, 6g/L calcium chloride, 30mg/L boric acid, 100mg/L manganese sulfate, 20mg/L zinc sulfate heptahydrate, 7.5mg/L potassium iodide, 2.5mg/L sodium molybdate dihydrate, 250 mu g/L copper sulfate, 250 mu g/L cobalt chloride hexahydrate, 100g/L potassium nitrate and the balance of water.
In the above method, the recombinant agrobacterium tumefaciens contains a spectinomycin resistance gene (which may be aada gene, and the nucleotide sequence of which is shown as sequence 2 in the sequence table), and the method further comprises a step of screening the infected explants with spectinomycin.
In the above method, the culturing the infected explant comprises the following steps:
transferring the infected explants to a co-culture medium, and co-culturing for 3-4d under a dark condition;
transferring the explants after co-culture into a recovery culture medium for recovery culture;
cutting off the root of the explant after the recovery culture, and transferring a human bud induction culture medium for screening induction;
and transferring the screened and induced explants to an elongation culture medium for elongation culture to obtain transformed plants.
Wherein the pH value of the bud induction culture medium is 5.6, the composition is that the concentration of a mixture of MS salt and B5 vitamin is 4.4g/L, the concentration of glucose is 20g/L, the concentration of calcium gluconate is 1.29g/L, the concentration of 6-BA is 1mg/L, the concentration of NAA is 0.1mg/L, the concentration of carbenicillin is 100mg/L, the concentration of cephamycin is 100mg/L, the concentration of spectinomycin is 100mg/L, 4g/L agar (can be replaced by other coagulants with proper amount), and the balance is water;
the pH of the elongation medium was 5.6 and consisted of: the mixture concentration of MS salt and B5 vitamin is 4.4g/L, the glucose concentration is 20g/L, the calcium gluconate concentration is 1.29g/L, the carbenicillin concentration is 100mg/L, the cephamycin concentration is 100mg/L, the spectinomycin concentration is 100mg/L, 4g/L agar (can be replaced by other coagulants with proper amount), and the balance is water;
the pH value of the co-culture medium is 5.4, and the co-culture medium comprises the following components: the CA mother liquor concentration is 10ml/L, the glucose concentration is 30g/L, the MES concentration is 4.2g/L, the vitamin B5 concentration is 0.1ml/L, the 6-BA concentration is 1mg/L, the NAA concentration is 0.1mg/L, the acetosyringone concentration is 0.2mM, the cysteine concentration is 200mg/ml, and the balance is water;
the recovery medium has a pH value of 5.6 and comprises the following components: the mixture concentration of MS salt and B5 vitamin was 4.4g/L, glucose concentration was 20g/L, calcium gluconate concentration was 1.29g/L, 6-BA concentration was 1mg/L, NAA concentration was 0.1mg/L, carbenicillin concentration was 100mg/L, cephamycin concentration was 100mg/L, 4g/L agar (which could be replaced with an appropriate amount of other coagulant), and the balance was water.
In the above method, the cotton may be upland cotton or sea island cotton. The upland cotton can be selected from any one of Zhongmiao 49, Zhongmiao 88, Zhongmiao 59, Baimiao No. 1 and TM-1. The sea island cotton may be selected from any one of the new sea 43 and the new sea 56.
The invention also provides application of the cotton genetic transformation method in cotton breeding.
The invention takes cotton mature seeds as materials, after soaking, peels off seed coats and cotyledons, exposes stem tip meristem, takes the stem tip meristem as a receptor, utilizes agrobacterium mediation, and adopts ultrasonic treatment to introduce exogenous genes into cotton genome, thereby obtaining transgenic cotton. The invention breaks through the restriction of genotype, can efficiently transform the stropanthus type upland cotton/sea island cotton variety in a shorter time, and solves the bottleneck problem of cotton genetic transformation. The invention does not need tissue culture, has low influence degree by the genotype, can quickly obtain transgenic cotton plants and has the following advantages: simple operation, short growth period (only 88 days are needed from seed soaking to transgenic plant obtaining), good repeatability, small restriction of receptor genotype, unified and simple transformation mode, high transformation efficiency (the transformation efficiency can reach 2.41-9.22%), and good genetic stability of the obtained transgenic cotton plant.
Drawings
FIG. 1 is a schematic diagram of a cotton stem tip transformation system in example 1 of the present invention. Wherein, the upper diagram of figure 1 is a flow chart of a high-efficiency cotton stem tip transformation system: taking the cotton embryo tip as an explant, and carrying out ultrasonic transformation. The treated explants produce adventitious buds containing the exogenous gene on a medium supplemented with a selection antibiotic, which in turn roots and regenerates transgenic plants. The lower graph of FIG. 1 is a photograph of the various stages of rapidly obtaining transgenic plants using shoot tip transformation techniques: in the figure, I is aseptically processed seeds; II, removing the stem tip meristem exposed by two cotyledons; III, treating the mixture of agrobacterium tumefaciens containing GFP genes and embryo tips by ultrasonic waves; IV, co-culturing the embryo tips after the dip dyeing treatment; v is GFP fluorescence detection after co-culture; VI, recovery culture; VII, screening and culturing; VIII is the appearance of spectinomycin resistant adventitious buds; IX is a transgenic bud; x is transgenic seedling.
FIG. 2 is a graph showing the results of detecting GFP in leaves, ovules, anthers and roots of transgenic positive cotton of the T0 generation in example 1 of the present invention.
FIG. 3 is a graph showing the results of stable inheritance of transgenic material in example 1 of the present invention. FIG. 3, Panel A, is a graph showing the results of detecting GFP in the stem (I), leaf (II), anther (III), petal (IV), 10d ovule and fiber (V), 20d ovule and fiber (VI) of transgenic cotton plants and wild type controls of generation T1, wherein GFP is the transgenic cotton plant and WT is the wild type control (Zhongmiao 49). FIG. 3B is a graph of the result of GFP detection in T2 generation seeds harvested from hybrid T1 generation transgenic cotton plants. FIG. 3C is a graph showing the result of GFP detection of T2 generation seeds harvested from a homozygous transgenic cotton plant.
FIG. 4 is a schematic diagram showing a comparative flow between the shoot tip transformation system and the somatic embryogenesis system in example 1 of the present invention.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.
The quantitative tests in the following examples, unless otherwise specified, were set up in triplicate and the results averaged.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The cotton institute 49 in the upland cotton variety in the following examples is a variety bred by the cotton research institute of the academy of agricultural sciences of china, and the approval number is national cotton 2004003, which can be purchased from the scientific and trade company of the gossypium hirsutum.
The cotton institute 88 in the upland cotton variety in the following examples is a variety bred by the cotton research institute of the academy of agricultural sciences of china, and is certified cotton 2013003, which can be purchased from the scientific trade company of the gossypium hirsutum.
The cotton institute 59 in the following examples is a variety selected by the cotton research institute of the academy of agricultural sciences of china, and can be purchased from the cotton institute of china kohami, and its approved number is zhe ju cotton 2007003.
The cotton variety "Bai Cotton" No. 1 in the examples below was examined for Country cotton 2009003, and was purchased from the Korea company of Mitsubishi.
The upland cotton variety TM-1 in the examples below was publicly available from the Cotton research institute of Chinese Agronomy academy of sciences.
The sea island cotton varieties Xinhai 43 and Xinhai 56 in the following examples are all products of Xinjiang Cotton seed industry Co.
In the following examples, the preparation method of 100mg/mL cefuroxime axetil solution is as follows (taking 100mL as an example): dissolving 10g of cefuroxime in 100ml of sterilized water, fully dissolving, filtering with 0.22um filter membrane for sterilization, subpackaging 1ml, and storing at-20 ℃.
In the following examples, a100 mg/mL carbenicillin solution was prepared (taking 100mL as an example): dissolving 10g carbenicillin in 100ml sterilized water, dissolving thoroughly, filtering with 0.22um filter membrane for sterilization, packaging into 1ml, and storing at-20 deg.C.
In the following examples, a100 mg/mL spectinomycin solution was prepared by the following method (taking 100mL as an example): dissolving 10g spectinomycin in 100ml sterilized water, dissolving completely, filtering with 0.22um filter membrane for sterilization, packaging into 1ml, and storing at-20 deg.C.
In the following examples, the preparation method of 100mM acetosyringone solution is as follows (taking 100mL as an example): 1.962g Acetosyringone (AS) was dissolved in 100ml dimethyl sulfoxide (DMSO), dissolved thoroughly, sterilized by filtration, and stored at-20 ℃.
In the following examples, the composition of the CA mother liquor is: 10g/L magnesium sulfate, 5.36g/L ammonium sulfate, 6g/L sodium phosphate monohydrate, 6g/L calcium chloride, 30mg/L boric acid, 100mg/L manganese sulfate, 20mg/L zinc sulfate heptahydrate, 7.5mg/L potassium iodide, 2.5mg/L sodium molybdate dihydrate, 250 mu g/L copper sulfate, 250 mu g/L cobalt chloride hexahydrate, 100g/L potassium nitrate and the balance of water. The preparation method of the CA mother solution comprises the following steps: 10g of magnesium sulfate, 5.36g of ammonium sulfate, 6g of sodium phosphate monohydrate, 6g of calcium chloride, 30mg of boric acid, 100mg of manganese sulfate, 20mg of zinc sulfate heptahydrate, 7.5mg of potassium iodide, 2.5mg of sodium molybdate dihydrate, 250. mu.g of copper sulfate, 250. mu.g of cobalt chloride hexahydrate and 100g of potassium nitrate were dissolved in dd water or ultrapure water to a constant volume of 1L.
In the following examples, the mixture of MS salts and B5 vitamins consisted of the following raw materials in parts by weight: 1900 parts of potassium nitrate, 1650 parts of ammonium nitrate, 170 parts of monopotassium phosphate, 598 parts of calcium nitrate dihydrate, 181 parts of anhydrous magnesium sulfate, 27.85 parts of ferrous sulfate heptahydrate, 37.3 parts of EDTA-na, 17.1 parts of manganese sulfate monohydrate, 0.83 part of potassium iodide, 6.2 parts of boric acid, 0.25 part of sodium molybdate dihydrate, 0.02 part of modified cobalt, 0.025 part of copper sulfate pentahydrate, 100 parts of inositol, 2 parts of glycine, 10.1 parts of VB, 60.5 parts of VB and 30.5 parts of VB.
In the following examples, the composition of the MSB liquid medium was: 4.4g/L of a mixture of MS salts and B5 vitamins (product of PhytoTech, Inc., type M404), 20g/L glucose, 1.29g/L calcium gluconate, pH 5.6, and balance water. The MSB liquid culture medium can be prepared by the following steps: 4.4g of a mixture of MS salt and vitamin B5 (M404), 20g of glucose and 1.29g of calcium gluconate are dissolved in deionized water to a constant volume of 1L, the pH value is adjusted to 5.6, and the mixture is sterilized at 121 ℃ for 20 min.
In the following examples, the composition of YP medium was: 5g/L NaCl, 5g/L yeast extract (yeast extract, product of OXOID Bio Inc., Cat. LP0021), 10g/L Tryptone (Tryptone, product of OXOID Bio Inc., Cat. LP0042), 15g/L Agar (Agar, product of Bio Inc., Cat. A100637), 0.2mM Acetosyringone (AS), and the balance water. The YP culture medium can be prepared by the following steps: 5g NaCl, 5g yeast extract, 10g tryptone, 15g agar, and 2ml 100mM acetosyringone solution, dissolving in deionized water to constant volume of 1L, and sterilizing at 121 deg.C for 20 min.
In the following examples, YP medium containing kanamycin and rifampicin was composed: 5g/L NaCl, 5g/L yeast extract (yeast extract), 10g/L Tryptone (Tryptone), 15g/L Agar (Agar), 0.2mM Acetosyringone (AS), kanamycin (Kan)50mg/L, rifampicin (rif)15mg/L, and the balance water. The YP culture medium can be prepared by the following steps: 5g NaCl, 5g yeast extract, 10g tryptone, 15g agar, and 2mL 100mM acetosyringone solution, dissolving in deionized water to constant volume of 1L, sterilizing at 121 deg.C for 20min, adding 1mL 50mg/mL kanamycin (Kan) and 0.3mL 50mg/mL rifampicin (rif) in a clean bench when the temperature of the culture medium is reduced to 50 deg.C, and mixing.
In the following examples, the composition of the infection medium (CAB) was: 10ml/L CA stock solution, 30G/L glucose, 4.2G/L MES (Sigma, cat # V900336), 0.1ml/L B5 vitamin (PhytoTech, West America, cat # G219), 1 mg/L6-BA, 0.1mg/L NAA, 0.2mM Acetosyringone (AS), pH 5.4, and the balance water. The preparation method of the infection culture medium can be as follows: 10ml of CA mother liquor, 30G of glucose, 4.2G of MES, 0.1ml of B5 vitamin (G219), 1mg of 6-BA, 0.1mg of NAA and 2ml of 100mM acetosyringone solution are dissolved in deionized water to be constant volume to 1L, the pH value is adjusted to be 5.4, and the solution is sterilized at 121 ℃ for 20 min.
In the following examples, the composition of the co-culture medium (CCM) was: 10ml/L CA stock solution, 30G/L glucose, 4.2G/L MES, 0.1ml/L B5 vitamin (G219), 1 mg/L6-BA, 0.1mg/L NAA, 0.2mM acetosyringone, 200mg/ml Cysteine (CYS), pH 5.4, and the balance water. The preparation method of the co-culture medium can be as follows: 10ml of CA mother liquor, 30G of glucose, 4.2G of MES, 0.1ml of B5 vitamin (G219), 1mg of 6-BA, 0.1mg of NAA, 2ml of 100mM acetosyringone solution and 4ml of 50mg/ml Cysteine (CYS) aqueous solution are dissolved in deionized water to be constant volume of 1L, the pH value is adjusted to be 5.4, and the mixture is sterilized at 121 ℃ for 20 min.
In the following examples, the composition of the recovery medium (R0) was: 4.4g/L mixture of MS salts and B5 vitamins (model M404), 20g/L glucose, 1.29g/L calcium gluconate, 4g/L Agar (Agar), 1 mg/L6-BA, 0.1mg/L NAA, 100mg/L carbenicillin, 100mg/L cephamycin, pH 5.6, and the balance water. The recovery medium (R0) may be prepared by: 4.4g MS salt and B5 vitamin mixture (M404), 20g glucose, 1.29g calcium gluconate, 4g agar, 1mg 6-BA, 0.1mg NAA, 100mg/ml carbenicillin solution 1ml, 100mg/ml cefuroxime 1ml, dissolved in deionized water to volume of 1L, adjusting pH to 5.6, 121 ℃ sterilization for 20 min.
In the following examples, the composition of the shoot induction medium (R1) was: 4.4g/L of a mixture of MS salts and B5 vitamins (model M404), 20g/L glucose, 1.29g/L calcium gluconate, 4g/L Agar (Agar), 1 mg/L6-BA, 0.1mg/L NAA, 100mg/L carbenicillin, 100mg/L cephamycin, 100mg/L spectinomycin, pH 5.6, and the balance water. The bud induction medium (R1) can be prepared by the following method: 4.4g of a mixture of MS salt and B5 vitamin (M404), 20g of glucose, 1.29g of calcium gluconate, 4g of agar, 1mg of 6-BA, 0.1mg of NAA, 1ml of a carbenicillin solution 100mg/ml, 1ml of a cephamycin solution 100mg/ml and 1ml of a spectinomycin solution 100mg/ml, dissolving in deionized water to a constant volume of 1L, adjusting the pH value to 5.6, and sterilizing at 121 ℃ for 20 min.
In the following examples, the composition of the elongation medium (E1) was: 4.4g/L of a mixture of MS salts and B5 vitamins (model M404), 20g/L glucose, 1.29g/L calcium gluconate, 4g/L Agar (Agar), 100mg/L carbenicillin, 100mg/L cephamycin, 100mg/L spectinomycin, pH 5.6, and the balance water. The elongation medium (E1) may be prepared by: 4.4g MS salt and B5 vitamin mixture (M404), 20g glucose, 1.29g calcium gluconate, 4g agar, 100mg/ml carbenicillin solution 1ml, 100mg/ml cefuroxime 1ml, 100mg/ml spectinomycin 1ml, dissolved in deionized water to 1L to adjust pH value to 5.6, 121 ℃ sterilization for 20 min.
The technical scheme of the present invention will be described below by taking the GFP gene as an example of the target DNA.
Example 1
The method of the invention is carried out according to the flow of the upper diagram in fig. 1, the photographs of the various stages being shown in the lower diagram of fig. 1, in particular as follows:
preparation of Agrobacterium
The fragment between the recognition sites for the restriction endonucleases BstEI and BstEII (small fragment including the recognition site for BstEI and the recognition site for BstEII) of the pCAMBIA3301 (product of Biovector) vector was replaced with the DNA (35S:: GFP gene) shown in sequence 1 of the sequence Listing, and the fragment between the recognition sites for the restriction endonucleases BstXI and PspXI (small fragment including the recognition site for BstXI and the recognition site for PspXI) of the pCAMBIA3301 vector was replaced with the DNA (35S:: aada gene) shown in sequence 2 of the sequence Listing, while keeping the other sequences of the pCAMBIA3301 vector unchanged, to obtain a recombinant expression vector of GFP protein and aada protein, which was designated as p 183343.
The recombinant vector p183343 is transferred into agrobacterium strain EHA105 to obtain agrobacterium carrying plasmid containing target gene (the target gene used in this example is green fluorescent protein GFP gene, the nucleotide sequence of which is shown in sequence 1 in the sequence table) and spectinomycin resistance gene (the spectinomycin resistance gene used in this example is aada gene, the nucleotide sequence of which is shown in sequence 2 in the sequence table), namely agrobacterium carrying plasmid containing target gene, and the agrobacterium carrying plasmid is named as Z113 and stored at-80 ℃.
Coating Z113 glycerol strain stored at-80 deg.C on YP culture medium containing kanamycin and rifampicin, culturing in 28 deg.C incubator for 2 days to obtain activated Agrobacterium, coating YP culture medium again one day before infection, and culturing in 28 deg.C incubator for 1 day. The agrobacterium on the plates was collected in a secondary inoculation loop and suspended in liquid infection medium (CAB). And blowing the bacterium block by using a sterile pipette until the agrobacterium cells are uniformly dispersed in the suspension, and measuring the agrobacterium suspension by using a spectrophotometer until the light absorption value OD660nm is about 0.9 to obtain Z113 suspension for later use.
S1 preparation of cotton genetic transformation receptor
The cotton varieties used in this example were Zhongmian cotton institute 49, Zhongmian cotton institute 88, Zhongmian cotton institute 59, Baimian No. 1 and TM-1. These varieties are both strongylus varieties that are severely genotype limited and cannot obtain regenerated shoots via somatic embryogenesis.
One day before infection, 300 delinted uncoated mature cotton seeds of each variety are placed in a sterile rooting tank or a blue-covered bottle, 200ML of alcohol with the volume percentage content of 75% is added, the mixture is shaken for 30s, waste liquid is discarded, 200ML of hydrogen peroxide with the volume percentage content of 6% is added, the waste liquid is discarded after shaking for 15min, the sterile water is washed for 4 times, MSB liquid culture medium 200ML is added, after overnight culture at 28 ℃ for 18-24 hours in the dark (the seeds germinate after 18-24 hours but do not yet show white), the seeds are clamped by tweezers, the seed coats are cut by a scalpel, the cotyledons are stripped under a dissecting mirror, and the shoot apical meristem at the lower part of the exposed leaf primordia is used as an acceptor experimental material for later use.
S2, infection and Co-cultivation
Parameter optimization for ultrasound processing
In order to optimize parameters of ultrasonic treatment, a shoot apical meristem at the lower part of a leaf primordium of a medium cotton institute 49 is used as a receptor explant for an experiment, the explant is placed into a sterile triangular flask added with 10ML infection culture medium, after 150 explants are treated in each flask, a staining culture medium is discarded, 15ML Z113 suspension is added, the explant is placed into an ultrasonic cleaning instrument, ultrasonic time is set to be 40s and 80s, ultrasonic frequency is set to be 40KHz and 100KHz, staining time is set to be 50min and 90min, rotating speed is set to be 80 revolutions per minute and 120 revolutions per minute, specific treatment combinations are shown in table 1, the repetition number is 3, the infected explants are cultured, and stem apical instantaneous conversion efficiency, cluster bud induction rate and conversion efficiency are counted.
The results are shown in Table 1, which shows that the ultrasonic time is 40-80s, the ultrasonic frequency is 40KHz-100KHz, the dip dyeing time is 50-90min, and the rotating speed is 80-120 revolutions per minute, so that the conversion effect is better. Wherein, all the treatment combinations with the ultrasonic time of 40s have the cluster bud induction rate and the transformation efficiency exceeding the corresponding treatment combinations with the ultrasonic time of 80 s; all the treatment combinations with the ultrasonic frequency of 40KHz, the cluster bud induction rate and the conversion efficiency exceed the corresponding treatment combinations with the ultrasonic frequency of 100 KHz.
TABLE 1 conversion efficiency of medium cottons 49 under different treatment conditions
S2.1 infection
The shoot apical meristem at the lower part of the leaf primordia prepared in step S1 is used as a recipient explant, and Agrobacterium carrying a plasmid containing a target gene is introduced into the explant, so that the T-DNA of the target gene carried by the Agrobacterium is inserted into the cotton genome.
The specific method comprises the following steps: the explants were placed in sterile Erlenmeyer flasks with 10ML infection medium added. After treating 150 explants per bottle with 100 times, discarding the staining medium, adding 15ML Z113 suspension, and placing into an ultrasonic cleaning instrument, wherein the ultrasonic treatment frequency is 40kHz and the time length is 40 s. Shaking in shaking table at room temperature for 50 min.
S2.2 Co-cultivation
After infection, the explants are placed on sterile filter paper to suck dry surface bacteria liquid, blown on a super clean workbench for 10min, placed in a co-culture medium, and co-cultured in the dark at 23 ℃ for 3-4 d.
S3, recovery culture
The co-cultured explants were transplanted into recovery medium, 50 explants were transferred to a petri dish, and radicles were inserted into the recovery medium. After culturing at 35 ℃ under a light cycle of 16 hours/8 hours, the cells were transferred to 25 ℃ under a light cycle of 16 hours/8 hours, and culturing was continued for 4 days.
S4 bud Induction and screening
After the culture was resumed, the explant was cut off at the root, transferred to a shoot induction medium and cultured at 25 ℃ under conditions where the photoperiod was 16 hours/8 hours in light/dark for 2 weeks for 3 subcultures.
S5, elongation culture
The explants were inserted into elongation medium and cultured at 25 ℃ under conditions where the photoperiod is 16 hours/8 hours in light/dark. Subculture is carried out once every 2 weeks, grafting or transplanting is carried out after the resistant buds are elongated, and the obtained product is the T0 generation transformed plant.
S6 identification of transgenic cotton plants
And transplanting the T0 generation transformed plants after rooting into a seedling culture substrate, performing conventional fertilizer and water management, waiting for the growth of the transformed plants, and taking T young leaves for PCR detection.
Respectively carrying out PCR amplification by using transgenic cotton DNA, plasmid and wild cotton DNA as templates; the primer pair aiming at the target gene consists of EGFP-F1 and EGFP-R1, and the primer pair aiming at the aadA gene consists of AADA-R1 and AADA-R1, and is synthesized by Shanghai's worker Limited.
EGFP-F1: 5-atcatggccgacaagcagaa-3 (shown as sequence 3 in the sequence table);
EGFP-R1: 5-tctcgttggggtctttgctc-3 (shown as a sequence 4 in a sequence table);
AADA-F1: 5-aatcttccccgtgacagcag-3 (shown as sequence 5 in the sequence table);
AADA-R1: 5-gtgatcgctgaggtctccac-3 (shown as a sequence 6 in a sequence table).
The PCR reaction program is: preheating at 98 ℃ for 10S; (98 ℃ 10S; 55 ℃ 30S; 72 ℃ 1min)30 cyclic reactions; extending for 5min at 72 ℃; storing at 4 ℃.
After the PCR reaction, 5. mu.l of the amplified product was electrophoresed in 1% agarose gel, observed under a Bio-Rad gel imaging system, and photographed. The specific primers are used for carrying out PCR amplification on the cotton plants obtained by screening to obtain a target strip with the size of about 500bp, which indicates that the GFP gene carried by the agrobacterium is successfully introduced into the cotton plants. The cotton plants with the target band about 500bp in size are T0 transgenic positive cotton.
The results are shown in Table 2, which shows that the method of the invention has good repeatability, small restriction of receptor genotypes, uniform and simple transformation mode and high transformation efficiency, the transformation efficiency of different varieties of upland cotton can reach 2.52-9.22%, and the transformation efficiency of different varieties of sea island cotton can also reach 0.64-1.59%.
TABLE 2 transformation efficiency of various cotton varieties
GFP green fluorescence signals were observed in all leaves, stems, fibers, anthers, petals and ovules of transgenic positive cotton of the T0 generation, wherein the results of measuring green fluorescence signals of transgenic positive cotton of the T0 generation of Mizhongmian 49 are shown in FIG. 2.
And (3) selfing the obtained T0 transgenic positive cotton plants respectively to obtain T1 transgenic cotton plants, and selfing the T1 transgenic plants to obtain T2 transgenic cotton seeds. And detecting GFP green fluorescence signals of organs of the T1 generation transgenic cotton plant and T2 generation transgenic cotton seeds, wherein GFP fluorescence photographs of the T1 generation transgenic cotton plant of the Zhongmian cotton institute 49 and the T2 generation transgenic cotton seeds are shown in figure 3, WT is 49 of the wild Zhongmian cotton institute, and other varieties obtain similar results.
The above results indicate that GFP signals were observed in leaves, stems, fibers, anthers, petals and ovules of transgenic cotton plants of the T1 generation, indicating that transgenic positive plants have stably integrated T-DNA, no significant chimera formation, stable inheritance of foreign genes to the next generation, segregation of the T1 population followed a mendelian ratio. Green fluorescence was observed in transgenic cotton seeds at T2 generation, indicating that the Green Fluorescent Protein (GFP) gene was stably transmitted to T2 generation.
The process comparison of the stem tip transformation system and the traditional somatic embryogenesis system is shown in figure 4, the method is simple to operate, a series of complicated processes such as culture medium preparation, callus proliferation, embryogenic callus induction, subculture, screening, regeneration, bud induction and root induction which are required in a complicated somatic embryogenesis culture process are not needed, the whole growth period is short, a transgenic plant is obtained after infection, the specific time required by each stage is shown in the left side of figure 4, the specific Co-culture (Co-culture)3d, the Recovery culture (Recovery culture)4d, the screening induction (i.e. bud induction)21d, the elongation culture (Shoot induction) 45d, the Rooting (Rooting)15d and the total 88d are needed, and the time required by the method is greatly shortened compared with the time required by the traditional tissue culture method.
The invention innovatively provides a simple, convenient, efficient and quick agrobacterium tumefaciens-mediated genetic transformation method for cotton stem tips, breaks through genotype limitation, can efficiently transform a stropanthus hirsutus/sea island cotton variety in a short time, and solves the bottleneck problem of cotton genetic transformation. The invention takes cotton mature seeds as materials, after soaking, peels off seed coats and cotyledons, exposes stem tip meristem, takes the stem tip meristem as a receptor, utilizes agrobacterium mediation, and adopts ultrasonic treatment to introduce exogenous genes into cotton genome, thereby obtaining transgenic cotton. The invention does not need tissue culture, has low influence degree by the genotype, can quickly obtain transgenic cotton plants and has the following advantages: simple operation, short growth period (only 88 days are needed from seed soaking to transgenic plant obtaining), good repeatability, small restriction of receptor genotype, unified and simple transformation mode, high transformation efficiency (the transformation efficiency can reach 2.41-9.22%), and good genetic stability of the obtained transgenic cotton plant.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced with equivalent parameters and conditions within a wide range without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In summary, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> Cotton research institute of Chinese academy of agricultural sciences
<120> agrobacterium tumefaciens-mediated cotton genetic transformation method
<130> GNCSY211872
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga 720
<210> 2
<211> 1017
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atggctcaga tcaggtcgat ggctcagggc attcagacgc tgtcgctcaa ctcctctaat 60
ctctccaaga cgcagaaggg gccgctcgtg tcgaactctc tcttcttcgg cagcaagaag 120
ctgacacaga tctctgccaa gtcactgggg gttttcaaga aggactcggt gctccgggtg 180
gtccgcaagt ccagcttccg catctcagct tccgtcgcta cagctgaggc tgaggccgtg 240
atcgctgagg tctccactca gctcagcgag gttgtgggcg tgatcgagag gcacctggag 300
ccaaccctcc tggctgtcca tctctacggg tcagcggttg atggtggcct gaagccccac 360
tccgacatcg atctcctggt tacagtgact gtccggctgg acgagaccac gaggagggct 420
ctcattaacg atctcctgga gaccagcgct tcgccaggcg agtccgagat cctcagggcg 480
gttgaggtga cgattgtcgt tcatgacgat atcattccat ggaggtaccc agctaagagg 540
gagctccagt tcggcgagtg gcagcgcaat gacatcctgg ccgggatttt cgagccagcg 600
acaatcgaca ttgatctggc tatcctcctg actaaggcta gggagcactc cgtcgctctg 660
gttggccctg ctgctgagga gctcttcgac ccagtccctg agcaggatct cttcgaggcc 720
ctgaacgaga ccctcacgct gtggaattct ccgcccgact gggctggcga tgagaggaat 780
gtggtcctca ccctgtcgcg catctggtac tctgctgtca cggggaagat tgctccaaag 840
gacgtggctg ctgattgggc gatggagagg ctgccagctc agtaccagcc tgtgatcctc 900
gaggctaggc aggcttacct gggccaggag gaggacaggc tcgcgtcccg ggctgatcag 960
ctggaggagt tcgttcatta cgtcaagggc gagattacta aggtcgttgg caagtga 1017
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<210> 4
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<213> Artificial Sequence (Artificial Sequence)
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tctcgttggg gtctttgctc 20
<210> 5
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<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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Claims (10)
1. A method for genetic transformation of cotton, comprising: the method comprises the steps of taking a shoot apical meristem at the lower part of a leaf primordium stripped from soaked cotton mature seeds as a genetic transformation receptor, infecting the genetic transformation receptor by recombinant agrobacterium tumefaciens containing target DNA under the condition of ultrasonic treatment to obtain an infected explant, and culturing the infected explant to obtain a transgenic cotton plant.
2. The method of claim 1, wherein: the ultrasonic power of the ultrasonic treatment is 72W, the ultrasonic frequency is 40-100kHz, and the ultrasonic time is 40-80 s.
3. The method according to claim 1 or 2, characterized in that: the soaking is to soak the cotton mature seeds with MSB liquid culture medium for 18-24 hours.
4. The method of claim 3, wherein: the MSB liquid culture medium has a pH value of 5.6 and comprises the following components: the mixture concentration of MS salt and B5 vitamin is 4.4g/L, the glucose concentration is 20g/L, the calcium gluconate concentration is 1.29g/L, and the rest is water; the mixture of the MS salt and the vitamin B5 consists of the following raw materials in parts by weight: 1900 parts of potassium nitrate, 1650 parts of ammonium nitrate, 170 parts of monopotassium phosphate, 598 parts of calcium nitrate dihydrate, 181 parts of anhydrous magnesium sulfate, 27.85 parts of ferrous sulfate heptahydrate, 37.3 parts of EDTA-na, 17.1 parts of manganese sulfate monohydrate, 0.83 part of potassium iodide, 6.2 parts of boric acid, 0.25 part of sodium molybdate dihydrate, 0.02 part of modified cobalt, 0.025 part of copper sulfate pentahydrate, 100 parts of inositol, 2 parts of glycine, 10.1 parts of VB, 60.5 parts of VB and 30.5 parts of VB.
5. The method of claim 1, wherein: the infection is carried out in an infection culture medium containing acetosyringone.
6. The method of claim 5, wherein: the pH value of the infection culture medium is 5.4, and the composition is as follows: the concentration of CA mother liquor is 10ml/L, the concentration of glucose is 30g/L, the concentration of MES is 4.2g/L, the concentration of B5 vitamin is 0.1ml/L, the concentration of 6-BA is 1mg/L, the concentration of NAA is 0.1mg/L, the concentration of acetosyringone is 0.2mM, and the balance is water; the CA mother liquor comprises the following components: 10g/L magnesium sulfate, 5.36g/L ammonium sulfate, 6g/L sodium phosphate monohydrate, 6g/L calcium chloride, 30mg/L boric acid, 100mg/L manganese sulfate, 20mg/L zinc sulfate heptahydrate, 7.5mg/L potassium iodide, 2.5mg/L sodium molybdate dihydrate, 250 mu g/L copper sulfate, 250 mu g/L cobalt chloride hexahydrate, 100g/L potassium nitrate and the balance of water.
7. The method of claim 1, wherein: the recombinant agrobacterium tumefaciens contains a spectinomycin resistance gene, and the method further comprises the step of screening the infected explants with spectinomycin.
8. The method according to any one of claims 1 to 7, wherein: the step of culturing the infected explant comprises the following steps:
transferring the infected explants to a co-culture medium, and co-culturing for 3-4d under a dark condition;
transferring the explants after co-culture into a recovery culture medium for recovery culture;
cutting off the root of the explant after the recovery culture, and transferring a human bud induction culture medium for screening induction;
and transferring the screened and induced explants to an elongation culture medium for elongation culture to obtain transformed plants.
9. The method of claim 8, wherein: the pH value of the bud induction culture medium is 5.6, the composition is that the concentration of a mixture of MS salt and B5 vitamin is 4.4g/L, the concentration of glucose is 20g/L, the concentration of calcium gluconate is 1.29g/L, the concentration of 6-BA is 1mg/L, the concentration of NAA is 0.1mg/L, the concentration of carbenicillin is 100mg/L, the concentration of cephamycin is 100mg/L, the concentration of spectinomycin is 100mg/L, 4g/L agar, and the balance is water;
the pH of the elongation medium was 5.6 and consisted of: the mixture concentration of MS salt and B5 vitamin is 4.4g/L, the glucose concentration is 20g/L, the calcium gluconate concentration is 1.29g/L, the carbenicillin concentration is 100mg/L, the cephamycin concentration is 100mg/L, the spectinomycin concentration is 100mg/L, 4g/L agar, and the rest is water;
the pH value of the co-culture medium is 5.4, and the co-culture medium comprises the following components: the CA mother liquor concentration is 10ml/L, the glucose concentration is 30g/L, the MES concentration is 4.2g/L, the vitamin B5 concentration is 0.1ml/L, the 6-BA concentration is 1mg/L, the NAA concentration is 0.1mg/L, the acetosyringone concentration is 0.2mM, the cysteine concentration is 200mg/ml, and the balance is water;
the recovery medium has a pH value of 5.6 and comprises the following components: the mixture concentration of MS salt and B5 vitamin is 4.4g/L, the glucose concentration is 20g/L, the calcium gluconate concentration is 1.29g/L, the 6-BA concentration is 1mg/L, the NAA concentration is 0.1mg/L, the carbenicillin concentration is 100mg/L, the cephamycin concentration is 100mg/L, 4g/L agar, and the rest is water.
10. Use of a method of genetic transformation of cotton as claimed in any one of claims 1 to 9 in cotton breeding.
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