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WO2023000167A1 - Procédé de transformation génétique de coton à médiation par agrobacterium - Google Patents

Procédé de transformation génétique de coton à médiation par agrobacterium Download PDF

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Publication number
WO2023000167A1
WO2023000167A1 PCT/CN2021/107336 CN2021107336W WO2023000167A1 WO 2023000167 A1 WO2023000167 A1 WO 2023000167A1 CN 2021107336 W CN2021107336 W CN 2021107336W WO 2023000167 A1 WO2023000167 A1 WO 2023000167A1
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concentration
weight
parts
cotton
medium
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李付广
许洁婷
葛晓阳
杨晓凤
王晔
陈艳丽
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Institute Of Cotton Reaserch Of Caas
Wimi Biotechnology (jiangsu) Co Ltd
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Institute Of Cotton Reaserch Of Caas
Wimi Biotechnology (jiangsu) Co Ltd
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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
    • 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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/60—Malvaceae, e.g. cotton or hibiscus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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)

Definitions

  • the invention relates to a method for genetic transformation of cotton in the field of biotechnology, in particular to a method for genetic transformation of cotton mediated by Agrobacterium.
  • Cotton is one of the world's most important sources of natural fiber and economic crops.
  • the completion of cotton genome sequencing has made it easier and faster to identify and isolate a large number of genes.
  • the genome editing technology that has emerged in recent years has provided simple and precise genetic improvement methods and has become a disruptive technological breakthrough in the field of life sciences.
  • the verification of gene function needs to transform these genes into cotton to verify the practicability of candidate genes.
  • the current conventional method of genetic transformation of cotton is to obtain transgenic plants through tissue culture after particle gun bombardment and Agrobacterium infiltration. This process usually takes about 10-11 months, which is not only time-consuming and laborious, but also only applicable to a few cotton varieties.
  • Gene editing requires a mature, stable, and efficient regeneration system.
  • the purpose of the present invention is to provide a cotton genetic transformation method with a low degree of dependence on the transformation receptor genotype.
  • the method for genetic transformation of cotton comprises that the stem apex meristem at the lower part of the leaf primordium stripped from soaked mature cotton seeds is used as a genetic transformation receptor, and the recombinant root cancer containing the target DNA is used under ultrasonic treatment conditions.
  • Agrobacterium infects the genetically transformed recipient to obtain infected explants, and cultures the infected explants to obtain transgenic cotton plants.
  • the culturing the infected explants does not include callus proliferation, embryogenic callus induction, callus subculture, callus selection, shoot induction and/or root induction.
  • the ultrasonic treatment has a frequency of 40-100 kHz and a duration of 40-80 s.
  • the preferred frequency is 40kHz and the duration is 40s.
  • the soaking is soaking the mature cotton seeds with MSB liquid medium for 18-24 hours.
  • the pH value of the MSB liquid medium is 5.6, and consists 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, and the rest are Water; the mixture of MS salt and vitamin B5 consists of the following raw materials by weight: 1900 parts by weight of potassium nitrate, 1650 parts by weight of ammonium nitrate, 170 parts by weight of potassium dihydrogen phosphate, 598 parts by weight of calcium nitrate dihydrate, anhydrous sulfuric acid 181 parts by weight of magnesium, 27.85 parts by weight of ferrous sulfate heptahydrate, 37.3 parts by weight of EDTA-na, 17.1 parts by weight of manganese sulfate monohydrate, 0.83 parts by weight of potassium iodide, 6.2 parts by weight of boric acid, 0.25 parts by weight of sodium molybdate dihydrate, modified 0.02 parts by weight of cobalt, 0.025 parts
  • the infection is carried out in an infection medium containing acetosyringone.
  • the pH value of the infection medium is 5.4, and it is composed of: the concentration of CA mother solution 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, 6 -BA concentration is 1mg/L, NAA concentration is 0.1mg/L, acetosyringone concentration is 0.2mM, all the other are water;
  • the composition of described 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 ⁇ g /L copper sulfate, 250 ⁇ g/L cobalt chloride
  • the recombinant Agrobacterium tumefaciens contains a spectinomycin resistance gene (it can be an aada gene, the nucleotide sequence of which is shown in sequence 2 in the sequence listing), and the method also includes the post-infection The explants were screened with spectinomycin.
  • said culturing the infected explants comprises the following steps:
  • the explants after selection and induction are transferred to elongation medium for elongation culture to obtain transformed plants.
  • the pH value of the bud induction medium is 5.6
  • the mixture concentration of the MS salt and B5 vitamin is 4.4g/L
  • the glucose concentration is 20g/L
  • the calcium gluconate concentration is 1.29g/L.
  • -BA concentration is 1mg/L
  • NAA concentration is 0.1mg/L
  • carbenicillin concentration is 100mg/L
  • cephalosporin concentration is 100mg/L
  • spectinomycin concentration is 100mg/L
  • 4g/L agar can be suitably amount of other coagulants to replace
  • the rest is water;
  • the pH value of described elongation medium is 5.6, is made up of: the mixture concentration of MS salt and B5 vitamin is 4.4g/L, and glucose concentration is 20g/L, and calcium gluconate concentration is 1.29g/L, carbenicillin Concentration is 100mg/L, and cephalosporin concentration is 100mg/L, and spectinomycin concentration is 100mg/L, and 4g/L agar (can be replaced by other coagulant of suitable amount), all the other are water;
  • the pH value of the co-culture medium is 5.4, consisting of: the concentration of the CA mother solution is 10ml/L, the concentration of glucose is 30g/L, the concentration of MES is 4.2g/L, the concentration of B5 vitamins is 0.1ml/L, 6- The concentration of BA is 1mg/L, the concentration of NAA is 0.1mg/L, the concentration of acetosyringone is 0.2mM, the concentration of cysteine is 200mg/ml, and the rest is water;
  • the pH value of the recovery medium is 5.6, and it consists of: the mixture concentration 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, and the concentration of 6-BA is 1mg /L, NAA concentration is 0.1mg/L, carbenicillin concentration is 100mg/L, cephalosporin concentration is 100mg/L, 4g/L agar (can be replaced by other coagulant of suitable amount), all the other are water.
  • the cotton can be upland cotton or sea island cotton.
  • the upland cotton can be selected from any one of Zhongmian 49, Zhongmian 88, Zhongmian 59, Baimian 1 and TM-1.
  • the sea-island cotton can be selected from any one of Xinhai 43 and Xinhai 56.
  • the invention also provides the application of the cotton genetic transformation method in cotton breeding.
  • the invention uses mature cotton seeds as materials, after soaking, peels off the seed coat and cotyledons, and exposes the shoot apex meristem, which is used as a receptor, mediated by Agrobacterium, and ultrasonic treatment is used to introduce exogenous genes into cotton Genome, so as to obtain transgenic cotton.
  • the invention breaks through the limitation of genotype, can efficiently transform stubborn upland cotton/sea-island cotton varieties in a short period of time, and solves the bottleneck problem of cotton genetic transformation.
  • the invention does not require tissue culture, is less affected by genotype, can quickly obtain transgenic cotton plants, and has the following advantages: simple operation, short growth cycle (from soaking seeds to obtaining transgenic plants, it only takes 88 days), good repeatability, and The somatic genotype is limited, the transformation method is uniform and convenient, the transformation efficiency is high (the transformation efficiency can reach 2.41-9.22%), and the obtained transgenic cotton plants have good genetic stability.
  • Figure 1 is a schematic diagram of the cotton shoot tip transformation system in Example 1 of the present invention.
  • the upper picture of Fig. 1 is a flow chart of a high-efficiency cotton stem tip transformation system: the cotton embryo tip is used as an explant for ultrasonic transformation.
  • the treated explants produce adventitious shoots containing the exogenous gene on the medium supplemented with selective antibiotics, and then take root and regenerate transgenic plants.
  • I is the aseptic treatment of seeds; II is the shoot tip meristem exposed by stripping off two cotyledons; III is ultrasonic treatment Mixture of Agrobacterium containing GFP gene and embryo tip; IV is co-cultivation of embryo tip after dipping treatment; V is GFP fluorescence detection after co-cultivation; VI is recovery culture; VII is screening culture; VIII is spectinomycin resistance Appearance of adventitious buds; IX, transgenic shoots; X, transgenic seedlings.
  • Fig. 2 is a graph showing the results of detecting GFP in leaves, ovules, anthers and roots of T0 generation transgenic positive cotton in Example 1 of the present invention.
  • Fig. 3 is a graph showing the result of stable inheritance of the transgenic material in Example 1 of the present invention.
  • the A picture of Fig. 3 is the stem (I), leaf (II), anther (III), petal (IV), ovule and fiber (V) of ovule and fiber (V) of T1 generation transgenic cotton plant and wild-type contrast, ovule of 20d and The results of detecting GFP in fiber (VI), in which GFP is a transgenic cotton plant, and WT is a wild-type control (Zhongmian Institute 49).
  • Figure 3 B shows the GFP detection results of the T2 generation seeds harvested from the heterozygous T1 generation transgenic cotton plants.
  • Panel C of Figure 3 is a graph of the GFP detection results of T2 generation seeds collected from homozygous transgenic cotton plants.
  • Fig. 4 is a schematic diagram of the flow comparison between the shoot tip transformation system and the somatic embryogenesis system in Example 1 of the present invention.
  • 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 glycosides.
  • the upland cotton variety Zhongmian 49 in the following examples is a variety bred by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.
  • the upland cotton variety Zhongmiansuo 88 in the following examples is a variety bred by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.
  • the cotton variety Zhongmiansuo 59 in the following examples is a variety bred by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.
  • the approval number of the upland cotton variety Baimian No. 1 in the following examples is National Approved Cotton 2009003, which can be purchased from China Cotton Research Institute Science and Trade Company.
  • sea-island cotton varieties Xinhai 43 and Xinhai 56 in the following examples are all products of Xinjiang Zhongmian Seed Industry Co., Ltd.
  • the preparation method of 100mg/ml cephalosporin solution is (taking 100mL as an example): take 10g cephalosporin, dissolve it in 100ml sterilized water, fully dissolve, filter and sterilize with a 0.22um filter membrane, and separate Pack 1ml and store at -20°C.
  • the preparation method of 100mg/ml carbenicillin solution is (taking 100mL as an example): take 10g carbenicillin, dissolve it in 100ml sterilized water, fully dissolve, 0.22um filter membrane filter to sterilize, divide Pack 1ml and store at -20°C.
  • the preparation method of 100mg/ml spectinomycin solution is (taking 100mL as an example): take 10g spectinomycin, dissolve it in 100ml sterilized water, fully dissolve, sterilize by 0.22um filter membrane, separate Pack 1ml and store at -20°C.
  • the preparation method of 100mM acetosyringone solution is (taking 100mL as an example): 1.962g acetosyringone (AS) must be dissolved in 100ml dimethyl sulfoxide (DMSO), fully dissolved, and sterilized by filtration. Aliquot 1ml and store at -20°C.
  • AS acetosyringone
  • DMSO dimethyl sulfoxide
  • the composition of 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 ⁇ g/L copper sulfate, 250 ⁇ g/L cobalt chloride hexahydrate, 100g/L potassium nitrate, and the rest is water.
  • the preparation method of CA mother liquor can be: 10g magnesium sulfate, 5.36g ammonium sulfate, 6g sodium phosphate monohydrate, 6g calcium chloride, 30mg boric acid, 100mg manganese sulfate, 20mg zinc sulfate heptahydrate, 7.5mg potassium iodide, 2.5mg molybdenum dihydrate Sodium sulfate, 250 ⁇ g copper sulfate, 250 ⁇ g cobalt chloride hexahydrate, 100g potassium nitrate, dissolve in dd water or ultrapure water, and make up to 1L.
  • the mixture of MS salt and B5 vitamins consists of the following raw materials by weight: 1900 parts by weight of potassium nitrate, 1650 parts by weight of ammonium nitrate, 170 parts by weight of potassium dihydrogen phosphate, 598 parts by weight of calcium nitrate dihydrate, no 181 parts by weight of magnesium sulfate water, 27.85 parts by weight of ferrous sulfate heptahydrate, 37.3 parts by weight of EDTA-na, 17.1 parts by weight of manganese sulfate monohydrate, 0.83 parts by weight of potassium iodide, 6.2 parts by weight of boric acid, 0.25 parts by weight of sodium molybdate dihydrate, 0.02 parts by weight of modified cobalt, 0.025 parts by weight of copper sulfate pentahydrate, 100 parts by weight of inositol, 2 parts by weight of glycine, 10.1 parts by weight of VB, 0.5 parts by weight of VB, and 30.5 parts by weight of
  • the composition of MSB liquid culture medium is: the mixture (PhytoTech (PhytoTech (Weimeijie) company product, model M404) of 4.4g/L MS salt and B5 vitamin), 20g/L glucose, 1.29g/L gluconic acid Calcium, pH 5.6, and the rest is water.
  • the preparation method of MSB liquid medium can be: 4.4g mixture of MS salt and B5 vitamin (M404), 20g glucose, 1.29g calcium gluconate, dissolve in deionized water to 1L, adjust pH value to 5.6, extinguish at 121°C Bacteria 20min.
  • the composition of the YP medium is: 5g/L NaCl, 5g/L yeast extract (yeast extract, product of OXOID biological company, item number LP0021), 10g/L tryptone (Tryptone, product of OXOID biological company, Product No. LP0042), 15 g/L agar (Agar, product of Sangon Company, product No. A100637), 0.2 mM acetosyringone (AS), and the rest is water.
  • yeast extract yeast extract, product of OXOID biological company, item number LP0021
  • 10g/L tryptone Teryptone, product of OXOID biological company, Product No. LP0042
  • 15 g/L agar Agar, product of Sangon Company, product No. A100637
  • AS 0.2 mM acetosyringone
  • the preparation method of YP medium is: 5g NaCl, 5g yeast extract, 10g tryptone, 15g agar, 2ml of 100mM acetosyringone solution, dissolved in deionized water to 1L, and sterilized at 121°C for 20min.
  • the composition of the YP medium containing kanamycin and rifampicin is: 5g/L NaCl, 5g/L yeast extract (yeast extract), 10g/L tryptone (Tryptone), 15g/L L agar (Agar), 0.2mM acetosyringone (AS), kanamycin (Kan) 50mg/L, rifampicin (rif) 15mg/L, the rest is water.
  • the preparation method of YP medium can be: 5g NaCl, 5g yeast extract, 10g tryptone, 15g agar, 2ml of 100mM acetosyringone solution, dissolved in deionized water to 1L, sterilized at 121°C for 20min, in the medium When the temperature drops to 50°C, add 1ml of 50mg/mL kanamycin (Kan) and 0.3ml of 50mg/mL rifampicin (rif) in an ultra-clean bench, and mix well.
  • Kan kanamycin
  • rifampicin rif
  • the composition of the infection medium is: 10ml/L CA mother solution, 30g/L glucose, 4.2g/L MES (product of sigma company, product number V900336), 0.1ml/L B5 vitamin (PhytoTech (Cimeijie) company product, article number G219), 1mg/L 6-BA, 0.1mg/L NAA, 0.2mM acetosyringone (AS), pH value 5.4, the rest is water.
  • the preparation method of the infection medium can be: 10ml CA mother solution, 30g glucose, 4.2g MES, 0.1ml B5 vitamin (G219), 1mg 6-BA, 0.1mg NAA, 2ml of 100mM acetosyringone solution, dissolved in deionized water Dilute to 1L, adjust the pH value to 5.4, and sterilize at 121°C for 20min.
  • composition of co-culture medium is: 10ml/L CA mother solution, 30g/L glucose, 4.2g/L MES, 0.1ml/L B5 vitamin (G219), 1mg/L 6-BA, 0.1mg/L NAA, 0.2mM acetosyringone, 200mg/ml cysteine (CYS), pH 5.4, the rest is water.
  • the preparation method of the co-culture medium can be: 10ml CA mother solution, 30g glucose, 4.2g MES, 0.1ml B5 vitamin (G219), 1mg 6-BA, 0.1mg NAA, 2ml 100mM acetosyringone solution, 50mg/ml cysteine Dissolve 4ml of acid (CYS) aqueous solution in deionized water to a volume of 1L, adjust the pH value to 5.4, and sterilize at 121°C for 20min.
  • CYS cysteine Dissolve 4ml of acid
  • the composition of recovery medium (R0) is: the mixture (model M404) of 4.4g/L MS salt and B5 vitamin, 20g/L glucose, 1.29g/L calcium gluconate, 4g/L agar ( Agar), 1mg/L 6-BA, 0.1mg/L NAA, 100mg/L carbenicillin, 100mg/L cephalosporin, pH 5.6, and the rest is water.
  • the preparation method of recovery medium (R0) can be: 4.4g mixture of MS salt and B5 vitamin (M404), 20g glucose, 1.29g calcium gluconate, 4g agar, 1mg 6-BA, 0.1mg NAA, 100mg/ml carboxylate 1ml of benzylpenicillin solution and 1ml of 100mg/ml cephalosporin solution were dissolved in deionized water to a volume of 1L, adjusted to pH 5.6, and sterilized at 121°C for 20min.
  • M404 MS salt and B5 vitamin
  • the composition of bud induction medium (R1) is: the mixture (model M404) of 4.4g/L MS salt and B5 vitamin, 20g/L glucose, 1.29g/L calcium gluconate, 4g/L agar (Agar), 1mg/L 6-BA, 0.1mg/L NAA, 100mg/L carbenicillin, 100mg/L cephalosporin, 100mg/L spectinomycin, pH 5.6, and the rest is water.
  • the preparation method of bud induction medium (R1) can be: 4.4g mixture of MS salt and B5 vitamin (M404), 20g glucose, 1.29g calcium gluconate, 4g agar, 1mg 6-BA, 0.1mg NAA, 100mg/ml Dissolve 1ml of carbenicillin solution, 1ml of 100mg/ml cephalosporin solution, and 1ml of 100mg/ml spectinomycin solution in deionized water to 1L, adjust the pH value to 5.6, and sterilize at 121°C for 20min.
  • M404 MS salt and B5 vitamin
  • the composition of elongation medium (E1) is: the mixture (model M404) of 4.4g/L MS salt and B5 vitamin, 20g/L glucose, 1.29g/L calcium gluconate, 4g/L agar (Agar), 100mg/L carbenicillin, 100mg/L cephalosporin, 100mg/L spectinomycin, pH value 5.6, all the other are water.
  • the preparation method of elongation medium (E1) can be: 4.4g mixture of MS salt and B5 vitamin (M404), 20g glucose, 1.29g calcium gluconate, 4g agar, 1ml of 100mg/ml carbenicillin solution, 100mg/ml Dissolve 1 ml of cephalosporin solution and 1 ml of 100 mg/ml spectinomycin solution in deionized water to 1 L, adjust the pH value to 5.6, and sterilize at 121°C for 20 minutes.
  • M404 MS salt and B5 vitamin
  • the recombinant vector p183343 was transformed into the Agrobacterium strain EHA105, and the recombinant vector containing the target gene (the target gene used in this example was the green fluorescent protein GFP gene, the nucleotide sequence of which was shown in sequence 1 in the sequence table) and spectinomycin resistance was obtained.
  • the Agrobacterium of the plasmid of the sex gene (the spectinomycin resistance gene used in this embodiment is aada gene, its nucleotide sequence is as shown in sequence 2 in the sequence table), that is, the Agrobacterium carrying the plasmid containing the gene of interest, named For Z113, store at -80°C.
  • the cotton varieties used in this embodiment are Zhongmiansuo 49, Zhongmiansuo 88, Zhongmiansuo 59, Baimian No. 1 and TM-1. These varieties are all obstinate varieties that are severely restricted by genotype and cannot obtain regenerated shoots through somatic embryogenesis.
  • the shoot apical meristem of the lower part of the leaf primordium of Zhongmian Institute 49 was used as the recipient explant for the experiment, and it was put into a sterile Erlenmeyer flask with 10ML infection medium, each bottle After treating 100-150 explants, discard the dipping medium, add 15ML Z113 suspension, put it into the ultrasonic cleaner, set the ultrasonic time to 40s and 80s, the ultrasonic frequency to 40KHz and 100KHz, and the dipping time to set Two kinds of 50min and 90min, the speed setting is 80 rpm and 120 rpm, the specific treatment combination is shown in Table 1, the number of repetitions is 3, the explants after the infection are cultured, and the instantaneous transformation efficiency of shoot tips and clustered buds are counted. Induction rate and transformation efficiency.
  • step S1 Use the shoot apical meristem at the lower part of the leaf primordia prepared in step S1 as the recipient explant, and insert the Agrobacterium containing the target gene plasmid 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 is as follows: put the explants into a sterile Erlenmeyer flask with 10ML infection medium. After treating 100-150 explants per bottle, discard the dipping medium, add 15ML Z113 suspension, and put it into an ultrasonic cleaner, the ultrasonic treatment frequency is 40kHz, and the duration is 40s. Place in a shaker and shake at room temperature for 50 min.
  • the explants were placed on sterile filter paper to blot the surface bacterial liquid, and blown on the ultra-clean workbench for 10 minutes, then put into the co-culture medium, and co-cultivated in the dark at 23°C for 3-4 days.
  • the explants after co-cultivation were transplanted into the recovery medium, 50 explants were transferred to a petri dish, and the radicles were inserted into the recovery medium. After culturing for 3 days at 35° C. under a light/dark photoperiod of 16 hours/8 hours, the cells were transferred to 25° C. and further cultured for 4 days under a light/dark photoperiod of 16 hours/8 hours.
  • the explants were cut off from the root, moved into the bud induction medium and cultured at 25°C under the conditions of light/darkness 16 hours/8 hours, subcultured once every 2 weeks, and co-cultured 3 times.
  • the explants were inserted into elongation medium and cultured at 25°C with a light/dark photoperiod of 16h/8h. Carry out subculture once every 2 weeks, and carry out grafting or transplanting after the resistant bud elongates, and what obtain is T0 generation transformation plant.
  • Transplant the transformed plants of the T0 generation into the seedling medium after rooting manage with conventional fertilizer and water, wait for their growth, and take the young leaves of T for PCR detection.
  • the transgenic cotton DNA, plasmid, and wild-type cotton DNA were used as templates for PCR amplification; the primer pair for the target gene consisted of EGFP-F1 and EGFP-R1, and the primer pair for the aadA gene consisted of AADA-R1 and AADA-R1 , synthesized by Shanghai Shenggong Co., Ltd.
  • EGFP-F1 5-atcatggccgacaagcagaa-3 (as shown in sequence 3 in the sequence listing);
  • EGFP-R1 5-tctcgttggggtctttgctc-3 (as shown in sequence 4 in the sequence listing);
  • AADA-F1 5-aatcttccccgtgacagcag-3 (as shown in sequence 5 in the sequence listing);
  • AADA-R1 5-gtgatcgctgaggtctccac-3 (shown in sequence 6 in the sequence listing).
  • the PCR reaction program was: preheating at 98°C for 10S; (98°C for 10S; 55°C for 30S; 72°C for 1min) 30 cycle reactions; extension at 72°C for 5min; storage at 4°C.
  • GFP green fluorescence signals can be observed in the leaves, stems, fibers, anthers, petals, and ovules of T0 transgenic positive cotton.
  • the results of measuring green fluorescent signal of T0 positive transgenic cotton of Zhongmian Institute 49 are shown in Figure 2.
  • the obtained T0 generation transgenic positive cotton plants are selfed respectively to obtain T1 generation transgenic cotton plants, and the T1 generation plants are selfed and harvested to obtain T2 generation transgenic cotton seeds.
  • Detect the GFP green fluorescence signal of each organ of the T1 generation transgenic cotton plant and the T2 generation transgenic cotton seed are shown in Figure 3, and WT is wild type Zhongmian Institute 49 and other varieties obtained similar results.
  • the present invention innovatively provides a simple, convenient, efficient and fast Agrobacterium-mediated genetic transformation method of cotton stem tips, which breaks through the genotype restriction and can efficiently transform stubborn upland cotton/sea island cotton varieties in a relatively short period of time.
  • the bottleneck problem of cotton genetic transformation is solved.
  • the invention uses mature cotton seeds as materials, after soaking, peels off the seed coat and cotyledons, and exposes the shoot apex meristem, which is used as a receptor, mediated by Agrobacterium, and ultrasonic treatment is used to introduce exogenous genes into cotton Genome, so as to obtain transgenic cotton.
  • the invention does not require tissue culture, is less affected by genotype, can quickly obtain transgenic cotton plants, and has the following advantages: simple operation, short growth cycle (from soaking seeds to obtaining transgenic plants, it only takes 88 days), good repeatability, and The somatic genotype is limited, the transformation method is uniform and convenient, the transformation efficiency is high (the transformation efficiency can reach 2.41-9.22%), and the obtained transgenic cotton plants have good genetic stability.
  • the invention provides a method for genetic transformation of cotton, which comprises the steps of using the shoot apical meristem at the lower part of the leaf primordium stripped from soaked mature cotton seeds as a genetic transformation receptor, and using The recombinant Agrobacterium tumefaciens infects the genetic transformation recipient to obtain infected explants, and cultures the infected explants to obtain transgenic cotton plants.
  • the invention has the advantages of simple operation, short growth period, good repeatability, less restriction of receptor genotype, uniform and convenient transformation mode, high transformation efficiency, good genetic stability of the obtained transgenic cotton plants and the like.

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Abstract

L'invention concerne un procédé de transformation génétique de coton. Le procédé comprend les étapes suivantes : utilisation comme récepteur de transformation génétique du méristème apical des pousses à la partie inférieure du primordium de la feuille obtenu par pelage à partir de graines de coton matures trempées, dans les conditions d'un traitement par ultrasons ; infestation du récepteur de transformation génétique en utilisant Agrobacterium tumefaciens recombiné contenant de l'ADN cible pour obtenir un explant de post-infestation ; et culture de l'explant de post-infestation pour obtenir un plant de coton transgénique. Le procédé de transformation génétique possède les avantages suivants : simplicité de fonctionnement, cycle de croissance court, bonne répétabilité, faible limitation du génotype, procédé de transformation unifié et simple, haute efficacité de transformation, bonne stabilité génétique du plant de coton transgénique obtenu, etc.
PCT/CN2021/107336 2021-07-20 2021-07-20 Procédé de transformation génétique de coton à médiation par agrobacterium Ceased WO2023000167A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116103337A (zh) * 2023-04-12 2023-05-12 中国热带农业科学院三亚研究院 一种农杆菌介导的西番莲遗传转化方法
CN117448370A (zh) * 2023-10-25 2024-01-26 安阳工学院 一种北艾遗传转化体系的构建方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6479287B1 (en) * 2000-05-01 2002-11-12 Mississippi State University Method for transformation of cotton and organogenic regeneration
US20040009601A1 (en) * 2002-07-15 2004-01-15 The Regents Of The University Of California Methods for the regeneration and transformation of cotton
CN101011027A (zh) * 2007-02-07 2007-08-08 华中农业大学 超声波辅助农杆菌转化棉花胚芽的方法
CN102719475A (zh) * 2011-03-30 2012-10-10 中国农业科学院生物技术研究所 一种利用棉花分生组织进行遗传转化的方法
CN104004783A (zh) * 2014-05-04 2014-08-27 河北农业大学 一种提高棉花转基因效率的方法
CN105274138A (zh) * 2015-11-03 2016-01-27 山西省农业科学院棉花研究所 一种提高棉花转基因效率的方法
CN107012162A (zh) * 2016-01-28 2017-08-04 中国种子集团有限公司 农杆菌介导的棉花胚尖快速转化方法
CN108486150A (zh) * 2018-04-20 2018-09-04 刘寒冬 一种棉花遗传转化方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6479287B1 (en) * 2000-05-01 2002-11-12 Mississippi State University Method for transformation of cotton and organogenic regeneration
US20040009601A1 (en) * 2002-07-15 2004-01-15 The Regents Of The University Of California Methods for the regeneration and transformation of cotton
CN101011027A (zh) * 2007-02-07 2007-08-08 华中农业大学 超声波辅助农杆菌转化棉花胚芽的方法
CN102719475A (zh) * 2011-03-30 2012-10-10 中国农业科学院生物技术研究所 一种利用棉花分生组织进行遗传转化的方法
CN104004783A (zh) * 2014-05-04 2014-08-27 河北农业大学 一种提高棉花转基因效率的方法
CN105274138A (zh) * 2015-11-03 2016-01-27 山西省农业科学院棉花研究所 一种提高棉花转基因效率的方法
CN107012162A (zh) * 2016-01-28 2017-08-04 中国种子集团有限公司 农杆菌介导的棉花胚尖快速转化方法
CN108486150A (zh) * 2018-04-20 2018-09-04 刘寒冬 一种棉花遗传转化方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GOULD, H. J. ET AL.: "Adaptation of Cotton Shoot Apex Culture to Agrobacterium-Mediated Transformation", PLANT MOLECULAR BIOLOGY REPORTER, vol. 16, 31 December 1998 (1998-12-31), pages 1 - 10, XP008017894, DOI: 10.1023/A:1007438104369 *
KESHAMMA E, ROHINI S, RAO K S, MADHUSUDHAN B, KUMAR M UDAYA, ROHINI ; S, RAO ; K S: "Tissue culture-independent In Planta Transformation strategy: an Agrobacterium tumefaciens-Mediated gene Transfer Method to overcome recalcitrance in cotton (Gossypium hirsutum L.)", THE JOURNAL OF COTTON SCIENCE, vol. 12, 1 January 2008 (2008-01-01), pages 264 - 272, XP093026065 *
WANG JING, NIE XIANG-XIANG, LI GUAN: "System Optimization of Agrobacterium-mediated Transformation of Cotton(Gossypium Hirsutum L.) in Xinjiang", XINJIANG DAXUE XUEBAO, XINJIANG DAXUE, URUMQI, CN, vol. 30, no. 2, 31 May 2013 (2013-05-31), CN , pages 218 - 223, XP093026068, ISSN: 1000-2839 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116103337A (zh) * 2023-04-12 2023-05-12 中国热带农业科学院三亚研究院 一种农杆菌介导的西番莲遗传转化方法
CN116103337B (zh) * 2023-04-12 2023-07-11 中国热带农业科学院三亚研究院 一种农杆菌介导的西番莲遗传转化方法
CN117448370A (zh) * 2023-10-25 2024-01-26 安阳工学院 一种北艾遗传转化体系的构建方法

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