WO2023000167A1 - Agrobacterium-mediated cotton genetic transformation method - Google Patents

Abstract

A cotton genetic transformation method. The method comprises using as a genetic transformation receptor the shoot apical meristem at the lower part of the leaf primordium obtained by peeling from soaked mature cotton seeds, under the condition of ultrasonic treatment, infesting the genetic transformation receptor by using recombinant Agrobacterium tumefaciens containing target DNA to obtain a post-infestation explant, and culturing the post-infestation explant to obtain a transgenic cotton plant. The genetic transformation method has the advantages of simple operation, short growth cycle, good repeatability, small genotype limitation, unified and simple transformation method, high transformation efficiency, good genetic stability of the obtained transgenic cotton plant, etc.

Description

A method for genetic transformation of cotton mediated by Agrobacterium technical field

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.

Background technique

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. However, the current popularized cotton varieties are severely restricted by their genotypes, and basically cannot carry out genetic transformation mediated by Agrobacterium; Due to the small number of regenerated seedlings, the vast majority of cotton varieties with important economic value have large genotype differences and poor regeneration performance, and are still difficult to transform. Therefore, it is necessary to establish a cotton genetic Transformation methods are of great importance.

invention disclosure

In view of the current situation that the genotype dependence of the transformation receptor is relatively high in the current cotton transgenic process, 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 provided by the present invention 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.

In the above method, the culturing the infected explants does not include callus proliferation, embryogenic callus induction, callus subculture, callus selection, shoot induction and/or root induction.

In the above method, 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.

In the above method, the soaking is soaking the mature cotton seeds with MSB liquid medium for 18-24 hours.

Wherein, 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 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 VB.

In the above method, the infection is carried out in an infection medium containing acetosyringone.

Wherein, 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, 250μg /L copper sulfate, 250μg/L cobalt chloride hexahydrate, 100g/L potassium nitrate, and the rest is water.

In the above method, 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.

In the above method, said culturing the infected explants comprises the following steps:

Transferring the infected explants into a co-culture medium, and co-cultivating them under dark conditions for 3-4 days;

Transferring the co-cultured explants into recovery medium for recovery culture;

Cut off the roots of the explants after the recovery culture, and move them into the bud induction medium for selection and induction;

The explants after selection and induction are transferred to elongation medium for elongation culture to obtain transformed plants.

Wherein, the pH value of the bud induction medium is 5.6, and the mixture concentration of the MS salt and B5 vitamin is 4.4g/L, the glucose concentration is 20g/L, and 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.

In the above method, 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.

Description of drawings

Figure 1 is a schematic diagram of the cotton shoot tip transformation system in Example 1 of the present invention. Among them, 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. The lower picture of Fig. 1 is the photos of various stages of rapidly obtaining transgenic plants by using the shoot tip transformation technology: in the figure, 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.

The best way to practice the invention

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

In the following examples, unless otherwise specified, the first 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 glycosides.

Quantitative experiments in the following examples, unless otherwise specified, were set up to repeat the experiments three times, and the results were averaged.

The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

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.

The upland cotton variety TM-1[1] in the following examples (please give the literature describing this variety) is available to the public from the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.

The sea-island cotton varieties Xinhai 43 and Xinhai 56 in the following examples are all products of Xinjiang Zhongmian Seed Industry Co., Ltd.

In the following examples, 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.

In the following examples, 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.

In the following examples, 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.

In the following examples, 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.

In the following examples, 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, 250μg/L copper sulfate, 250μg/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, 250μg copper sulfate, 250μg cobalt chloride hexahydrate, 100g potassium nitrate, dissolve in dd water or ultrapure water, and make up to 1L.

In the following examples, 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 VB.

In following embodiment, 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.

In the following examples, 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. 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.

In the following examples, 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.

In the following examples, the composition of the infection medium (CAB) 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.

In the following examples, the composition of co-culture medium (CCM) 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.

In following embodiment, 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.

In the following examples, 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.

In the following examples, 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.

The technical scheme of the present invention will be described below by taking the green fluorescent protein GFP gene as the target DNA as an example.

Example 1

Carry out the method of the present invention according to the flow process of upper figure in Fig. 1, the photo of each stage is shown in the lower figure of Fig. 1, specifically as follows:

Agrobacterium preparation

Replace the fragment between the restriction endonuclease BstEI and the BstEII recognition site of the pCAMBIA3301 (Biovector company product) carrier with the DNA (35S::GFP gene) shown in sequence 1 in the sequence listing (comprising the recognition site of BstEI and the BstEII small fragment including the recognition site), and replace the fragment between the restriction endonuclease BstXI and the PspXI recognition site of the pCAMBIA3301 vector with the DNA (35S::aada gene) shown in sequence 2 in the sequence listing (including the fragment of BstXI recognition site and PspXI recognition site), keeping other sequences of the pCAMBIA3301 vector unchanged, the recombinant expression vector of GFP protein and aada protein was obtained, named p183343.

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.

Spread the Z113 glycerol bacteria stored at -80°C on the YP medium containing kanamycin and rifampicin, and culture them in a 28°C incubator for 2 days to obtain activated Agrobacterium, and spread the YP medium again the day before the infection , and cultivated in a 28°C incubator for 1 day. The next day, the Agrobacteria on the plate were collected with an inoculation loop and suspended in a liquid infection medium (CAB). Blow and beat the bacteria block with a sterile pipette until the Agrobacterium cells are evenly dispersed in the suspension, measure the Agrobacterium suspension with a spectrophotometer, the light absorption value OD660nm is about 0.9, and obtain the Z113 suspension for later use.

S1. Cotton Genetic Transformation Receptor Preparation

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 day before the infection, take 300 mature cotton seeds that have been delinted and uncoated for each variety, put them in a sterile rooting jar or a blue cap bottle, add 200ml of alcohol with a volume percentage of 75%, shake for 30s, and discard the waste liquid , add 200ML of hydrogen peroxide with a volume percentage of 6%, shake for 15 minutes, discard the waste liquid, rinse with sterile water 4 times, add 200ML of MSB liquid medium, and cultivate overnight at 28°C for 18-24 hours in the dark (18-24 hours After the seeds germinated, but not yet white), the seeds were clamped with tweezers, the seed coat was cut with a scalpel, and the cotyledons were peeled off under a dissecting microscope, and the shoot apical meristem at the lower part of the exposed leaf primordium was used as the receptor experimental material ,spare.

S2. Infection and co-cultivation

Parameter optimization for sonication

In order to optimize the parameters of ultrasonic treatment, 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.

The results are shown in Table 1, which shows that the ultrasonic time is 40-80s, the ultrasonic frequency is 40KHz-100KHz, the dipping time is 50-90min, and the rotating speed is within the range of 80-120 revolutions per minute, all of which can have a good conversion effect. Among them, for all the treatment combinations with ultrasonic time of 40s, the induction rate and transformation efficiency of clustered buds exceeded the corresponding treatment combination with ultrasonic time of 80s; for all the treatment combinations with ultrasonic frequency of 40KHz, the induction rate and transformation efficiency of clustered buds exceeded the ultrasonic frequency of 100KHz corresponding processing combination.

Table 1. The transformation efficiency of Zhongmian Institute 49 under different treatment conditions

S2.1 Infection

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.

S2.2 Co-cultivation

After infection, 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.

S3, recovery culture

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.

S4, bud induction and screening

After resuming the culture, 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.

S5, elongation culture

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.

S6. Identification of transgenic cotton plants

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.

After the PCR reaction was completed, 5 μl of the amplified product was taken, electrophoresed in 1% agarose gel electrophoresis, observed and photographed with a Bio-Rad gel imaging system. The cotton plants obtained by screening were amplified by PCR using specific primers, and a target band with a size of about 500 bp was obtained, indicating that the GFP gene carried by Agrobacterium had been successfully introduced into the cotton plants. Cotton plants with a target band of about 500 bp in size are transgenic positive cotton of the T0 generation.

The results are shown in Table 2, showing that the method of the present invention has good repeatability, small restriction of receptor genotype, unified and convenient transformation mode, high transformation efficiency, and the transformation efficiency of different varieties of upland cotton can reach 2.52-9.22%. The conversion efficiency can also reach 0.64-1.59%.

Table 2. Transformation efficiency of each variety of cotton

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. Among them, the GFP fluorescence photos of the T1 generation transgenic cotton plant and the T2 generation transgenic cotton seed of the China Cotton Research Institute 49 are shown in Figure 3, and WT is wild type Zhongmian Institute 49 and other varieties obtained similar results.

The above results indicated that GFP signals were observed in the leaves, stems, fibers, anthers, petals, and ovules of T1 transgenic cotton plants, indicating that the transgenic positive plants had stably integrated T-DNA, no obvious chimera formation, and stable exogenous genes. Inherited to the next generation, the segregation of the T1 population follows Mendelian ratios. Green fluorescence was observed in the transgenic cotton seeds of the T2 generation, indicating that the green fluorescent protein (GFP) gene had been stably transmitted to the T2 generation.

See Figure 4 for the process flow comparison between the shoot tip transformation system of the present invention and the traditional somatic embryogenesis system. The method of the present invention is simple to operate and does not require the preparation of medium, callus proliferation, and embryogenesis necessary in the cumbersome somatic embryogenesis culture process. A series of tedious processes such as callus induction, subculture, screening, regeneration, bud induction, root induction, etc., the entire growth cycle is short, and the specific time required for each stage from infection to obtaining transgenic plants is shown on the left side of Figure 4. Co-culture (Co-culture) 3d, recovery culture (Recovery culture) 4d, screening induction (i.e. bud induction stage, Shoot induction) 21d, elongation culture (Shoot elongation) 45d, rooting (Rooting) 15d, a total of only 88d, Compared with the traditional tissue culture method, the time required is greatly shortened.

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 present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be implemented within a wider range under equivalent parameters and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application includes any changes, uses or improvements to the present invention, including changes made with conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

industrial application

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.

Claims (13)

1. A method for genetic transformation of cotton, characterized in that: the method comprises taking the stem apical meristem at the lower part of the leaf primordia stripped from soaked mature cotton seeds as a genetic transformation receptor, and using the DNA recombination Agrobacterium tumefaciens infects the genetic transformation recipient to obtain infected explants, and cultures the infected explants to obtain transgenic cotton plants.
2. The method according to claim 1, characterized in that: the ultrasonic power of the ultrasonic treatment is 72W, the ultrasonic frequency is 40-100kHz, and the ultrasonic time is 40-80s.
3. The method according to claim 1 or 2, characterized in that: the soaking is soaking the mature cotton seeds with MSB liquid medium for 18-24 hours.
4. The method according to claim 3, characterized in that: the pH value of the MSB liquid culture medium is 5.6, consisting of: the mixture concentration of MS salt and B5 vitamin is 4.4g/L, the glucose concentration is 20g/L, grape Calcium acid concentration is 1.29g/L, and all the other are water; The mixture of described MS salt and vitamin B5 is made up of the raw material of following weight part: Potassium nitrate 1900 weight parts, ammonium nitrate 1650 weight parts, potassium dihydrogen phosphate 170 weight parts, 598 parts by weight of calcium nitrate dihydrate, 181 parts by weight of anhydrous magnesium sulfate, 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 VB6, and 30.5 parts by weight of VB30.5 parts by weight.
5. The method according to claim 1, characterized in that: the infection is carried out in an infection medium containing acetosyringone.
6. The method according to claim 5, characterized in that: the pH value of the infection medium is 5.4, consisting of: the concentration of CA mother solution is 10ml/L, the concentration of glucose is 30g/L, and the concentration of MES is 4.2g/L , B5 vitamin concentration 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, 250μg/L copper sulfate, 250μg/L cobalt chloride hexahydrate, 100g/L potassium nitrate, and the rest is water.
7. The method according to claim 1, characterized in that: the recombinant Agrobacterium tumefaciens contains a spectinomycin resistance gene, and the method also includes screening the explants after the infection with spectinomycin step.
8. The method according to any one of claims 1-7, wherein said culturing the infected explants comprises the following steps:

   Transferring the infected explants into a co-culture medium, and co-cultivating them under dark conditions for 3-4 days;

   Transferring the co-cultured explants into recovery medium for recovery culture;

   Cut off the roots of the explants after the recovery culture, and move them into the bud induction medium for selection and induction;

   The explants after selection and induction are transferred to elongation medium for elongation culture to obtain transformed plants.
9. The method according to claim 8, characterized in that: the pH value of the bud induction medium is 5.6, the concentration of the mixture consisting of the MS salt and B5 vitamin is 4.4g/L, and 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 cephalosporin is 100mg/L, and the concentration of spectinomycin is 100mg/L, 4g/L agar, the rest is water.
10. The method according to claim 8, characterized in that: the pH value of the elongation medium is 5.6, consisting of: the mixture concentration of MS salt and B5 vitamin is 4.4g/L, the glucose concentration is 20g/L, The concentration of calcium gluconate is 1.29g/L, the concentration of carbenicillin is 100mg/L, the concentration of cephalosporin is 100mg/L, the concentration of spectinomycin is 100mg/L, 4g/L agar, and the rest is water.
11. The method according to claim 8, characterized in that: the co-culture medium has a pH value of 5.4 and consists of: the concentration of the CA mother liquor is 10ml/L, the concentration of glucose is 30g/L, and the concentration of MES is 4.2g/L L, B5 vitamin concentration is 0.1ml/L, 6-BA concentration is 1mg/L, NAA concentration is 0.1mg/L, acetosyringone concentration is 0.2mM, cysteine concentration is 200mg/ml, and the rest is water.
12. The method according to claim 8, characterized in that: the recovery medium has a pH value of 5.6 and consists of: a mixture concentration of MS salt and B5 vitamins of 4.4g/L, a glucose concentration of 20g/L, and calcium gluconate Concentration is 1.29g/L, 6-BA concentration is 1mg/L, NAA concentration is 0.1mg/L, carbenicillin concentration is 100mg/L, cephalosporin concentration is 100mg/L, 4g/L agar, the rest is water .
13. Application of the method for genetic transformation of cotton according to any one of claims 1-12 in cotton breeding.

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