Peanut stem tip genetic transformation method and application thereof

Abstract

The invention relates to a peanut stem tip genetic transformation method and application thereof, and belongs to the technical field of biology. The invention provides a peanut genetic transformation method which comprises the following steps: using vacuum negative pressure treatment to assist in infecting peanut explants with agrobacterium liquid, then co-culturing, recovering culture and screening culture to obtain genetic transformation explants, performing rooting culture on the genetic transformation explants to obtain seedlings, and performing rooting culture on the seedlings. And then transplanting into a matrix and culturing to obtain a peanut genetic transformation plant. By optimizing peanut genetic transformation parameters, the transgenic peanut plant is successfully prepared, the genetic transformation efficiency is improved, and an important genetic basis is provided for gene function research and new germplasm material creation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a peanut stem tip genetic transformation method and application thereof. Background Art

[0002] Peanut (Arachis hypogaea L.) is an important cash crop and oilseed. With rising living standards, demand for peanuts is growing, and the peanut industry continues to expand. Traditional breeding methods are struggling to meet domestic market demand. my country urgently needs to conduct transgenic technology research to increase peanut yield, improve quality, and cultivate peanuts with desirable traits such as disease and pest resistance, thereby enhancing peanut productivity and market competitiveness. Efficient and stable genetic transformation systems are crucial for gene function research and molecular breeding, and Agrobacterium-mediated genetic transformation remains the preferred method for many plants. However, current peanut transgenic technology systems still suffer from serious challenges, including low transformation efficiency, genotype limitations, poor reproducibility, false positives, and mosaicism. Compared to crops like rice, wheat, and corn, peanut transgenic breeding still lags far behind in scale and transformation efficiency.

[0003] Based on this, the present invention is proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a peanut stem tip genetic transformation method and application thereof, so as to solve the problem of low genetic transformation efficiency of peanut in the prior art.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a peanut genetic transformation method, comprising the following steps:

[0007] (1) Mixing peanut explants with Agrobacterium bacterial solution, vacuum-treating for 4-6 minutes, and shaking-culturing for 45-55 minutes to obtain infected explants;

[0008] (2) inoculating the infected explants into a co-cultivation solution with a pH of 5.2 to 5.6, and culturing in the dark at 26 to 30° C. for 2 to 4 days to obtain co-cultivated explants;

[0009] (3) inoculating the co-cultured explants into a recovery medium with a pH value of 5.5 to 5.8 for recovery culture for 5 to 9 days to obtain recovered explants;

[0010] (4) The explants after recovery culture are inoculated into a screening culture medium with a pH value of 5.5 to 5.8 and cultured for 4 to 8 weeks to obtain genetically transformed explants, and the genetically transformed explants are inoculated into a rooting culture medium with a pH value of 5.6 to 5.8 and cultured for 14 to 16 days to obtain genetically transformed seedlings, and the genetically transformed seedlings are transplanted into a substrate and cultured to obtain genetically transformed peanut plants.

[0011] Preferably, the peanut explant in step (1) is the complete embryonic stem tip of a peanut seed.

[0012] Preferably, the Agrobacterium in step (1) is Agrobacterium tumefaciens EHA105;

[0013] The OD of the Agrobacterium culture solution 600 The value is 0.8~1.2.

[0014] Preferably, the pressure of the vacuum negative pressure treatment in step (1) is 0.1 to 0.3 MPa;

[0015] The rotation speed of the shaking culture is 60-100 rpm.

[0016] Preferably, the co-cultivation solution in step (2) uses water as a solvent and includes the following components at the following final concentrations:

[0017] Potassium nitrate 900-1100 mg / L, ammonium sulfate 50.6-56.6 mg / L, sodium dihydrogen phosphate 55-65 mg / L, magnesium sulfate 95-105 mg / L, calcium chloride 55-65 mg / L, thiamine hydrochloride 0.5-1.5 mg / L, niacin 5-15 mg / L, pyridoxine hydrochloride 5-15 mg / L, potassium iodide 0.364-0.964 mg / L, boric acid 2.96-6.96 mg / L, manganese sulfate 15.84-19.84 mg / L, zinc sulfate 4.88-8.88 mg / L, sodium molybdate 0.1-0.3 mg / L, copper sulfate 0.01-0.03 mg / L, cobalt chloride 0.01-0.03 mg / L and 2-(N-morpholino)ethanesulfonic acid 38,000-40,000 mg / L.

[0018] Preferably, the recovery medium in step (3) uses water as a solvent and includes the following components at the following final concentrations:

[0019] Ammonium nitrate 390-410 mg / L, potassium sulfate 940-960 mg / L, potassium dihydrogen phosphate 160-180 mg / L, magnesium sulfate 360-380 mg / L, calcium nitrate 390-410 mg / L, calcium chloride 94-98 mg / L, manganese sulfate 20-24 mg / L, zinc sulfate 7.6-9.6 mg / L, boric acid 5.2-7.2 mg / L, copper sulfate 0.15-0.35 mg / L, sodium molybdate 0.15-0.35 mg / L, ferrous sulfate 26.3-28.3 mg / L, disodium EDTA 36.3-38.3 mg / L, thiamine hydrochloride 0.5-1.5 mg / L, pyridoxine hydrochloride 8-12 mg / L, niacin 8-12 mg / L, inositol 90-110 mg / L, glycine 1-3 mg / L, cytokinin 6-BA 4-6 mg / L, thidiazuron 0.1-0.3 mg / L, sucrose 23-27 g / L, plant gel 2-4 g / L, carbenicillin 240-260 mg / L.

[0020] Preferably, the recovery culture method in step (3) is to first culture at a temperature of 33-37°C for 2-4 days, and then culture at a temperature of 23-27°C for 3-5 days;

[0021] The photoperiod of the recovery culture is 15 to 17 hours of light treatment and 7 to 9 hours of darkness treatment every day.

[0022] Preferably, the screening medium in step (4) uses water as a solvent and includes the following components at the following final concentrations:

[0023] Ammonium nitrate 390-410 mg / L, potassium sulfate 940-960 mg / L, potassium dihydrogen phosphate 160-180 mg / L, magnesium sulfate 360-380 mg / L, calcium nitrate 390-410 mg / L, calcium chloride 94-98 mg / L, manganese sulfate 20-24 mg / L, zinc sulfate 7.6-9.6 mg / L, boric acid 5.2-7.2 mg / L, copper sulfate 0.15-0.35 mg / L, sodium molybdate 0.15-0.35 mg / L, ferrous sulfate 26.3-28.3 mg / L, EDT Disodium A 36.3-38.3 mg / L, thiamine hydrochloride 0.5-1.5 mg / L, pyridoxine hydrochloride 8-12 mg / L, niacin 8-12 mg / L, inositol 90-110 mg / L, glycine 1-3 mg / L, 2-(N-morpholino)ethanesulfonic acid 2-6 g / L, cytokinin 6-BA 4-6 mg / L, thidiazuron 0.1-0.3 mg / L, sucrose 23-27 g / L, plant gel 2-4 g / L, carbenicillin 240-260 mg / L, spectinomycin 120-130 mg / L;

[0024] The screening culture temperature is 23-27° C., and the photoperiod is 15-17 hours of light treatment and 7-9 hours of darkness treatment per day.

[0025] Preferably, the rooting medium in step (4) uses water as a solvent and includes the following components at final concentrations:

[0026] Ammonium nitrate 1600-1700 mg / L, potassium sulfate 1850-1950 mg / L, potassium dihydrogen phosphate 160-180 mg / L, magnesium sulfate 360-380 mg / L, calcium nitrate 430-450 mg / L, manganese sulfate 21.3-23.3 mg / L, zinc sulfate 7.6-9.6 mg / L, boric acid 5.2-7.2 mg / L, copper sulfate 0.015-0.035 mg / L, sodium molybdate 0.15-0.35 mg / L, potassium iodide 0 .73~0.93mg / L, cobalt chloride 0.015~0.035mg / L, sodium iron EDTA 35.7~37.7mg / L, thiamine hydrochloride 0.05~0.15mg / L, niacin 0.3~0.7mg / L, pyridoxine hydrochloride 0.3~0.7mg / L, inositol 90~110mg / L, glycine 1~3mg / L, auxin IBA 0.5~1.5mg / L, sucrose 20~40g / L, agar 7.3~9.3g / L;

[0027] The temperature of the rooting culture is 23-27° C., and the photoperiod is 15-17 hours of light treatment and 7-9 hours of darkness treatment per day.

[0028] The present invention provides the application of the peanut genetic transformation method in cultivating high-yield and high-quality transgenic peanuts.

[0029] The present invention has the following technical effects and advantages:

[0030] The present invention provides a peanut genetic transformation method, comprising the following steps: using ultrasound and vacuum negative pressure treatment to assist the infection of peanut explants with Agrobacterium bacteria, followed by co-cultivation, recovery culture, and screening culture to obtain genetically transformed explants, rooting the genetically transformed explants to obtain seedlings, and then transplanting them into a substrate for culture to obtain genetically transformed peanut plants. By optimizing the genetic transformation parameters of peanuts, the present invention successfully produced transgenic peanut plants, improving the efficiency of genetic transformation and providing an important genetic foundation for gene function research and the creation of new germplasm materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Figure 1 is the structure diagram of expression vector M1-4;

[0032] Figure 2 The growth of intact embryonic stem tip explants of different peanut varieties in recovery medium with different 6-BA concentrations;

[0033] Figure 3 is the average number of axillary buds of semi-embryo stem tip explants of different peanut varieties grown on recovery medium with different 6-BA concentrations;

[0034] Figure 4 The average number of axillary buds of intact embryonic stem tip explants of different peanut varieties grown on recovery medium with different 6-BA concentrations;

[0035] Figure 5 The growth of intact embryonic stem tip explants of different peanut varieties in recovery medium with different 6-KT concentrations;

[0036] Figure 6 The average number of axillary buds of semi-embryo stem tip explants of different peanut varieties grown on recovery medium with different 6-KT concentrations;

[0037] Figure 7 The average number of axillary buds of intact embryonic stem tip explants of different peanut varieties grown on recovery medium with different 6-KT concentrations;

[0038] Figure 8 The growth of intact embryonic stem tip explants of different peanut varieties in recovery medium with different TDZ concentrations;

[0039] Figure 9 is the average number of axillary buds of semi-embryo stem tip explants of different peanut varieties grown on recovery medium with different TDZ concentrations;

[0040] Figure 10 The average number of axillary buds of intact embryonic stem tip explants of different peanut varieties grown on recovery medium with different TDZ concentrations;

[0041] Figure 11 The growth of explants on screening medium with different spectinomycin concentrations;

[0042] Figure 12 The growth status of the explants in the shoot tip explants was evaluated by different antibiotic types and concentrations;

[0043] Figure 13 is the GUS transient expression rate in the semi-embryo tip explants infected with different concentrations of Agrobacterium tumefaciens EHA105 bacterial solution;

[0044] Figure 14 is the GUS transient expression rate of intact embryonic stem tip explants infected with different concentrations of Agrobacterium tumefaciens EHA105 bacterial solution;

[0045] Figure 15 is the GUS transient expression rate of intact embryonic stem tip explants infected with different vacuum treatment pressures;

[0046] Figure 16is the GUS transient expression rate of intact embryonic stem tip explants at different infection times;

[0047] Figure 17 is the GUS transient expression rate of intact embryonic stem tip explants at different co-culture times;

[0048] Figure 18 genetically transforming peanut plants for color changes;

[0049] Figure 19 GUS staining results of peanut plant leaves with different treatments;

[0050] Figure 20 This is the gel electrophoresis diagram of peanut genetic transformation positive plants. DETAILED DESCRIPTION

[0051] The present invention provides a peanut genetic transformation method, comprising the following steps:

[0052] (1) Mixing peanut explants with Agrobacterium bacterial solution, vacuum-treating for 4-6 minutes, and shaking-culturing for 45-55 minutes to obtain infected explants;

[0053] The vacuum negative pressure treatment time is preferably 5 minutes; the shaking culture time is preferably 50 minutes;

[0054] (2) inoculating the infected explants into a co-cultivation solution with a pH of 5.2 to 5.6, and culturing in the dark at 26 to 30° C. for 2 to 4 days to obtain co-cultivated explants;

[0055] The pH value of the co-cultivation solution is preferably 5.4; the temperature of the dark culture is preferably 28° C.; and the time of the dark culture is preferably 3 days;

[0056] (3) inoculating the co-cultured explants into a recovery medium with a pH value of 5.5 to 5.8 for recovery culture for 5 to 9 days to obtain recovered explants;

[0057] The pH value of the recovery culture medium is preferably 5.7; the number of days of the recovery culture is preferably 7 days;

[0058] (4) inoculating the recovered explants into a screening medium with a pH value of 5.5 to 5.8 for screening and culturing for 4 to 8 weeks to obtain genetically transformed explants, inoculating the genetically transformed explants into a rooting medium with a pH value of 5.6 to 5.8 for rooting and culturing for 14 to 16 days to obtain genetically transformed seedlings, and transplanting the genetically transformed seedlings into a substrate for culturing to obtain genetically transformed peanut plants;

[0059] The pH value of the screening culture medium is preferably 5.7; the screening culture time is preferably 6 weeks, and the screening culture medium is replaced once every 2 weeks; the pH value of the rooting culture medium is preferably 5.7; and the rooting culture time is preferably 15 days.

[0060] In the present invention, the peanut explant in step (1) is a complete embryonic stem tip of a peanut seed, preferably a complete embryonic stem tip of a Huayu 39 seed.

[0061] In the present invention, the Agrobacterium in step (1) is Agrobacterium tumefaciens EHA105, preferably Agrobacterium tumefaciens EHA105 containing a recombinant vector of the target gene;

[0062] The OD of the Agrobacterium culture solution 600 The value is 0.8 to 1.2, preferably 1.

[0063] In the present invention, the pressure of the vacuum negative pressure treatment in step (1) is 0.1 to 0.3 MPa, preferably 0.2 MPa;

[0064] The rotation speed of the shaking culture is 60-100 rpm, preferably 80 rpm.

[0065] In the present invention, the co-cultivation solution in step (2) uses water as a solvent and includes the following components at the following final concentrations:

[0066] Potassium nitrate 900-1100 mg / L, preferably 1000 mg / L; ammonium sulfate 50.6-56.6 mg / L, preferably 53.6 mg / L; sodium dihydrogen phosphate 55-65 mg / L, preferably 60 mg / L; magnesium sulfate 95-105 mg / L, preferably 100 mg / L; calcium chloride 55-65 mg / L, preferably 60 mg / L; thiamine hydrochloride 0.5-1.5 mg / L, preferably 1 mg / L; nicotinic acid 5-15 mg / L, preferably 10 mg / L; pyridoxine hydrochloride 5-15 mg / L, preferably 10 mg / L; potassium iodide 0.364-0.964 mg / L, preferably 0.664 mg / L; boric acid 2.96-6.96 mg / L, preferably 4.96 mg / L; manganese sulfate 15.84-19.84 mg / L, preferably 17.84 mg / L; zinc sulfate 4.88-8.88 mg / L, preferably 6.88 mg / L; sodium molybdate 0.1-0.3 mg / L, preferably 0.2 mg / L; copper sulfate 0.01-0.03 mg / L, preferably 0.02 mg / L; cobalt chloride 0.01-0.03 mg / L, preferably 0.02 mg / L; 2-(N-morpholino)ethanesulfonic acid 38,000-40,000 mg / L, preferably 39,000 mg / L.

[0067] In the present invention, the recovery medium in step (3) uses water as a solvent and includes the following components at final concentrations:

[0068] Ammonium nitrate 390-410 mg / L, preferably 400 mg / L; potassium sulfate 940-960 mg / L, preferably 950 mg / L; potassium dihydrogen phosphate 160-180 mg / L, preferably 170 mg / L; magnesium sulfate 360-380 mg / L, preferably 370 mg / L; calcium nitrate 390-410 mg / L, preferably 400 mg / L; calcium chloride 94-98 mg / L, preferably 96 mg / L; manganese sulfate 20-24 mg / L, preferably 22 mg / L; zinc sulfate 7.6-9.6 mg / L, preferably 8.6 mg / L; boric acid 5.2-7.2 mg / L, preferably 6.2 mg / L; copper sulfate 0.15-0.35 mg / L, preferably 0.25 mg / L; sodium molybdate 0.15-0.35 mg / L, preferably 0.25 mg / L; ferrous sulfate 26.3-28.3 mg / L, preferably 27.3 mg / L; disodium EDTA 36.3-38.3 mg / L, preferably 37.3 mg / L; thiamine hydrochloride 0.5-1.5 mg / L, preferably 1 mg / L; pyridoxine hydrochloride 8-12 mg / L, preferably 10 mg / L; niacin 8-12 mg / L, preferably 10 mg / L; inositol 90-110 mg / L, preferably 100 mg / L; glycine 1-3 mg / L, preferably 2 mg / L; cytokinin 6-BA 4-6 mg / L, preferably 5 mg / L; thidiazuron 0.1-0.3 mg / L, preferably 0.2 mg / L; sucrose 23-27 g / L, preferably 25 g / L; plant gel 2-4 g / L, preferably 3 g / L; carbenicillin 240-260 mg / L, preferably 250 mg / L.

[0069] In the present invention, the method for recovery culture in step (3) is to first culture at a temperature of 33-37°C for 2-4 days, and then culture at a temperature of 23-27°C for 3-5 days; preferably, to first culture at a temperature of 35°C for 3 days, and then culture at a temperature of 25°C for 4 days;

[0070] The photoperiod of the recovery culture is 15 to 17 hours of light treatment and 7 to 9 hours of dark treatment per day; preferably 16 hours of light treatment and 8 hours of dark treatment per day.

[0071] In the present invention, the screening culture medium in step (4) uses water as a solvent and includes the following components at the following final concentrations:

[0072] Ammonium nitrate 390-410 mg / L, preferably 400 mg / L; potassium sulfate 940-960 mg / L, preferably 950 mg / L; potassium dihydrogen phosphate 160-180 mg / L, preferably 170 mg / L; magnesium sulfate 360-380 mg / L, preferably 370 mg / L; calcium nitrate 390-410 mg / L, preferably 400 mg / L; calcium chloride 94-98 mg / L, preferably 96 mg / L; manganese sulfate 2 0-24 mg / L, preferably 22 mg / L; zinc sulfate 7.6-9.6 mg / L, preferably 8.6 mg / L; boric acid 5.2-7.2 mg / L, preferably 6.2 mg / L; copper sulfate 0.15-0.35 mg / L, preferably 0.25 mg / L; sodium molybdate 0.15-0.35 mg / L, preferably 0.25 mg / L; ferrous sulfate 26.3-28.3 mg / L, preferably 27.3 mg / L; E Disodium DTA 36.3-38.3 mg / L, preferably 37.3 mg / L; Thiamine hydrochloride 0.5-1.5 mg / L, preferably 1 mg / L; Pyridoxine hydrochloride 8-12 mg / L, preferably 10 mg / L; Nicotinic acid 8-12 mg / L, preferably 10 mg / L; Inositol 90-110 mg / L, preferably 100 mg / L; Glycine 1-3 mg / L, preferably 2 mg / L; 2-(N-morpholino)ethanesulfonic acid 2- 6g / L, preferably 4g / L; cytokinin 6-BA 4-6mg / L, preferably 5mg / L; thidiazuron 0.1-0.3mg / L, preferably 0.2mg / L; sucrose 23-27g / L, preferably 25g / L; phytogels 2-4g / L, preferably 3g / L; carbenicillin 240-260mg / L, preferably 250mg / L; spectinomycin 120-130mg / L, preferably 125mg / L;

[0073] The screening culture temperature is 23-27° C., preferably 25° C.; the photoperiod is 15-17 hours of light treatment and 7-9 hours of dark treatment per day, preferably 16 hours of light treatment and 8 hours of dark treatment per day.

[0074] In the present invention, the rooting medium in step (4) uses water as a solvent and includes the following components at final concentrations:

[0075] Ammonium nitrate 1600-1700 mg / L, preferably 1650 mg / L; potassium sulfate 1850-1950 mg / L, preferably 1900 mg / L; potassium dihydrogen phosphate 160-180 mg / L, preferably 170 mg / L; magnesium sulfate 360-380 mg / L, preferably 370 mg / L; calcium nitrate 430-450 mg / L, preferably 440 mg / L; manganese sulfate 21.3-23.3 mg / L, preferably 22.3 mg / L; zinc sulfate 7.6-9.6 mg / L, preferably 8.6 mg / L; boric acid 5.2-7.2 mg / L, preferably 6.2 mg / L; copper sulfate 0.015-0.035 mg / L, preferably 0.025 mg / L; sodium molybdate 0.15-0.35 mg / L, preferably 0.025 mg / L; potassium iodide 0.73-0.93 mg / L, preferably 0.83 mg / L; cobalt chloride 0.015-0.035 mg / L, preferably 0.025 mg / L; sodium ferric EDTA 35.7-37.7 mg / L, preferably 36.7 mg / L; thiamine hydrochloride 0.05-0.15 mg / L, preferably 0.1 mg / L; nicotinic acid 0.3-0.7 mg / L, preferably 0.5 mg / L; pyridoxine hydrochloride 0.3-0.7 mg / L, preferably 0.5 mg / L; inositol 90-110 mg / L, preferably 100 mg / L; glycine 1-3 mg / L, preferably 2 mg / L; auxin IBA 0.5-1.5 mg / L, preferably 1 mg / L; sucrose 20-40 g / L, preferably 30 g / L; agar 7.3-9.3 g / L, preferably 8.3 g / L;

[0076] The temperature of the rooting culture is 23-27° C., and the photoperiod is 15-17 hours of light treatment and 7-9 hours of darkness treatment per day.

[0077] The present invention provides the application of the peanut genetic transformation method in cultivating high-yield and high-quality transgenic peanuts.

[0078] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0079] In the embodiment, Huayu 39, Huayu 22, and Huayu 60 seeds were harvested and stored by the Peanut Laboratory of the College of Agriculture of South China Agricultural University. Escherichia coli DH5α and Agrobacterium tumefaciens competent cells EHA105 were purchased from Shanghai Weidi Biotechnology Co., Ltd.; the recombinant expression vector M1-4 was obtained from the Cotton Research Institute of the Chinese Academy of Agricultural Sciences. The structure of the recombinant expression vector M1-4 is as follows: Figure 1 shown.

[0080] Example 1: Preparation of Agrobacterium tumefaciens culture medium containing recombinant vector expressing RUBY gene and GUS gene

[0081] The GUS gene, or β-glucuronidase gene (β-glucuronidase), is present in the genome of certain bacteria such as Escherichia coli (E. coli). This gene encodes β-glucuronidase (GUS for short), a hydrolase that catalyzes the hydrolysis of many β-glucosides. GUS histochemical detection can clearly observe the localization distribution and content differences of the GUS gene in various parts of the plant, thereby understanding the expression pattern and level of exogenous genes in the plant. No background activity of glucuronidase is detected in the vast majority of plants, so the GUS gene is widely used in gene regulation research, especially as a reporter gene for transgenic plants; the RUBY gene is a synthetic gene that encodes an enzyme that synthesizes betalains. It is an excellent reporter gene for monitoring transformation events and gene expression levels because the transgenic cells are bright red and visible to the naked eye. RUBY has been successfully used in the genetic transformation of rice, Arabidopsis and cotton. This application uses the recombinant expression vector M1-4 containing the GUS gene and the RUBY gene to transform into Agrobacterium tumefaciens for detecting Agrobacterium-transformed peanuts.

[0082]

[0083]

[0084] Preparation of working solution:

[0085] 1. The recombinant expression vector M1-4 was transformed into Escherichia coli DH5α competent cells to obtain recombinant bacteria, which were inoculated into LB liquid culture medium and cultured in a 37°C incubator overnight to obtain a bacterial solution.

[0086] 2. Use the FastPure PLasmidMini Kit-BOX2 kit to extract the plasmid of the recombinant bacteria in the bacterial solution according to the instructions.

[0087] 3. The extracted plasmid was transformed into Agrobacterium tumefaciens EHA105 competent cells using the freeze-thaw method to obtain Agrobacterium tumefaciens EHA105 containing the recombinant plasmid as follows:

[0088] (1) Take out the competent Agrobacterium cells stored at -80℃, wait for them to partially melt at room temperature for a while, and insert them into ice when they are in an ice-water mixture state.

[0089] (2) Take 10 μL of plasmid and add it to Agrobacterium tumefaciens EHA105 competent cells, and mix it by hand at the bottom of the tube.

[0090] (3) Place on ice for 5 min, freeze in liquid nitrogen for 5 min, bath in 37°C water for 5 min, and then bath in ice for 5 min.

[0091] (4) Add 700 μL of antibiotic-free LB liquid culture medium and culture at 28°C with shaking at 180 rpm for 3 hours to obtain culture medium.

[0092] (5) Remove the culture medium, centrifuge at 6000 rpm for 1 min, and collect the bacteria.

[0093] (6) Discard the excess supernatant on a clean bench, and take 100 μL of the supernatant to gently pipette and resuspend the bacterial mass to mix.

[0094] (7) Spread the mixed liquid on an LB plate containing antibiotics (final concentration of 50 mg / L Kan and 16 mg / L Rif), place it upside down in a 28°C incubator and culture for 3 days.

[0095] (8) Pick a single colony with resistance on the LB plate and culture it in LB liquid culture medium with a final concentration of 50 mg / L Kan and a final concentration of 50 mg / L R Fif until the culture medium becomes turbid to obtain Agrobacterium tumefaciens EHA105 culture medium. Mix the culture medium with 50% glycerol at a volume ratio of 1:1 and add it to a 1.5 mL sterile centrifuge tube. Quickly freeze it with liquid nitrogen and store it in a -80°C refrigerator for later use.

[0096] Example 2: Screening of peanut genetic transformation conditions

[0097] 1. Preparation of Experimental Materials

[0098] Preparation of co-culture solution: Add potassium nitrate, ammonium sulfate, sodium dihydrogen phosphate, magnesium sulfate, calcium chloride, thiamine hydrochloride, nicotinic acid, pyridoxine hydrochloride, potassium iodide, boric acid, manganese sulfate, zinc sulfate, sodium molybdate, copper sulfate, cobalt chloride, and 2-(N-morpholino)ethanesulfonic acid to 1 L of water and mix well to make the final concentration of potassium nitrate 1000 mg / L, the final concentration of ammonium sulfate 53.6 mg / L, the final concentration of sodium dihydrogen phosphate 60 mg / L, the final concentration of magnesium sulfate 100 mg / L, the final concentration of calcium chloride 60 mg / L, the final concentration of thiamine hydrochloride 1 mg / L, and the final concentration of nicotinic acid 100 mg / L. The final concentration of 4-nitropropene was 10 mg / L, the final concentration of pyridoxine hydrochloride was 10 mg / L, the final concentration of potassium iodide was 0.664 mg / L, the final concentration of boric acid was 4.96 mg / L, the final concentration of manganese sulfate was 17.84 mg / L, the final concentration of zinc sulfate was 6.88 mg / L, the final concentration of sodium molybdate was 0.2 mg / L, the final concentration of copper sulfate was 0.02 mg / L, the final concentration of cobalt chloride was 0.02 mg / L, and the final concentration of 2-(N-morpholino)ethanesulfonic acid was 39000 mg / L. The pH value was adjusted to 5.4 with HCl to obtain a co-culture solution for later use.

[0099] Preparation of recovery medium: Add ammonium nitrate, potassium sulfate, potassium dihydrogen phosphate, magnesium sulfate, calcium nitrate, calcium chloride, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, ferrous sulfate, disodium EDTA, thiamine hydrochloride, pyridoxine hydrochloride, niacin, inositol, glycine, cytokinin 6-BA, sucrose, phytosterol, cephalosporin, and carbenicillin to 1 L of water and mix well to make the final concentration of ammonium nitrate 400 mg / L, the final concentration of potassium sulfate 950 mg / L, the final concentration of potassium dihydrogen phosphate 170 mg / L, the final concentration of magnesium sulfate 370 mg / L, the final concentration of calcium nitrate 400 mg / L, the final concentration of calcium chloride 96 mg / L, the final concentration of manganese sulfate 22 mg / L, the final concentration of zinc sulfate 8.6 mg / L, and the final concentration of boric acid 6. 0.2 mg / L, the final concentration of copper sulfate is 0.25 mg / L, the final concentration of sodium molybdate is 0.25 mg / L, the final concentration of ferrous sulfate is 27.8 mg / L, the final concentration of disodium EDTA is 37.3 mg / L, the final concentration of thiamine hydrochloride is 1 mg / L, the final concentration of pyridoxine hydrochloride is 10 mg / L, the final concentration of niacin is 10 mg / L, the final concentration of inositol is 100 mg / L, the final concentration of glycine is 2 mg / L, the final concentration of cytokinin 6-BA is 1 mg / L, the final concentration of sucrose is 25 g / L, the final concentration of phytogel is 3 g / L, the final concentration of cephalosporin is 200 mg / L, and the final concentration of carbenicillin is 200 mg / L. HCl is used to adjust the pH to 5.7 to obtain a recovery medium for standby use.

[0100] Preparation of screening medium: Add ammonium nitrate, potassium sulfate, potassium dihydrogen phosphate, magnesium sulfate, calcium nitrate, calcium chloride, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, ferrous sulfate, disodium EDTA, thiamine hydrochloride, pyridoxine hydrochloride, niacin, inositol, glycine, 2-(N-morpholino)ethanesulfonic acid, cytokinin 6-BA, cytokinin 6-KT, sucrose, phytogel, cephalosporin, carbenicillin and spectinomycin to 1 L of water and mix well. The final concentration of ammonium nitrate is 400 mg / L, the final concentration of potassium sulfate is 950 mg / L, the final concentration of potassium dihydrogen phosphate is 170 mg / L, the final concentration of magnesium sulfate is 370 mg / L, the final concentration of calcium nitrate is 400 mg / L, the final concentration of calcium chloride is 96 mg / L, the final concentration of manganese sulfate is 22 mg / L, the final concentration of zinc sulfate is 8.6 mg / L, the final concentration of boric acid is 6.2 mg / L, and the final concentration of copper sulfate is 0. 25 mg / L, the final concentration of sodium molybdate is 0.25 mg / L, the final concentration of ferrous sulfate is 27.3 mg / L, the final concentration of disodium EDTA is 37.3 mg / L, the final concentration of thiamine hydrochloride is 1 mg / L, the final concentration of pyridoxine hydrochloride is 10 mg / L, the final concentration of niacin is 10 mg / L, the final concentration of inositol is 100 mg / L, the final concentration of glycine is 2 mg / L, the final concentration of 2-(N-morpholino)ethanesulfonic acid is The final concentration of the culture medium is 4 g / L, the final concentration of the cytokinin 6-BA is 0.5 mg / L, the final concentration of the cytokinin 6-KT is 1 mg / L, the final concentration of sucrose is 25 g / L, the final concentration of the phytogel is 3 g / L, the final concentration of cephalosporin is 200 mg / L, the final concentration of carbenicillin is 200 mg / L, and the final concentration of spectinomycin is 125 mg / L. The pH value is adjusted to 5.7 with HCl to obtain a screening medium for later use.

[0101] Preparation of rooting medium: Add ammonium nitrate, potassium sulfate, potassium dihydrogen phosphate, magnesium sulfate, calcium nitrate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, potassium iodide, cobalt chloride, sodium iron EDTA, thiamine hydrochloride, niacin, pyridoxine hydrochloride, inositol, glycine, auxin, sucrose, and agar to 1 L of water and mix well to make the final concentration of ammonium nitrate 1650 mg / L, the final concentration of potassium sulfate 1900 mg / L, the final concentration of potassium dihydrogen phosphate 170 mg / L, the final concentration of magnesium sulfate 370 mg / L, the final concentration of calcium nitrate 440 mg / L, the final concentration of manganese sulfate 22.3 mg / L, the final concentration of zinc sulfate 8.6 mg / L, and the final concentration of boric acid 6.2 mg / L. The final concentration of copper sulfate is 0.025 mg / L, the final concentration of sodium molybdate is 0.25 mg / L, the final concentration of potassium iodide is 0.83 mg / L, the final concentration of cobalt chloride is 0.025 mg / L, the final concentration of sodium ferric EDTA is 36.7 mg / L, the final concentration of thiamine hydrochloride is 0.1 mg / L, the final concentration of niacin is 0.5 mg / L, the final concentration of pyridoxine hydrochloride is 0.5 mg / L, the final concentration of inositol is 100 mg / L, the final concentration of glycine is 2 mg / L, the final concentration of auxin IBA is 1 mg / L, the final concentration of sucrose is 30 g / L, and the final concentration of agar is 8.3 g / L. The pH value is adjusted to 5.7 with HCl to obtain a rooting medium for later use.

[0102] Preparation of Agrobacterium tumefaciens EHA105 bacterial solution: 1 mL of the Agrobacterium tumefaciens EHA105 bacterial solution prepared in Example 1 was inoculated into 50 mL of YEB liquid culture medium and activated in a shaker at 28°C and 180 rpm to obtain an OD 600 =1.0 of Agrobacterium tumefaciens EHA105 bacterial solution.

[0103] 2. Peanut Genetic Transformation Methods

[0104] (1) Select healthy and plump peanut seeds, peel off the shells and leave the kernels, mix 40 mL of 30% hydrogen peroxide with 200 mL of sterile water to obtain a disinfectant solution, place the peanut kernels in the disinfectant solution and soak them in a constant temperature incubator at 28°C for 18 hours, pour out the disinfectant solution, rinse the seeds three times with sterile water, and then dry them with sterile filter paper. In a sterile environment, peel off the peanut seed coat, remove the excess cotyledons, and peel off the complete embryonic stem tip explant of the peanut seed;

[0105] Peanut seed intact embryonic stem tip explants were mixed with Agrobacterium tumefaciens EHA105 working solution, treated under vacuum at a pressure of 0.3 MPa for 4 minutes, and then cultured at a shaking speed of 60 rpm for 45 minutes to obtain infected explants;

[0106] (2) The infected explants were inoculated into the prepared co-culture solution and cultured in the dark at 28°C for 3 days to obtain co-cultured explants;

[0107] (3) The co-cultured explants were inoculated into the prepared recovery medium and cultured under light conditions for 16 h and darkness conditions for 8 h per day. The explants were first cultured at 35°C for 3 days and then at 25°C for 4 days to obtain the recovered explants.

[0108] (4) The explants after recovery culture were inoculated into the prepared screening culture medium for culture. The culture temperature during the culture period was 25°C, the light treatment was 16 hours and the dark treatment was 8 hours per day, and the screening culture medium was replaced once every 2 weeks. After 6 weeks of culture, genetically transformed explants were obtained. The genetically transformed explants were inoculated into the prepared rooting culture medium for rooting culture for 15 days to obtain genetically transformed seedlings. The genetically transformed seedlings were transplanted into a mixed soil matrix with a mass ratio of vermiculite: peat soil of 3:1 to obtain genetically transformed peanut plants.

[0109] 3. Screening of peanut shoot tip regeneration system conditions

[0110] 3.1 Screening of recovery medium

[0111] Preparation of peanut explants: Select healthy and plump peanut seeds from Huayu 39, Huayu 22, and Huayu 60 respectively, peel off the shells and leave the kernels, mix 40 mL of 30% hydrogen peroxide with 200 mL of sterile water to obtain a disinfectant solution, place the peanut kernels in the disinfectant solution and soak in a constant temperature incubator at 28°C for 18 hours, pour out the disinfectant solution, rinse the seeds 3 times with sterile water, and then dry the moisture with sterile filter paper, peel off the peanut seed coat in a sterile environment, remove the excess cotyledons and partially peel off to obtain complete embryonic stem tip explants of Huayu 39, Huayu 22, and Huayu 60 peanut seeds, and use a sterile scalpel to cut the outer longitudinal axis of the complete embryonic stem tip of Huayu 39, Huayu 22, and Huayu 60 peanut seeds to obtain half-embryonic stem tip explants of Huayu 39, Huayu 22, and Huayu 60 seeds.

[0112] 3.1.1 Effects of different 6-BA concentrations on peanut axillary bud induction

[0113] The concentration of 6-BA in the recovery medium was adjusted to obtain recovery medium with final 6-BA concentrations of 0.5 mg / L, 1 mg / L, 1.5 mg / L, 3 mg / L, and 5 mg / L, respectively. The complete embryonic stem tip explants and half-embryonic stem tip explants of peanut varieties Huayu 39, Huayu 22, and Huayu 60 were inoculated into the recovery medium with different 6-BA concentrations, respectively. The culture was carried out for 10 days, and the growth of the complete embryonic stem tip explants of different peanut varieties in the recovery medium with different 6-BA concentrations was observed. The results are shown in FIG. Figure 2As shown in the figure, A represents Huayu 39, B represents Huayu 22, and C represents Huayu 60. The average number of axillary buds of semi-embryo stem tip explants of different peanut varieties on recovery medium with different 6-BA concentrations was determined. The results are shown in Figure 3 The average number of axillary buds of intact embryonic stem tip explants of different peanut varieties grown on recovery medium with different 6-BA concentrations is shown in Figure 4 The calculation formula for the average number of axillary buds is as follows:

[0114] Average number of axillary buds = number of differentiated axillary buds / number of inoculated explants

[0115] according to Figures 2 to 4It can be seen that Huayu 39 has the fastest growth and differentiation speed among the five concentrations of 6-BA, and grows well. The leaves all expand and become larger, the plants elongate and develop, and the explant height is medium; the proto-embryonic part of the explant of Huayu 22 develops and swells, the diameter is smaller near the main stem, the bottom is relatively swollen, the stem nodes are slender, the leaves are mostly curled and the leaflets are closed, and the plant is taller; the base of the explant of Huayu 60 is swollen, the leaves on the main stem are mostly expanded round leaves, and the plant height is the most advantageous among the three varieties. The growth of Huayu 39 in culture media containing different 6-BA concentrations showed little difference, but the higher the 6-BA concentration, the more leaves differentiated. The average number of axillary buds of the semi-embryo tips of Huayu 39 on 6-BA recovery media with concentrations of 0.5 mg / L, 1 mg / L, 1.5 mg / L, 3 mg / L, and 5 mg / L were 1.17, 1.5, 1.4, 1.3, and 1.93, respectively; the average number of axillary buds differentiated from the complete embryo tips were 1.5, 1.57, 1.77, 1.8, and 2.07, respectively. When the 6-BA concentration was 5 mg / L, both the semi-embryo and complete embryo tips of Huayu 39 obtained the highest average number of axillary buds, and the explants could almost differentiate into about 2 axillary buds. The average number of axillary buds differentiated from the semi-embryo tips of Huayu 22 cultured at different 6-BA concentrations was 1.37, 1.87, 1.43, 1.77, and 1.8, respectively. The average number of axillary buds differentiated from the intact embryo tips was 1.8, 1.7, 1.67, 1.83, and 1.7, respectively. The average number of axillary buds differentiated from the semi-embryo tips of Huayu 22 cultured at different 6-BA concentrations was similar between the intact and semi-embryo tips. The highest average number of axillary buds, 1.87, was obtained from the semi-embryo tips at 1 mg / L 6-BA, while the optimal average number of axillary buds, 1.83, was obtained from the intact embryo tips at 3 mg / L 6-BA. The average number of axillary buds differentiated from the semi-embryo tips of Huayu 60 cultured at the five 6-BA concentrations was 1, 1.47, 1.23, 1.43, and 1.7, respectively. The average number of axillary buds differentiated from the intact embryo tips at the five 6-BA concentrations was 1, 1.73, 1.47, 1.23, 1.43, and 1.73, respectively. The two types of Huayu 60 stem apex explants exhibited the highest average axillary bud number, 1.7, on the 5 mg / L 6-BA medium. Overall, the three varieties' stem apex explants were able to differentiate normally on the 6-BA recovery medium, with an average of 1 to 2 axillary buds, with similar numbers. Peanut varieties Huayu 39 and Huayu 60 achieved the highest average axillary bud number on the 5 mg / L 6-BA recovery medium. Huayu 39 demonstrated the best differentiation results, surpassing the other two varieties in terms of the maximum number of differentiated buds.

[0116] 3.1.2 Effects of different 6-KT concentrations on peanut axillary bud induction

[0117] 6-BA in the recovery medium was replaced with 6-KT, and the concentration of 6-KT in the recovery medium was adjusted to obtain recovery medium with final 6-KT concentrations of 0.5 mg / L, 1 mg / L, 1.5 mg / L, 3 mg / L, and 5 mg / L, respectively. Whole embryonic stem tip explants and half-embryonic stem tip explants of peanut varieties Huayu 39, Huayu 22, and Huayu 60 were inoculated into recovery medium with different 6-KT concentrations, cultured for 10 days, and the growth of whole embryonic stem tip explants of different peanut varieties in recovery medium with different 6-KT concentrations was observed. The results are shown in FIG. Figure 5 As shown in the figure, A represents Huayu 39, B represents Huayu 22, and C represents Huayu 60. The average number of axillary buds of semi-embryo stem tip explants of different peanut varieties on recovery medium with different 6-KT concentrations was determined. The results are shown in Figure 6 The average number of axillary buds of intact embryonic stem tip explants of different peanut varieties grown on recovery medium with different 6-KT concentrations is shown in Figure 7 shown.

[0118] according to Figures 5 to 7The differentiation of Huayu 22 plants was similar to that of Huayu 39, with a swollen base, multiple leaves, but some curled, and significantly taller than Huayu 39, with a more slender plant overall. The leaves of Huayu 60 explants were sparser and dark green than those of Huayu 22, and the plant height was almost identical, with a more swollen base. The average number of axillary buds in the semi- and complete embryonic stem tips of Huayu 39 grown on 6-KT at 0.5 mg / L, 1 mg / L, 1.5 mg / L, 3 mg / L, and 5 mg / L concentrations was 1.27, 1.33, 1.27, 1.13, and 1.77, respectively, and 1.2, 1.23, 1.27, 1.6, and 1.7, respectively. The average axillary bud numbers of the semi-embryo tips of Huayu 22 cultured at five different 6-KT concentrations were 1.4, 1.47, 1.3, 1.57, and 1.6, respectively; the average axillary bud numbers of the intact embryo tips were 1.2, 1.3, 1.17, 1.37, and 1.6, respectively. The average axillary bud numbers of the semi-embryo tips of Huayu 60 cultured at different 6-BA concentrations were similar, while the average axillary bud numbers of the intact embryo tips reached 1, 1.03, 1, 1.07, and 1.13, respectively. Both types of Huayu 39 shoot apex explants achieved their respective optimal average axillary bud numbers of 1.7 at 5 mg / L 6-KT. Both the semi-embryo tips and the intact embryo tips of Huayu 22 cultured at 5 mg / L 6-KT also achieved the same highest average axillary bud number of 1.6, respectively. Huayu 60 semi-embryo tip explants produced the highest average axillary bud number (1.3) at both 3 mg / L and 5 mg / L 6-KT concentrations. Completely embryonic stem tips also produced an average of 1.13 axillary buds at 5 mg / L. All three varieties' stem tip explants were able to differentiate into axillary buds normally on recovery medium containing 6-KT, with the number of differentiated axillary buds ranging from 1 to 2. Both types of stem tip explants from the three peanut varieties produced the highest average axillary bud number when induced at the higher 6-KT concentration (5 mg / L).

[0119] 3.1.3 Effects of different thidiazuron (TDZ) concentrations on peanut axillary bud induction

[0120] TDZ was used to replace 6-BA in the recovery medium, and then the concentration of TDZ in the recovery medium was adjusted to obtain recovery medium with final TDZ concentrations of 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1 mg / L, and 2 mg / L, respectively. The complete embryonic stem tip explants and half-embryonic stem tip explants of peanut varieties Huayu 39, Huayu 22, and Huayu 60 were inoculated into the recovery medium with different TDZ concentrations and cultured for 10 days. The growth of the complete embryonic stem tip explants of different peanut varieties in the recovery medium with different TDZ concentrations was observed. The results are shown in FIG. Figure 8As shown in the figure, A represents Huayu 39, B represents Huayu 22, and C represents Huayu 60. The average number of axillary buds of semi-embryo stem tip explants of different peanut varieties on recovery medium with different TDZ concentrations was determined. The results are shown in Figure 9 The average number of axillary buds of intact embryonic stem tip explants of different peanut varieties grown on recovery medium with different TDZ concentrations is shown in Figure 10 shown.

[0121] according to Figures 8 to 10 As shown, Huayu 39, induced by TDZ, developed multiple buds, which further developed into multiple axillary buds, achieving a medium plant height. Compared to Huayu 39, Huayu 22 exhibited a larger basal bud, with a larger embryonic node at the base of the stem. The plant then developed a cluster of axillary buds with sharp, small leaves, resulting in a medium plant height. Huayu 60 explants showed similar differentiation to Huayu 22, with a longer embryonic node at the base and sparser axillary bud leaves compared to Huayu 22. Most of the leaves on the main stem were dark green, and the plant height was slightly higher than that of Huayu 22. The average number of axillary buds in Huayu 39 semi-embryonic stem apex explants at five TDZ concentrations (0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1 mg / L, and 2 mg / L) was 2.43, 2.9, 2.1, 2.17, and 1.83, respectively. The average number of axillary buds in the intact embryonic stem apex was 2.73, 2.5, 2.9, 2.77, and 2.47, respectively. The average number of axillary buds obtained from Huayu 22 stem apex explants induced under five different TDZ concentrations was 2.6, 2.7, 2.73, 2.07, and 2, and 2.2, 2.1, 2.17, 2.17, and 2.07, respectively. The average number of axillary buds obtained from Huayu 60 semi-embryonic stem apex explants induced under different TDZ concentrations was 2.3, 2.03, 1.87, 2.53, and 2. The average number of axillary buds obtained from intact stem apex explants induced under different TDZ concentrations reached 2.67, 1.9, 2.67, 2.7, and 2.27, respectively. Compared with 6-BA and 6-KT, the average number of axillary buds induced by TDZ generally reached 2, ranging from 2 to 3. The optimal average axillary bud number obtained from Huayu 39 stem apex explants was 2.9. Half-embryonic stem apex exhibited the best axillary bud differentiation at a TDZ concentration of 0.2 mg / L, while full-embryonic stem apex achieved the average number of axillary buds at a TDZ concentration of 0.5 mg / L. Half-embryonic stem apex of Huayu 22 exhibited the best axillary bud differentiation ability at a TDZ concentration of 0.2 mg / L, with an average number of 2.7 axillary buds. The average number of axillary buds obtained from full-embryonic stem apex explants was almost identical, with full-embryonic stem apex differentiation slightly superior to the other concentrations at TDZ concentrations of 0.1 mg / L. Both types of Huayu 60 stem apex explants achieved the optimal average axillary bud number when treated with 1 mg / L TDZ recovery medium. Huayu 39 also achieved a higher average axillary bud number than the other two cultivars at different TDZ concentrations.

[0122] Based on the results of axillary bud induction by 6-BA, 6-KT, and TDZ across the three cultivars, TDZ was found to be particularly effective in promoting axillary bud differentiation. While 6-BA and 6-KT generally induced only one axillary bud, TDZ generally produced two to three, demonstrating superior axillary bud differentiation ability compared to 6-BA and 6-KT. However, TDZ-induced plants were less pronounced in height, being shorter than those induced by 6-BA or 6-KT. Given the universal adaptability of 6-BA and 6-KT for axillary bud induction across the three peanut cultivars, with 6-BA producing slightly more axillary buds than 6-KT, a combination of 6-BA and TDZ was used as a growth hormone in the recovery medium. Among the three cultivars, Huayu 39 achieved the best differentiation response under all three hormone concentrations. This suggests that Huayu 39 is more susceptible to hormone-induced differentiation than Huayu 22 and Huayu 60, demonstrating its suitability for explant differentiation and growth. Finally, it was decided to use the explants of Huayu 39 for differentiation growth, and 6-BA with a final concentration of 5 mg / L and TDZ with a final concentration of 0.2 mg / L were selected in the recovery culture medium as hormones to induce differentiation.

[0123] 3.2 Determination of screening medium

[0124] 3.2.1 Effects of different concentrations of resistance screening agents on explant screening efficiency

[0125] The concentration of spectinomycin (SPE) in the screening medium was adjusted to obtain screening medium with a final spectinomycin concentration of 100 mg / L, 125 mg / L, and 150 mg / L, respectively. Uninfected half-embryonic stem tip explants of Huayu 39 were inoculated into screening medium with different spectinomycin concentrations. The culture was subcultured three times at a temperature of 25°C and a light-dark cycle of 16 h light / 8 h dark. The growth of the explants on the screening medium with different spectinomycin concentrations was observed. The results are shown in FIG. Figure 11 shown.

[0126] according to Figure 11 As can be seen, at a spectinomycin concentration of 100 mg / L, most plants began to show albinism symptoms, but some plants remained green and viable. At this concentration, spectinomycin failed to completely screen out normal negative plants. At the other two spectinomycin concentrations, almost all plants that grew and differentiated turned white, and non-resistant seedlings could not survive normally. Spectinomycin concentrations of 125 mg / L and 150 mg / L in the screening medium were both effective in screening resistant plants. Therefore, 125 mg / L spectinomycin was selected as the screening concentration for resistance screening.

[0127] 3.2.2 Antibacterial sensitivity test

[0128] Antibacterial agents are often used to remove residual Agrobacterium after co-cultivation or to inhibit the growth of Agrobacterium to reduce contamination of explants during the transformation process.

[0129] During the preparation of the screening culture medium, cefotaxime and carbenicillin were not added to obtain a screening culture medium without antibacterial agents. Then, cefotaxime (Cef), carbenicillin (Carb) and timentin (Tim) were added separately as antibacterial agents, and the concentrations were adjusted to obtain screening culture media with final cefotaxime concentrations of 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, and 250 mg / L, screening culture media with final carbenicillin concentrations of 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, and 250 mg / L, and screening culture media with final timentin concentrations of 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, and 250 mg / L.

[0130] The semi-embryo stem tip explants of Huayu 39 that had been infected and co-cultured were inoculated into screening medium containing different concentrations of cefotaxime, screening medium containing different concentrations of carbenicillin, and screening medium containing different concentrations of timentin, respectively. The culture temperature was 25°C, and the light treatment was 16 hours and the dark treatment was 8 hours per day. The screening medium was changed once every 2 weeks. After 6 weeks of culture, genetically transformed explants were obtained. The effects of different antibiotic types and concentrations on the growth of the explants in the stem tip explants were observed. The results are as follows: Figure 12 As shown in the figure, A represents cefotaxime, B represents carbenicillin, and C represents timentin. The effects of different antibiotic types and concentrations on the bacterial infection and growth of shoot apex explants were measured, and the results are shown in Table 1.

[0131] Table 1 Effects of different antibiotic types and concentrations on bacterial infection and growth of shoot apex explants

[0132]

[0133] according to Figure 12As shown in Table 1, Agrobacterium is very sensitive to cefotaxime (Cef), and 50 mg / L can inhibit the contamination of Agrobacterium. Cefotaxime has a very significant inhibitory effect on Agrobacterium, but it also has a very obvious damaging effect on explants. As the concentration of cefotaxime increases, the growth of explants begins to be inhibited. At a 150 mg / L cefotaxime concentration, explant growth was inhibited. Carbenicillin (Carb) exhibited a favorable effect on explant growth, and even high concentrations did not inhibit explant growth. Shoot tip explants grown under different carbenicillin concentrations showed normal growth. Carbenicillin at 250 mg / L effectively inhibited Agrobacterium contamination, while explants cultured under the other four concentrations were contaminated. Timentin (Tim) had a minimal toxic effect on explants. Explants grew normally in culture medium containing different concentrations of Timentin, showing no inhibition. Low concentrations of Timentin failed to inhibit plant contamination. Like carbenicillin, Timentin's effective antibacterial concentration was 250 mg / L. Based on the later growth of the explants, shoot tip explants grown most vigorously in the screening medium containing 250 mg / L carbenicillin. Therefore, 250 mg / L carbenicillin was selected as the antibacterial agent in the transformation system.

[0134] In summary, when using peanut explants to construct regeneration system plants, the optimal recovery medium formula is: using water as the solvent, containing the following components at the final concentrations:

[0135] Ammonium nitrate 400mg / L, potassium sulfate 950mg / L, potassium dihydrogen phosphate 170mg / L, magnesium sulfate 370mg / L, calcium nitrate 400mg / L, calcium chloride 96mg / L, manganese sulfate 22mg / L, zinc sulfate 8.6mg / L, boric acid 6.2mg / L, copper sulfate 0.25mg / L, sodium molybdate 0.25mg / L, ferrous sulfate 27.3mg / L, disodium EDTA 37.3mg / L, thiamine hydrochloride 1mg / L, pyridoxine hydrochloride 10mg / L, niacin 10mg / L, inositol 100mg / L, glycine 2mg / L, cytokinin 6-BA 5mg / L, thidiazuron 0.2mg / L, sucrose 25g / L, plant gel 3g / L, carbenicillin 250mg / L.

[0136] The optimal screening medium formulation is: water as the solvent, containing the following components at the final concentrations:

[0137] Ammonium nitrate 400mg / L, potassium sulfate 950mg / L, potassium dihydrogen phosphate 170mg / L, magnesium sulfate 370mg / L, calcium nitrate 400mg / L, calcium chloride 96mg / L, manganese sulfate 22mg / L, zinc sulfate 8.6mg / L, boric acid 6.2mg / L, copper sulfate 0.25mg / L, sodium molybdate 0.25mg / L, ferrous sulfate 27.3mg / L, disodium EDTA 37.3mg / L, thiamine hydrochloride 1mg / L, pyridoxine hydrochloride 10mg / L, niacin 10mg / L, inositol 100mg / L, glycine 2mg / L, 2-(N-morpholino)ethanesulfonic acid 4g / L, cytokinin 6-BA 5mg / L, thidiazuron 0.2mg / L, sucrose 25g / L, plant gel 3g / L, carbenicillin 250mg / L, spectinomycin 125mg / L.

[0138] 4. Screening of Agrobacterium-mediated genetic transformation conditions

[0139] 4.1 Effect of different Agrobacterium tumefaciens EHA105 bacterial concentrations on transformation efficiency

[0140] Adjust the concentration of the Agrobacterium tumefaciens EHA105 bacterial solution prepared in Example 1 to obtain OD 600 The half-embryo tip explants and the complete embryonic stem tip explants of Huayu 39 were mixed with Agrobacterium tumefaciens EHA105 bacterial solutions of different concentrations, which were 0.2, 0.4, 0.8, 1.0, and 1.5, respectively. The explants were then treated with vacuum negative pressure for 5 minutes at a pressure of 0.5 MPa and shaken for 50 minutes to obtain the infected explants. 50 explants were treated each time and GUS staining was performed on the explants. The GUS transient expression rates of the half-embryo tip explants and the complete embryonic stem tip explants after infection were counted.

[0141] The GUS staining method is as follows: prepare the GUS staining solution according to the instructions of the GUS staining kit (Beijing Solebow Technology Co., Ltd., catalog number: G3060), take an appropriate amount of GUS staining solution, completely immerse the explant in the GUS staining solution, and place it at 30°C for 12 hours. Pour off the GUS staining solution, wash and decolorize with 70% alcohol three times until the negative control material turns white, and then observe whether blue spots appear on the explant. If blue spots appear, it means that the plant is GUS-stained, and then calculate the GUS transient expression rate. The calculation formula of the GUS transient expression rate is as follows:

[0142] GUS transient expression rate (%) = number of GUS-stained plants / number of inoculated explants × 100%.

[0143] The transient expression rate of GUS in the semi-embryo tip explants infected with different concentrations of Agrobacterium tumefaciens EHA105 Figure 13As shown in Figure 2, the GUS transient expression rates of intact embryonic stem tip explants infected with different concentrations of Agrobacterium tumefaciens EHA105 bacterial solution are as follows: Figure 14 shown.

[0144] according to Figure 13 It can be seen that the GUS transient expression results of explants infected with different bacterial solution concentrations are different. When the bacterial solution concentration is too low, the transient conversion rate of the explant is not high. As the bacterial solution concentration increases, the semi-embryo tip explant obtains the highest GUS transient expression result when the bacterial solution concentration is 1.0, reaching 64%. As the bacterial solution concentration continues to increase, the GUS transient conversion rate begins to decrease. Too high or too low bacterial solution concentration will affect the explant conversion rate. When the bacterial solution concentration is appropriate, the conversion efficiency can reach the optimal level. The experimental results show that OD 600 =1.0 is the optimal bacterial infection concentration for semi-embryo explants.

[0145] according to Figure 14 It can be seen that, like the semi-embryo tip explants, the complete embryo tip explants are also affected by the bacterial solution concentration in the same way. When the bacterial solution concentration is too low, the explant transient conversion rate is also low. As the bacterial solution concentration increases, the GUS transient expression rate of the explants begins to gradually increase. 600 =1.0, the highest transient expression rate of 71.33% was obtained, and the bacterial concentration OD 600 =1.5, the transient expression rate began to decline. The experimental results showed that the optimal bacterial infection concentration of the intact embryonic stem tip explant was OD 600 = 1.0. Under the optimal infection treatment with the same bacterial solution concentration, the GUS transient expression ability of the complete embryonic stem tip explants was better than that of the half embryonic stem tip explants.

[0146] 4.2 Effect of different vacuum treatment pressures on conversion efficiency

[0147] The intact embryonic stem tip explants of Huayu 39 were mixed with OD 600 =1.0 Agrobacterium tumefaciens EHA105 bacterial solution, respectively, under the conditions of vacuum negative pressure of 0.2MPa, 0.5MPa, and 0.8MPa for 5 minutes, shake culture for 50 minutes, and obtain infected explants. Each treatment was repeated 3 times, with 50 explants treated each time. The explants were stained with GUS, and the GUS transient expression rate of the intact embryonic stem tip explants after infection was counted. The results of the GUS transient expression rate of the intact embryonic stem tip explants infected with different vacuum treatment pressures are shown in Figure 2. Figure 15 shown.

[0148] according to Figure 15It can be seen that when the vacuum pressure was 0.2MPa, the GUS transient expression rate of the explants after 5 minutes of treatment reached 76%, and when the vacuum pressure was 0.5MPa, the transient expression rate of the explants after 5 minutes of treatment was 72%. Both transient expression rates were significantly higher than the results after the vacuum pressure was 0.8MPa. When the vacuum pressure was 0.8MPa, the transient conversion rate of the explants after 5 minutes of treatment was only 50%. Within the same vacuum treatment time, as the negative pressure intensity increased, the GUS transient expression rate showed a downward trend. Therefore, it is speculated that excessively high negative pressure intensity may damage the explants and affect the transient infection effect of the explants. Therefore, when the shoot apex explants were vacuumed with a negative pressure of 0.2MPa for 5 minutes, the explants achieved the best infection effect.

[0149] 4.3 Effect of different infection times on transformation efficiency

[0150] The intact embryonic stem tip explants of Huayu 39 were mixed with OD 600 = 1.0 Agrobacterium tumefaciens EHA105 bacterial solution, vacuum negative pressure treatment at a pressure of 0.5 MPa for 5 minutes, shaking culture for 10 minutes, 50 minutes, and 120 minutes respectively, to obtain infected explants. Each treatment was repeated 3 times, with 50 explants treated each time. The explants were stained with GUS, and the GUS transient expression rate of the intact embryonic stem tip explants after infection was counted. The GUS transient expression rate results of the intact embryonic stem tip explants at different infection times are shown in FIG. Figure 16 shown.

[0151] according to Figure 16 It can be seen that when the bacterial liquid infection time is 10 minutes, the transient expression rate of GUS in the explant is only 51.33%. When the explant infection time is increased to 50 minutes, the transient expression rate of GUS in the explant increases to 60.67%. After the explants are shaken for 120 minutes, the transient conversion rate drops to 44%. Too short or too long infection time is not conducive to the transient infection of the explant. Too long shaking time may affect the activity of the explant, and too short time may not allow the bacterial liquid to effectively infect the explant. Therefore, choosing a suitable infection time can help with transient conversion. In the experiment, the explant achieved the highest transient infection efficiency when the infection time was 50 minutes, and 50 minutes can be selected as the shaking culture time for the explant.

[0152] 4.4 Effect of different co-culture times on transformation efficiency

[0153] The intact embryonic stem tip explants of Huayu 39 were mixed with OD 600= 1.0 Agrobacterium tumefaciens EHA105 bacterial solution, vacuum negative pressure treatment at a pressure of 0.5 MPa for 5 minutes, shaking culture for 50 minutes to obtain infected explants, the infected explants were inoculated into the prepared co-cultivation solution and cultured in the dark at 28°C for 2 days, 3 days, 4 days and 5 days to obtain co-cultivated explants, each treatment was repeated 3 times, 50 explants were treated each time, the explants were stained with GUS, and the GUS transient expression rate of the intact embryonic stem tip explants after infection was counted. The GUS transient expression rate results of the intact embryonic stem tip explants with different co-cultivation times are shown in Figure 2. Figure 17 shown.

[0154] according to Figure 17 As can be seen, the GUS transient transformation rate gradually increases with increasing co-culture time. However, Agrobacterium can also overproduce with increasing culture time, toxic to the recipient material, and affect transformation of the explants. The optimal GUS transient expression effect in explants is achieved when the co-culture time is 3 days, so the optimal co-culture time is 3 days.

[0155] In summary, combined with the GUS transient expression results of each treatment, this experiment obtained the optimized transformation process: using the concentration OD 600 =1.0 Agrobacterium tumefaciens EHA105 bacterial liquid was used to infect the intact embryonic stem tip, and vacuum negative pressure treatment was carried out at a pressure of 0.2 MPa for 5 minutes, followed by shaking culture at 80 rpm for 50 minutes to obtain the infected explants, which were then inoculated into the co-culture solution and co-cultured for 3 days.

[0156] Example 3: Peanut genetic transformation

[0157] 1. Peanut genetic transformation

[0158] (1) The complete embryonic stem tip explant of Huayu 39 was mixed with OD 600 =1.0, mixed with Agrobacterium tumefaciens EHA105 bacterial solution, treated under vacuum negative pressure at a pressure of 0.2 MPa for 5 min, and then cultured at 80 rpm for 50 min to obtain infected explants;

[0159] (2) The infected explants were inoculated into the co-cultivation solution prepared in Example 2 and cultured in the dark at 28° C. for 3 days to obtain co-cultivated explants;

[0160] (3) The co-cultured explants were inoculated into the recovery medium obtained by screening in Example 2 for recovery culture. During the recovery culture period, the explants were exposed to light for 16 hours and dark for 8 hours per day. The explants were first cultured at a temperature of 35°C for 3 days and then cultured at a temperature of 25°C for 4 days to obtain recovered explants.

[0161] (4) The explants after recovery culture were inoculated into the screening culture medium obtained by screening in Example 3 for screening culture. The culture temperature during the screening culture was 25° C., the light treatment was 16 h and the dark treatment was 8 h every day, and the screening culture medium was replaced once every 2 weeks. After 6 weeks of culture, genetically transformed explants were obtained. The genetically transformed explants were inoculated into the prepared rooting culture medium for rooting culture for 15 days to obtain genetically transformed seedlings. The genetically transformed seedlings were transplanted into a mixed soil matrix with a mass ratio of vermiculite to peat soil of 3:1 to obtain genetically transformed peanut plants.

[0162] 2. Identification and Detection of Genetically Transformed Peanut Plants

[0163] 2.1. GUS detection of genetically transformed peanut plants

[0164] The peanut genetic transformation method of "1. Peanut genetic transformation" was used to obtain the peanut genetic transformation plants and the color of the plants was observed. The results are as follows: Figure 18 As shown, the left side of the figure shows negative genetic transformation plants, and the right side shows positive genetic transformation plants.

[0165] according to Figure 18 It can be seen that the positive genetic transformation plants are red, and the negative genetic transformation plants have no color change, indicating that the method of "1. Peanut Genetic Transformation" successfully introduced the RUBY gene into the peanut plants.

[0166] Cut the leaves of the positive genetically transformed plants, put them into GUS staining solution for GUS staining, place them at 30℃ for 12 hours, pour out the GUS staining solution, wash and decolorize them with 70% alcohol three times until the green tissue is decolorized to colorless, and use the leaves of normal peanut plants as the control to observe the different leaf colors. The results are as follows: Figure 19 As shown, the left side of the figure shows the leaves of a normal peanut plant, and the right side shows the leaves of the peanut genetically transformed plant obtained in Example 3.

[0167] according to Figure 19 It can be seen that blue spots appeared on the leaves of the peanut transformed plants obtained in "1. Peanut Genetic Transformation", indicating that the genetic transformation method in "1. Peanut Genetic Transformation" successfully constructed peanut transformed plants containing the GUS gene.

[0168] 2.2 PCR detection of genetically transformed peanut plants

[0169] According to the instructions of the Plant Genomic DNA Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat. No. DP305), DNA of the six peanut genetic transformation-positive plants obtained in "1. Peanut Genetic Transformation" was extracted. Based on the sequences of the GUS gene and the RUBY gene, corresponding primer pairs were designed. Then, PCR amplification reactions were performed using the extracted DNA as templates and the primer pairs, respectively, to obtain PCR amplification products. The PCR amplification products were detected by agarose gel electrophoresis. The results are shown as follows: Figure 20 As shown in the figure, lane P represents the M1-4 recombinant expression vector, H2O represents water, T-1 to T-6 represent 6 peanut genetically transformed positive plants, the primer pair sequences of the GUS gene and the RUBY gene are shown in Table 2, the PCR reaction system is shown in Table 3, and the PCR amplification program is shown in Table 4.

[0170] Table 2 Primer pair sequences for GUS and RUBY genes

[0171]

[0172] Table 3 PCR reaction system

[0173]

[0174]

[0175] Table 4 PCR amplification program

[0176]

[0177] according to Figure 20 As can be seen, some peanut plants that were positive for genetic transformation showed single bands around 359 bp (RUBY) and 638 bp (GUS), which are consistent with the target fragment sizes. The color, GUS staining, and PCR identification results of the peanut plants were consistent. This indicates that the genetic transformation method described in "1. Peanut Genetic Transformation" successfully constructed peanut plants containing the GUS and RUBY genes.

[0178] 2.3 Determination of transformation efficiency of peanut genetic transformation

[0179] Using the method in "1. Peanut Genetic Transformation", 1000 intact stem tip explants of peanut seeds were genetically transformed to obtain genetically transformed plants. All the genetically transformed peanut plants were then tested by PCR. The results showed that among the genetically transformed peanut plants prepared using the method in "1. Peanut Genetic Transformation", 5 plants were positive for both the GUS gene and the RUBY gene, with a transformation rate of 0.5%.

[0180] As can be seen from the above examples, the present invention provides a method for genetic transformation of peanuts, comprising the following steps: using ultrasound and vacuum negative pressure treatment to assist the infection of peanut explants with Agrobacterium tumefaciens, followed by co-cultivation, recovery culture, and screening culture to obtain genetically transformed explants; rooting the genetically transformed explants to obtain seedlings; and then transplanting them into a substrate for culture to obtain genetically transformed peanut plants. By optimizing the genetic transformation parameters of peanuts, the present invention successfully produced transgenic peanut plants, improving the efficiency of genetic transformation and providing an important genetic foundation for gene function research and the creation of new germplasm materials.

[0181] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A peanut genetic transformation method, characterized in that: The steps include: (1) Mixing peanut explants with Agrobacterium bacterial solution, vacuum-treating for 4-6 minutes, and shaking-culturing for 45-55 minutes to obtain infected explants; (2) inoculating the infected explants into a co-cultivation solution with a pH of 5.2 to 5.6, and culturing in the dark at 26 to 30° C. for 2 to 4 days to obtain co-cultivated explants; (3) inoculating the co-cultured explants into a recovery medium with a pH value of 5.5 to 5.8 for recovery culture for 5 to 9 days to obtain recovered explants; (4) The explants after recovery culture are inoculated into a screening culture medium with a pH value of 5.5 to 5.8 and cultured for 4 to 8 weeks to obtain genetically transformed explants, and the genetically transformed explants are inoculated into a rooting culture medium with a pH value of 5.6 to 5.8 and cultured for 14 to 16 days to obtain genetically transformed seedlings, and the genetically transformed seedlings are transplanted into a substrate and cultured to obtain genetically transformed peanut plants.

2. The peanut genetic transformation method according to claim 1, characterized in that The peanut explant in step (1) is the complete embryonic stem tip of a peanut seed.

3. The peanut genetic transformation method according to claim 1, characterized in that The Agrobacterium in step (1) is Agrobacterium tumefaciens EHA105; The OD of the Agrobacterium culture solution 600 The value is 0.8~1.

2.

4. The peanut genetic transformation method according to claim 1, characterized in that The pressure of the vacuum negative pressure treatment in step (1) is 0.1-0.3 MPa; The rotation speed of the shaking culture is 60-100 rpm.

5. The peanut genetic transformation method according to claim 1, characterized in that The co-cultivation solution in step (2) uses water as a solvent and includes the following components at the following final concentrations: Potassium nitrate 900-1100 mg / L, ammonium sulfate 50.6-56.6 mg / L, sodium dihydrogen phosphate 55-65 mg / L, magnesium sulfate 95-105 mg / L, calcium chloride 55-65 mg / L, thiamine hydrochloride 0.5-1.5 mg / L, niacin 5-15 mg / L, pyridoxine hydrochloride 5-15 mg / L, potassium iodide 0.364-0.964 mg / L, boric acid 2.96-6.96 mg / L, manganese sulfate 15.84-19.84 mg / L, zinc sulfate 4.88-8.88 mg / L, sodium molybdate 0.1-0.3 mg / L, copper sulfate 0.01-0.03 mg / L, cobalt chloride 0.01-0.03 mg / L and 2-(N-morpholino)ethanesulfonic acid 38,000-40,000 mg / L.

6. The peanut genetic transformation method according to claim 1, characterized in that The recovery medium in step (3) uses water as a solvent and includes the following components at the following final concentrations: Ammonium nitrate 390-410 mg / L, potassium sulfate 940-960 mg / L, potassium dihydrogen phosphate 160-180 mg / L, magnesium sulfate 360-380 mg / L, calcium nitrate 390-410 mg / L, calcium chloride 94-98 mg / L, manganese sulfate 20-24 mg / L, zinc sulfate 7.6-9.6 mg / L, boric acid 5.2-7.2 mg / L, copper sulfate 0.15-0.35 mg / L, sodium molybdate 0.15-0.35 mg / L, ferrous sulfate 26.3-28.3 mg / L, disodium EDTA 36.3-38.3 mg / L, thiamine hydrochloride 0.5-1.5 mg / L, pyridoxine hydrochloride 8-12 mg / L, niacin 8-12 mg / L, inositol 90-110 mg / L, glycine 1-3 mg / L, cytokinin 6-BA 4-6 mg / L, thidiazuron 0.1-0.3 mg / L, sucrose 23-27 g / L, plant gel 2-4 g / L, carbenicillin 240-260 mg / L.

7. The peanut genetic transformation method according to claim 1, characterized in that The method for recovering the culture in step (3) is to first culture at a temperature of 33-37°C for 2-4 days, and then culture at a temperature of 23-27°C for 3-5 days; The photoperiod of the recovery culture is 15 to 17 hours of light treatment and 7 to 9 hours of darkness treatment every day.

8. The peanut genetic transformation method according to claim 1, characterized in that The screening medium in step (4) uses water as a solvent and includes the following components at final concentrations: Ammonium nitrate 390-410 mg / L, potassium sulfate 940-960 mg / L, potassium dihydrogen phosphate 160-180 mg / L, magnesium sulfate 360-380 mg / L, calcium nitrate 390-410 mg / L, calcium chloride 94-98 mg / L, manganese sulfate 20-24 mg / L, zinc sulfate 7.6-9.6 mg / L, boric acid 5.2-7.2 mg / L, copper sulfate 0.15-0.35 mg / L , sodium molybdate 0.15-0.35 mg / L, ferrous sulfate 26.3-28.3 mg / L, disodium EDTA 36.3-38.3 mg / L, thiamine hydrochloride 0.5-1.5 mg / L, pyridoxine hydrochloride 8-12 mg / L, niacin 8-12 mg / L, inositol 90-110 mg / L, glycine 1-3 mg / L, 2-(N-morpholino)ethanesulfonic acid 2-6 g / L, cytokinin 6-BA 4-6 mg / L, thidiazuron 0.1-0.3 mg / L, sucrose 23-27 g / L, plant gel 2-4 g / L, carbenicillin 240-260 mg / L, spectinomycin 120-130 mg / L; The screening culture temperature is 23-27° C., and the photoperiod is 15-17 hours of light treatment and 7-9 hours of darkness treatment per day.

9. The peanut genetic transformation method according to claim 1, characterized in that The rooting medium in step (4) uses water as a solvent and includes the following components at final concentrations: Ammonium nitrate 1600-1700 mg / L, potassium sulfate 1850-1950 mg / L, potassium dihydrogen phosphate 160-180 mg / L, magnesium sulfate 360-380 mg / L, calcium nitrate 430-450 mg / L, manganese sulfate 21.3-23.3 mg / L, zinc sulfate 7.6-9.6 mg / L, boric acid 5.2-7.2 mg / L, copper sulfate 0.015-0.035 mg / L, sodium molybdate 0.15-0.35 mg / L, potassium iodide 0 .73~0.93mg / L, cobalt chloride 0.015~0.035mg / L, sodium iron EDTA 35.7~37.7mg / L, thiamine hydrochloride 0.05~0.15mg / L, niacin 0.3~0.7mg / L, pyridoxine hydrochloride 0.3~0.7mg / L, inositol 90~110mg / L, glycine 1~3mg / L, auxin IBA 0.5~1.5mg / L, sucrose 20~40g / L, agar 7.3~9.3g / L; The temperature of the rooting culture is 23-27° C., and the photoperiod is 15-17 hours of light treatment and 7-9 hours of darkness treatment per day.

10. Use of the peanut genetic transformation method according to any one of claims 1 to 9 in cultivating high-yield, high-quality transgenic peanuts.

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