Method for efficient regeneration and genetic transformation of young leaves of peanut variety

Abstract

The invention belongs to the technical field of plant tissue culture and rapid propagation, and particularly relates to a method for efficient regeneration and genetic transformation of young leaves of a peanut variety. The method mainly comprises the following steps: disinfecting, soaking and germinating seeds, preparing transformed bacteria, obtaining and cutting sterile young leaves, obtaining agrobacterium-infected young leaves, inducing and extending adventitious buds, identifying transformed plants, performing rooting culture, and performing acclimatization and transplanting. According to the method, for the Lu Hua No.3 peanut variety, peanut young leaves serve as explants, only 6-BA and NAA are added into an induction culture medium, the adventitious bud induction efficiency can be 95% or above, and the probability of generating loose lump and snowflake-shaped calluses is small; the method is simple to operate and short in seedling time, sterile seedlings are obtained in a short time by using young leaves under in-vitro culture according to the characteristics of cell totipotency, and a foundation is laid for efficient tissue culture and genetic transformation of peanuts.

Description

Technical field:

[0001] The invention belongs to the technical field of plant tissue culture and rapid propagation, and in particular relates to a method for efficient regeneration and genetic transformation of young leaves of peanut varieties. Technical background:

[0002] Peanut is an important oil crop with high yield and strong adaptability. It has a long history of cultivation and economic benefits around the world. At present, cultivated peanuts are facing problems such as difficulty in breaking through yield, serious pests and diseases, and narrow quality adaptability. These problems seriously restrict the development of the peanut industry. In order to solve these problems, variety improvement has become one of the key ways. Peanut is an allotetraploid self-pollinating crop. The traditional peanut breeding method mainly relies on artificial hybrid breeding, but this method is inefficient and limited by the scarcity of available genetic resources, the close linkage between excellent resistance gene sources and undesirable traits, and the incompatibility of interspecific hybridization. Therefore, the use of bioengineering technology can improve breeding efficiency and provide technical support for the molecular design of peanuts. Through genetic engineering technology, the selected excellent genes can be introduced into peanuts, providing a new way for peanut breeding and germplasm innovation. The establishment of an efficient regeneration system and genetic transformation technology can accelerate the peanut breeding process and innovate a large number of germplasm resources, which is of great significance to peanut variety improvement and gene function research.

[0003] So far, a variety of peanut parts have been used as explants to obtain regenerated plants, including roots, stems, leaves, young embryos, anthers, pericarps, cotyledons and protoplasts. The efficiency of peanut tissue culture has always been highly dependent on genotypes, and the regeneration efficiency varies greatly between different varieties, and the regeneration efficiency is not high. A successful peanut regeneration system depends on genotype, explant type, plant hormone type, etc. Once these factors change, they will have a certain degree of impact on the regeneration system. Summary of the invention:

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art. The present invention uses the peanut variety "Luhua No. 3" to provide a method for efficient tissue culture regeneration and genetic transformation of peanut young leaves. The operation is simple and efficient, and provides a basis for peanut genetic transformation and molecular breeding.

[0005] In order to achieve the above object, the present invention provides a method for efficiently regenerating young leaves of peanut varieties, which specifically comprises the following steps:

[0006] (a) Seed disinfection and seed soaking for germination: Select full and mature peanut seeds for disinfection and sterilization, then soak them in sterile water for 12-14 hours, drain the seeds, peel off the inner seed coat, and place them in germination medium for 5 days;

[0007] (b) Obtaining and cutting sterile young leaves: Take out the peanut seedlings after germination for 5 days, place them on a sterilized culture dish, cut the unexpanded young leaves, separate the four leaflets, cut two horizontal cuts in the middle of the leaflets, and spread the leaflets flat on the induction medium;

[0008] (c) Induction and elongation of adventitious buds: Young leaves are continuously cultured in the induction medium, and adventitious buds are separated at 12-18 days. The culture medium is then replaced with the same induction medium to maintain the elongation of the adventitious buds. This step is repeated every 15 days.

[0009] (d) Rooting culture: When the adventitious buds grow to 2-3 cm, separate the adventitious buds with good growth and place them in rooting medium, subculture every 15 days;

[0010] (e) Hardening and transplanting: When the adventitious roots are 3-4 cm long and the upper part of the roots is 4-5 cm high, open the culture bottle cap to allow the plants to adapt to the external environment. During this period, observe the seedlings at all times and replenish water to the seedlings in a timely manner in the form of spray. After 2-3 days, transplant them into a pot containing sterilized nutrient soil.

[0011] Furthermore, the peanut variety is "Luhua No. 3".

[0012] Furthermore, the germination medium is MS medium (4.4 g / L) + agar powder (6.5 g / L) + sucrose (30 g / L), and the pH value is 5.84; the induction medium is MS medium (4.4 g / L) + agar powder (6.5 g / L) + sucrose (30 g / L) + 6-BA (4 mg / L) + NAA (0.8 mg / L), and the pH value is 5.84; the rooting medium is MS medium (4.4 g / L) + agar powder (6.5 g / L) + sucrose (30 g / L) + NAA (1.4 mg / L), and the pH value is 5.84.

[0013] Furthermore, the specific steps of seed sterilization are as follows: select full and mature peanut seeds, peel off the pods, soak and shake in 75% alcohol in a sterilized culture bottle for 1 minute, then wash with sterilized water 3-4 times, each time for 10 seconds; then soak and sterilize the peanuts in 0.1% mercuric chloride for 15 minutes, and rinse with sterilized water 4-5 times, each time for 2 minutes. It should be noted that if the seeds are old and there is a lot of dust on the surface of the pods, they need to be soaked in mercuric chloride for 18 minutes.

[0014] Furthermore, the culture conditions for the whole process are as follows: the culture temperature is 22°C, the light culture is 16 hours, and the dark culture is 8 hours per day.

[0015] Experiments show that the young leaf efficient regeneration method established by the present invention can achieve the regeneration of peanut tissue culture seedlings within two months.

[0016] The present invention also provides a method for efficient genetic transformation of young leaves of peanut varieties, comprising the following steps:

[0017] (a) Seed disinfection and seed soaking for germination: Select full and mature peanut seeds for disinfection and sterilization, then soak them in sterile water for 12-14 hours, drain the seeds, peel off the inner seed coat, and place them in germination medium for 5 days;

[0018] (b) Preparation of transformant bacteria: The plasmid pCAMBIA2300-35S-OCS carrying the peanut purple seed coat gene AhTc1 was transformed into the competent E. coli DH5α, and the plasmid was extracted. After PCR amplification, agarose gel electrophoresis was performed to verify it, and Agrobacterium competent GV3101 was transformed. After the transformation operation, 100 μl of the revived bacterial solution was applied to the solid LB medium containing rifampicin Rif (50 mg / L) and kanamycin Kan resistance (50 mg / L), and cultured in the dark for 2 to 3 days. Pick a single colony and shake the bacteria. When the bacterial solution is turbid (OD value 1.0-1.2), take 1 μl, and verify the bacterial solution by agarose gel electrophoresis after PCR amplification. The target fragment size is 750 bp. After verification, add sterilized 50% glycerol and mix it with the bacterial solution in a ratio of 1:1, and place it at -80℃ for bacteria preservation;

[0019] The transformed Agrobacterium was streaked on a culture plate of resistant LB solid medium, cultured in the dark at 28°C for 36 hours, and then a single colony was picked and shaken at 200 rpm at 28°C for 16 hours; until the bacterial solution reached the logarithmic growth phase (OD value 1.0-1.2), the bacterial solution was centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the solution was added to the bacterial resuspension solution and resuspended to an OD value of 0.5. Acetosyringone AS (100 μmol / L) dissolved in dimethyl sulfoxide (DMSO) was added to the resuspension solution.

[0020] (c) Obtaining sterile young leaves: Take out the peanut seedlings after germination for 5 days, place them on a sterilized culture dish, cut the unexpanded young leaves, separate the four leaflets, and make two horizontal cuts in the middle of the leaflet.

[0021] (d) Obtaining young leaves infected with Agrobacterium: The explant infection adopts the leaf disc transformation method. In an ultra-clean workbench, the cut peanut young leaves are placed in the Agrobacterium cell resuspension solution, and shaken and cultured in a shaker at 28°C and 180 rpm for about 30 minutes. After that, the excess bacterial solution is absorbed with sterile filter paper and placed in the co-culture medium for dark culture for 4 days.

[0022] (e) Induction of adventitious buds: After the co-cultivation, the explants in the culture dish are transferred to an adventitious bud induction medium for subsequent cultivation, the number of adventitious buds is observed and recorded, and the adventitious bud induction medium is replaced in a timely manner; after the adventitious buds grow up, they are transferred to a subculture medium for subculture;

[0023] (f) Identification of transformed plants: Grind the young leaves of the infected seedlings into powder, extract DNA using the SteadyPurePlant Genomic DNA Extraction Kit (AG, Qingdao, China), use the plasmid DNA containing the target gene as a positive control and the untransformed plant as a negative control, perform PCR amplification, and detect the positive rate. Then, use the SteadyPurePlant RNAExtraction Kit (AG, Qingdao, China) to extract RNA from the peanut tissue culture seedling gene regeneration plant of the positive transgenic plant, reverse transcribe it into cDNA, and perform qRT-PCR relative expression measurement to complete the establishment of the peanut tissue culture seedling transgenic system;

[0024] (g) Rooting culture: When the adventitious buds grow to 2-3 cm, separate the adventitious buds with good growth and place them in rooting medium, subculture every 15 days;

[0025] (h) Hardening and transplanting: When the adventitious roots are 3-4 cm long and the upper part of the roots is about 4-5 cm high, open the culture bottle cap to allow the plants to adapt to the external environment. During this period, observe the seedlings at all times and replenish water to the seedlings in a timely manner in the form of spray. After 2-3 days, transplant them into a pot containing sterilized nutrient soil.

[0026] Furthermore, the tissue culture peanut variety is "Luhua No. 3".

[0027] Further, the germination medium is MS medium (4.4 g / L) + agar powder (6.5 g / L) + sucrose (30 g / L), with a pH value of 5.84; the resistance LB solid medium is yeast powder (5 g / L) + tryptone (10 g / L) + sodium chloride (10 g / L) + agar powder (15 g / L) + Kan (50 mg / L) + Rif (50 mg / L); the resistance LB liquid medium is yeast powder ( 5g / L)+tryptone (10g / L)+sodium chloride (10g / L)+Kan (50mg / L)+Rif (50mg / L); the bacterial resuspension was MS medium (4.4g / L)+sucrose (30g / L)+6-BA (4.5mg / L)+NAA (0.7mg / L)+AS (100μmol / L), with a pH value of 5.84; the co-culture medium was MS medium (4.4g / L) + agar powder (6.5g / L) + sucrose (30g / L) + 6-BA (4.5mg / L) + NAA (0.7mg / L) + Kan (50mg / L) + Rif (50mg / L) + AS (100μmol / L), pH value is 5.84; adventitious bud induction medium is MS medium (4.4g / L) + agar powder (6.5g / L) + sucrose (30g / L) + 6-BA (4m g / L)+NAA(0.8mg / L)+Kan(75mg / L)+Cef(50mg / L)+Tim(50mg / L), pH value is 5.84; rooting medium is MS medium (4.4g / L)+agar powder (6.5g / L)+sucrose (30g / L)+NAA(1.4mg / L)+Kan(75mg / L)+Cef(50mg / L), pH value is 5.84. Kan is kanamycin, Rif is rifampicin, AS is acetosyringone, Cef is cefoxime, and Tim is timentin.

[0028] Furthermore, the specific steps of seed sterilization are as follows: select full and mature peanut seeds, peel off the pods and shells, soak and shake in 75% alcohol in a sterilized culture bottle for 1 minute, and then wash with sterilized water 3-4 times, each time for 10 seconds. After soaking and sterilizing peanuts in 0.1% mercuric chloride for 15 minutes, rinse with sterilized water 4-5 times, each time for 2 minutes. It should be noted that if the seeds are old and there is a lot of dust on the surface of the shell, they need to be soaked in mercuric chloride for 18 minutes.

[0029] Furthermore, the culture conditions for the whole process are culture temperature of 22°C, light culture for 16h and dark culture for 8h.

[0030] The primers used for PCR amplification at the DNA level in the present invention are AhTc1-F / R, AhTc1-F: 5'-CCAGGCTTTACACTTTATGC-3'; AhTc1-R: 3'-GCGATTAAGTTGGGTAACGC-5'; the PCR reaction system is: template DNA 1μl, H2O 7μl, primer AhTc1-F 1μl, primer AhTc1-R 1μl, 2×Buffer 10μl; the PCR reaction process is: pre-denaturation at 94°C for 30s, then denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 1min, 35 cycles, and finally extension at 72°C for 2min.

[0031] By applying the technical solution of the present invention and utilizing the above peanut tissue culture method and steps, the regeneration system obtained from peanuts is suitable for Agrobacterium transformation.

[0032] Compared with the prior art, the present invention is aimed at the Luhua No. 3 peanut variety, uses peanut young leaves as explants, and only adds 6-BA and NAA to the induction medium, which can make the adventitious bud induction efficiency above 95%, and the probability of producing loose mass and snowflake-shaped callus tissue is small; the method is simple to operate and has a short seedling time. According to the characteristics of cell totipotency, young leaves are used to obtain sterile seedlings in a short time under in vitro culture, laying a foundation for efficient tissue culture and genetic transformation of peanuts. Description of the drawings:

[0033] Figure 1 The present invention relates to a plasmid pCAMBIA2300-35S-OCS schematic diagram, which carries the peanut purple seed coat gene AhTc1 and the selection marker gene NPTII (kanamycin resistance).

[0034] Figure 2 This is a diagram of electrophoresis detection of the plasmid and bacterial solution involved in the present invention; wherein 1, 2, 3, and 4 are transformed Agrobacterium, and Escherichia coli is Escherichia coli carrying the AhTc1 target gene.

[0035] Figure 3 This is a schematic diagram of the young leaf tissue culture process of Example 1 Luhua No. 3 involved in the present invention, wherein a is Luhua 3 germinated for 5 days; b is the young leaves of Luhua 3 co-cultured for 4 days; c is the young leaf explants in the adventitious bud induction medium; dg is the continuous subculture process of the young leaf explants; h is the adventitious buds cut off and placed in the rooting medium for 7 days; i is the root system of h.

[0036] Figure 4Figure 1 is a transgenic detection diagram of the young leaves of Luhua No. 3, where a is the PCR agarose gel electrophoresis gel diagram of the plants transformed with the young leaves of Luhua No. 3, 1-11 are randomly selected transgenic plants, and 5 transformed seedlings have the same size as the target band, namely No. 2, No. 3, No. 6, No. 8 and No. 10; b is the qRT-PCR result of the transformed seedlings; c is the DNA sequencing result of the positive plants, the yellow horizontal line is the plasmid sequence, and the red horizontal line is the AhTc1 sequence. Specific implementation method:

[0037] The present invention is further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0038] Embodiment 1:

[0039] This embodiment relates to a method for efficiently regenerating young leaves of peanut varieties, which specifically includes the following steps:

[0040] (a) Screening of peanut varieties: Through tissue culture screening of different peanut varieties, a peanut variety "Luhua No. 3" with a germination rate and adventitious bud induction rate of 100% was selected;

[0041] (b) Seed disinfection and soaking for germination: Select full and mature peanut seeds for disinfection and sterilization, specifically: select full and mature peanut seeds, peel off the pod shells, soak and shake in 75% alcohol in a sterile culture bottle for 1 minute, and then wash with sterile water 3-4 times, each time for 10 seconds; then soak and sterilize the peanuts in 0.1% mercuric chloride for 15 minutes, and then rinse with sterile water 4-5 times, each time for 2 minutes. It should be noted that if the seeds are old and there is a lot of dust on the surface of the shell, they need to be soaked in mercuric chloride for 18 minutes; then place the disinfected and sterilized seeds in sterile water for 12-14 hours, drain the seeds, peel off the inner seed coat, place them in germination medium, and culture for 5 days; the germination medium is MS medium (4.4g / L) + agar powder (6.5g / L) + sucrose (30g / L), with a pH value of 5.84;

[0042] (c) Obtaining and cutting sterile young leaves: Peanut seedlings were taken out after germination for 5 days, placed on sterile culture dishes, and unexpanded young leaves were cut. The four leaflets were separated, and two cuts were made in the middle of the leaflets. The leaflets were spread flat on the induction medium; the induction medium was MS medium (4.4 g / L) + agar powder (6.5 g / L) + sucrose (30 g / L) + 6-BA (4 mg / L) + NAA (0.8 mg / L), with a pH of 5.84;

[0043] (d) Induction and elongation of adventitious buds: After 12-18 days of continuous culture in the induction medium, the young leaves gradually become larger and thicker, and callus tissue is formed. Then, dense small protrusions appear on the surface of the swollen protrusions. At this time, the adventitious buds are separated and replaced with the same induction medium to maintain the elongation of the adventitious buds. This step is subcultured every 15 days; the induction efficiency of adventitious buds is more than 95%;

[0044] (e) Rooting culture: When the adventitious buds grow to 2-3 cm, separate the adventitious buds with good growth and place them in rooting medium, subculture every 15 days; the rooting medium is MS medium (4.4 g / L) + agar powder (6.5 g / L) + sucrose (30 g / L) + NAA (1.4 mg / L), with a pH of 5.84;

[0045] (f) Hardening and transplanting: When the adventitious roots are 3-4 cm long and the upper part of the roots is about 4-5 cm high, open the culture bottle cap to allow the plants to adapt to the external environment. During this period, observe the seedlings at all times and replenish water to the seedlings in time in the form of spray. After 2-3 days, transplant them into a pot containing sterilized nutrient soil.

[0046] The whole process culture conditions of peanut leaflet regeneration in this example are as follows: the culture temperature is 22° C., the light culture is 16 h and the dark culture is 8 h every day.

[0047] Embodiment 2:

[0048] This embodiment relates to a method for efficient genetic transformation of young leaves of a peanut variety, the peanut variety being Luhua No. 3, comprising the following steps:

[0049] (a) Seed disinfection and soaking for germination: Select full and mature peanut seeds for disinfection and sterilization. The specific steps are as follows: Select full and mature peanut seeds, peel off the pods and shells, soak and shake in 75% alcohol in a sterilized culture bottle for 1 minute, and then wash with sterile water 3-4 times, each time for 10 seconds. After soaking and sterilizing peanuts in 0.1% mercuric chloride for 15 minutes, rinse with sterile water 4-5 times, each time for 2 minutes. It should be noted that if the seeds are old and there is a lot of dust on the surface of the shell, they need to be soaked in mercuric chloride for 18 minutes; soak the disinfected and sterilized seeds in sterile water for 12-14 hours, drain the seeds, peel off the inner seed coat, place them in germination medium, and culture for 5 days; the germination medium is MS medium (4.4g / L) + agar powder (6.5g / L) + sucrose (30g / L), with a pH value of 5.84;

[0050] (b) Preparation of transformant bacteria: The plasmid pCAMBIA2300-35S-OCS carrying the peanut purple seed coat gene AhTc1 was transformed into the competent Escherichia coli DH5α, and the plasmid was extracted and verified by agarose gel electrophoresis after PCR amplification. The Agrobacterium competent GV3101 was transformed. After the transformation operation was completed, 100 μl of the resuscitated bacterial solution was spread on a solid LB medium containing Rif (50 mg / L) and Kan resistance (50 mg / L), and cultured in the dark for 2 to 3 days; a single colony was picked and shaken for culture. When the bacterial solution became turbid (OD value 1.0-1.2), 1 μl was taken and the bacterial solution was verified by PCR and agarose gel electrophoresis. The results are as follows: Figure 2 As shown; the PCR primers on the above DNA level are AhTc1-F / R, AhTc1-F: 5'-CCAGGCTTTACACTTTATGC-3'; AhTc1-R: 3'-GCGATTAAGTTGGGTAACGC-5'; the target fragment size is 750bp, after verification, sterilized 50% glycerol is added to the bacterial solution in a 1:1 ratio and then placed at -80℃ for bacterial preservation;

[0051] The transformed Agrobacterium was streaked on a culture plate containing LB solid medium with Kan resistance and Rif resistance. After culturing in the dark at 28°C for 36 hours, a single colony was picked and propagated in LB liquid medium at 28°C at 200 rpm for 16 hours until the bacterial solution reached the logarithmic growth phase (OD value 1.0-1.2). The bacterial solution was centrifuged at 4000 rpm for 5 minutes and the supernatant was discarded. The bacterial bodies were added to the resuspension solution and resuspended to an OD value of 0.5. The resuspension solution was added with DM SO dissolved AS (100 μmol / L); the resistance LB solid culture medium is yeast powder (5 g / L) + tryptone (10 g / L) + sodium chloride (10 g / L) + agar powder (15 g / L) + Kan (50 mg / L) + Rif (50 mg / L); the resistance LB liquid culture medium is yeast powder (5 g / L) + tryptone (10 g / L) + sodium chloride (10 g / L) + Kan (50 mg / L) + Rif (50 mg / L);

[0052] (c) Obtaining sterile young leaves: Take out the peanut seedlings after germination for 5 days, place them on a sterilized culture dish, cut the unexpanded young leaves, separate the four leaflets, and make two horizontal cuts in the middle of the leaflet;

[0053] (d) Obtaining young leaves infected with Agrobacterium: The leaf disc transformation method was used for explant infection. The cut young peanut leaves were placed in a heavy suspension of Agrobacterium in a clean bench and cultured in a shaker at 28°C and 180 rpm for about 30 min. The excess bacterial solution was then dried with sterile filter paper and placed in a co-culture medium (Kan, Rif 50mg / L; AS100μmol / L) in dark culture for 4d; the bacterial resuspension is MS medium (4.4g / L) + sucrose (30g / L) + 6-BA (4.5mg / L) + NAA (0.7mg / L) + AS (100μmol / L), and the pH value is 5.84; the co-culture medium is MS medium (4.4g / L) + agar powder (6.5g / L) + sucrose (30g / L) + 6-BA (4.5mg / L) + NAA (0.7mg / L) + Kan (50mg / L) + Rif (50mg / L) + AS (100μmol / L), and the pH value is 5.84;

[0054] (e) Induction of adventitious buds: After the co-cultivation, the explants in the culture dish were transferred to an induction medium to induce adventitious buds for subsequent cultivation. The induction medium contained antibiotics Kan 75 mg / L, Cef 50 mg / L, and Tim 50 mg / L. The number of adventitious buds was observed and recorded, and the adventitious bud induction medium was replaced in a timely manner. After the adventitious buds grew up, they were transferred to an induction medium for subculture. The induction medium was MS medium (4.4 g / L) + agar powder (6.5 g / L) + sucrose (30 g / L) + 6-BA (4 mg / L) + NAA (0.8 mg / L) + Kan (75 mg / L) + Cef (50 mg / L) + Tim (50 mg / L), and the pH value was 5.84. The addition of antibiotics was to prevent excessive growth of Agrobacterium.

[0055] (f) Identification of transformed plants: Young leaves of infected seedlings were ground into powder and DNA was extracted using the SteadyPurePlant Genomic DNAExtraction Kit (AG, Qingdao, China). DNA was sequenced and PCR amplification and agarose gel assay were performed using plasmid DNA containing the target gene as a positive control (P) and untransformed plants as a negative control (WT) to determine whether the exogenous gene was integrated into the genomic DNA of the plant. The primers used for PCR were AhTc1-F / R. The positive rate was tested. The results are shown in Table 2. Figure 4 a; the SteadyPure Plant RNA Extraction Kit (AG, Qingdao, China) was used to extract RNA from the positive peanut tissue culture seedling gene regeneration plants, reverse transcribed into cDNA, and qRT-PCR was performed to measure the relative expression of AhTc1 in the infected seedlings. The results are shown in Figure 4As shown in b, the establishment of the transgenic system of peanut tissue culture seedlings is completed;

[0056] The PCR reaction system at the DNA level is: 1 μl of template DNA, 7 μl of H2O, 1 μl of primer AhTc1-F, 1 μl of primer AhTc1-R, and 10 μl of 2×Buffer; the PCR reaction process is: pre-denaturation at 94°C for 30 s, then denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 35 cycles, and finally extension at 72°C for 2 min.

[0057] In gel electrophoresis, the specific bands amplified by the transformed plants and the positive control were consistent, and the negative control (untransformed plants) did not amplify any bands, indicating that the exogenous gene had been integrated into the genomic DNA of the plants, and the PCR positive detection rate of the transformed plants was about 45.5%, indicating that the success rate of the genetic transformation method of the present invention was 45.5%.

[0058] The qRT-PCR process was performed according to the instructions. After the RNA was extracted, gDNA was removed and reverse transcription was performed simultaneously. The reaction conditions were 37°C for 15 min, 85°C for 5 s, and 4°C Hold. The obtained cDNA was directly subjected to qRT-PCR analysis and the operation was continued according to the instructions.

[0059] (g) Rooting culture: When the adventitious buds grow to 2-3 cm, separate the adventitious buds with good growth and place them in rooting medium, subculture every 15 days; the rooting medium is MS medium (4.4 g / L) + agar powder (6.5 g / L) + sucrose (30 g / L) + NAA (1.4 mg / L) + Kan (75 mg / L) + Cef (50 mg / L), and the pH value is 5.84.

[0060] (h) Hardening and transplanting: When the adventitious roots are 3-4 cm long and the upper part of the roots is about 4-5 cm high, open the culture bottle cap to allow the plants to adapt to the external environment. During this period, observe the seedlings at all times and replenish water to the seedlings in a timely manner in the form of spray. After 2-3 days, transplant them into a pot containing sterilized nutrient soil.

[0061] Furthermore, the culture conditions for the whole process are culture temperature of 22°C, light culture for 16h and dark culture for 8h.

[0062] In this example, the pCAMBIA2300-35S-OCS plasmid carrying the peanut purple seed coat gene AhTc1 and the selection marker gene NPTII (kanamycin resistance) was transferred into Agrobacterium tumefaciens GV3101 competent cells to perform Agrobacterium-mediated peanut genetic transformation.

[0063] By applying the technical solution of the present invention and utilizing the above peanut tissue culture method and steps, the regeneration system obtained from peanuts is suitable for Agrobacterium transformation.

Claims

1. A method for efficiently regenerating young leaves of peanut varieties, characterized in that: The specific steps include the following: (a) Seed disinfection and seed soaking for germination: Select full and mature peanut seeds for disinfection and sterilization, then soak them in sterile water for 12-14 hours, drain the seeds, peel off the inner seed coat, and place them in germination medium for 5 days; (b) Obtaining and cutting sterile young leaves: Take out the peanut seedlings after germination for 5 days, place them on a sterilized culture dish, cut the unexpanded young leaves, separate the four leaflets, cut two horizontal cuts in the middle of the leaflets, and spread the leaflets flat on the induction medium; (c) Induction and elongation of adventitious buds: Young leaves are continuously cultured in the induction medium, and adventitious buds are separated at 12-18 days. The culture medium is then replaced with the same induction medium to maintain the elongation of the adventitious buds. This step is repeated every 15 days. (d) Rooting culture: When the adventitious buds grow to 2-3 cm, separate the adventitious buds with good growth and place them in rooting medium, subculture every 15 days; (e) Hardening and transplanting: When the adventitious roots are 3-4 cm long and the upper part of the roots is 4-5 cm high, open the culture bottle cap to allow the plants to adapt to the external environment. During this period, observe the seedlings at all times and replenish water to the seedlings in a timely manner in the form of spray. After 2-3 days, transplant them into a pot containing sterilized nutrient soil.

2. The method for efficient regeneration of young leaves of peanut varieties according to claim 1, characterized in that: The peanut variety is "Luhua No. 3".

3. The method for efficient regeneration of young leaves of peanut varieties according to claim 1, characterized in that: The germination medium is MS medium 4.4 g / L + agar powder 6.5 g / L + sucrose 30 g / L, and the pH value is 5.84; the induction medium is MS medium 4.4 g / L + agar powder 6.5 g / L + sucrose 30 g / L + 6-BA 4 mg / L + NAA 0.8 mg / L, and the pH value is 5.84; the rooting medium is MS medium 4.4 g / L + agar powder 6.5 g / L + sucrose 30 g / L + NAA 1.4 mg / L, and the pH value is 5.

84.

4. The method for efficient regeneration of young leaves of peanut varieties according to claim 1, characterized in that: The specific steps for seed sterilization are: select full and mature peanut seeds, peel off the pods, soak and shake in 75% alcohol in a sterilized culture bottle for 1 minute, then wash with sterilized water 3-4 times, each time for 10 seconds; then soak and sterilize the peanuts in 0.1% mercuric chloride for 15 minutes, and rinse with sterilized water 4-5 times, each time for 2 minutes. It should be noted that if the seeds are old and there is a lot of dust on the surface of the pods, they need to be soaked in mercuric chloride for 18 minutes.

5. The method for efficient regeneration of young leaves of peanut varieties according to claim 1, characterized in that: The culture conditions for the whole process are: the culture temperature is 22°C, the light culture is 16 hours and the dark culture is 8 hours every day.

6. A method for efficient genetic transformation of young leaves of peanut varieties, characterized in that: It includes the following steps: (a) Seed disinfection and seed soaking for germination: Select full and mature peanut seeds for disinfection and sterilization, then soak them in sterile water for 12-14 hours, drain the seeds, peel off the inner seed coat, and place them in germination medium for 5 days; (b) Preparation of transformant bacteria: The plasmid pCAMBIA2300-35S-OCS carrying the peanut purple seed coat gene AhTc1 was transformed into the competent Escherichia coli DH5α, and the plasmid was extracted and verified by agarose gel electrophoresis after PCR amplification. Then, the competent Agrobacterium GV3101 was transformed according to the steps in the competent instructions. After transformation, PCR amplification and agarose gel electrophoresis were performed to verify the bacterial solution. After verification, sterilized 50% glycerol was added to the bacterial solution in a ratio of 1:1 and the mixture was placed at -80°C for bacterial preservation; The transformed Agrobacterium was streaked on a plate of resistant LB solid medium, cultured in the dark at 28°C for 36 hours, and then a single colony was picked and shaken at 200 rpm at 28°C for 16 hours; when the bacterial solution reached the logarithmic growth phase, the bacterial solution was centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the solution was added to the bacterial resuspension solution and resuspended to an OD value of 0.5; (c) Obtaining sterile young leaves: Take out the peanut seedlings after germination for 5 days, place them on a sterilized culture dish, cut the unexpanded young leaves, separate the four leaflets, and make two horizontal cuts in the middle of the leaflet; (d) Obtaining young leaves infected with Agrobacterium: The explant infection adopts the leaf disc transformation method. In an ultra-clean workbench, the cut peanut young leaves are placed in the Agrobacterium cell resuspension solution, and shaken and cultured in a shaker at 28°C and 180 rpm for about 30 minutes. After that, the excess bacterial solution is absorbed with sterile filter paper and placed in the co-culture medium for dark culture for 4 days. (e) Induction of adventitious buds: After the co-cultivation, the explants in the culture dish are transferred to an adventitious bud induction medium for subsequent cultivation, the number of adventitious buds is observed and recorded, and the adventitious bud induction medium is replaced in a timely manner; after the adventitious buds grow up, they are transferred to a subculture medium for subculture; (f) Identification of transformed plants: Grind the young leaves of the infected seedlings into powder, extract DNA using the SteadyPure Plant Genomic DNA Extraction Kit, use the plasmid DNA containing the target gene as a positive control and the untransformed plant as a negative control, perform PCR amplification, detect the positive rate, and then use the SteadyPure Plant RNA Extraction Kit to extract RNA from the peanut tissue culture seedling gene regeneration plant of the positive transgenic plant, reverse transcribe it into cDNA, and perform qRT-PCR relative expression measurement to complete the establishment of the peanut tissue culture seedling transgenic system; (g) Rooting culture: When the adventitious buds grow to 2-3 cm, separate the adventitious buds with good growth and place them in rooting medium, subculture every 15 days; (h) Hardening and transplanting: When the adventitious roots are 3-4 cm long and the upper part of the roots is about 4-5 cm high, open the culture bottle cap to allow the plants to adapt to the external environment. During this period, observe the seedlings at all times and replenish water to the seedlings in a timely manner in the form of spray. After 2-3 days, transplant them into a pot containing sterilized nutrient soil.

7. The method for efficient genetic transformation of young leaves of peanut varieties according to claim 6, characterized in that: The tissue culture peanut variety is "Luhua No. 3".

8. The method for efficient genetic transformation of young leaves of peanut varieties according to claim 6, characterized in that: The germination medium is 4.4 g / L MS medium + 6.5 g / L agar powder + 30 g / L sucrose, with a pH value of 5.84; the resistance LB solid medium is 5 g / L yeast powder + 10 g / L tryptone + 10 g / L sodium chloride + 15 g / L agar powder + 50 mg / L Kan + 50 mg / L Rif; the resistance LB liquid medium is 5 g / L yeast powder + 10 g / L tryptone + 10 g / L sodium chloride + 50 mg / L Kan + 50 mg / L Rif; the bacterial resuspension is 4.4 g / L MS medium + 30 g / L sucrose + 6-BA 4.5mg / L)+NAA0.7mg / L+AS100μmol / L, pH value is 5.84; co-cultivation medium is MS medium 4.4g / L+agar powder 6.5g / L+sucrose 30g / L+6-BA4.5mg / L+NAA0.7mg / L+Kan50mg / L+Rif 50mg / L+AS100μmol / L, pH value is 5.84; adventitious bud induction medium is MS medium 4.4g / L+agar powder 6.5g / L+sucrose 30g / L+6-BA 4mg / L+NAA 0.8mg / L+Kan 75mg / L+Cef 50mg / L+Tim 50mg / L, pH value is 5.84; rooting medium is MS medium 4.4g / L+agar powder 6.5g / L+sucrose 30g / L+NAA 1.4mg / L+Kan75mg / L+Cef50mg / L, pH value is 5.84, wherein Kan is kanamycin, Rif is rifampicin, AS is acetosyringone, Cef is ceftriaxone, and Tim is timentin.

9. The method for efficient genetic transformation of young leaves of peanut varieties according to claim 6, characterized in that: The primers used for PCR amplification at the DNA level are AhTc1-F / R, the sequence of AhTc1-F is: 5'-CCAGGCTTTACACTTTATGC-3'; the sequence of AhTc1-R is: 3'-GCGATTAAGTTGGGTAACGC-5'; the PCR reaction system is: template DNA 1μl, H2O7μl, primer AhTc1-F 1μl, primer AhTc1-R 1μl, 2×Buffer 10μl; the PCR reaction process is: pre-denaturation at 94°C for 30s, then denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 1min, 35 cycles, and finally extension at 72°C for 2min.

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