Culture medium for efficient genetic transformation of kidney beans and genetic transformation method

Abstract

The invention provides a culture medium for efficient genetic transformation of kidney beans and a genetic transformation method, and belongs to the field of plant genetic transformation. The culture medium for efficient genetic transformation of kidney beans comprises a co-culture medium and a bud induction culture medium. The preparation method comprises the following steps: dissolving aluminum nitrate nonahydrate and magnesium nitrate hexahydrate, mixing and shearing with a sodium hydroxide solution, centrifuging, washing, adding deionized water, heating, reacting to prepare nano-carrier particles, and supporting titanium dioxide columns among layers of the nano-carrier particles by using tetrabutyl titanate as a raw material through a sol-gel method to prepare the nano-carrier particles. Compared with the prior art, the composite carrier has the advantages that the interlayer spacing of nano-carrier particles is obviously increased, more space is provided for loading ascorbic acid, and the effect of improving the ascorbic acid loading rate is further achieved; therefore, the anti-oxidation persistence of the prepared co-culture medium and bud induction culture medium can be improved.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic transformation, and in particular to a culture medium for efficient genetic transformation of kidney beans and a genetic transformation method. Background Art

[0002] The common bean is one of the most important edible legumes cultivated worldwide, boasting rich nutritional and economic value. However, traditional breeding methods for improving common bean varieties suffer from long cycles and low efficiency. With the development of biotechnology, genetic transformation techniques have provided a new avenue for rapid improvement of common beans. By introducing exogenous genes into bean cells, they can impart desirable traits such as disease resistance and stress tolerance, thereby improving yield and quality.

[0003] Genetic transformation technology is a key tool in modern plant molecular biology research and crop improvement. It involves introducing target genes into plant cells through Agrobacterium-mediated or direct gene bombardment, where they integrate into the plant genome, ultimately yielding transgenic plants. For beans, Agrobacterium-mediated genetic transformation is one of the most commonly used methods, favored for its high efficiency and ease of use.

[0004] Existing culture media usually directly add antioxidants such as ascorbic acid to reduce oxidative damage to explants during the culture process. However, antioxidants are generally unstable and easily decompose and become ineffective during the culture process, and cannot provide continuous antioxidant effects, resulting in low genetic transformation efficiency of kidney beans.

[0005] Therefore, it is necessary to propose a culture medium and a genetic transformation method for efficient genetic transformation of kidney beans that can continuously resist oxidation. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention aims to provide a culture medium and a genetic transformation method for efficient genetic transformation of kidney beans.

[0007] A culture medium for efficient genetic transformation of kidney beans, comprising a co-culture medium and a sprout induction culture medium;

[0008] The co-culture medium includes: MS medium, 20 g / L sucrose, 7 g / L agar, 40-50 mg / L acetosyringone and 0.6-0.8 g / L composite antioxidant;

[0009] The bud induction medium includes: MS medium, 20 g / L sucrose, 7 g / L agar, 100 mg / L kanamycin and 0.6-0.8 g / L compound antioxidant.

[0010] Furthermore, the preparation steps of the composite antioxidant are as follows:

[0011] drying the nanocarrier particles at 100-110° C. to a constant weight to obtain dry nanocarrier particles;

[0012] Tetrabutyl titanate was added to anhydrous ethanol in a volume ratio of 1:(8-10), stirred thoroughly to dissolve, and then ammonia water was added to adjust the pH to 8-9, and the mixture was allowed to stand for aging for 20-24 hours to obtain a titanium dioxide sol;

[0013] Immerse the dried nanocarrier particles in the titanium dioxide sol, ultrasonicate for 20-30 minutes, and stir for 6-8 hours to obtain a suspension;

[0014] The suspension is filtered, and the filter residue is dried at 60-80°C for 8-10 hours, and then placed in a muffle furnace and heat-treated at 450-550°C for 2-4 hours under nitrogen protection to obtain a composite carrier;

[0015] Ascorbic acid was dissolved in deionized water to prepare a 0.1 mol / L ascorbic acid aqueous solution, and then the composite carrier was added at a solid-liquid ratio of 1 g: (40-50) mL. After ultrasonic loading for 20-30 minutes, the composite antioxidant was obtained by filtration and drying.

[0016] Furthermore, the preparation steps of the nanocarrier particles are as follows:

[0017] Add sodium hydroxide to deionized water at a solid-liquid ratio of 1 g: (25-35) mL and stir thoroughly to dissolve to obtain a sodium hydroxide solution;

[0018] Add aluminum nitrate nonahydrate and magnesium nitrate hexahydrate into deionized water at a solid-liquid ratio of 1 g: (1.8-2.2) g: (13-15) mL, stir thoroughly to dissolve, and obtain a mixed solution;

[0019] The mixed solution was added to the sodium hydroxide solution while stirring, and then placed in a high-speed shearing machine. After shearing for 60-80 seconds, the mixture was centrifuged at a speed of 8000-9000 r / min for 8-10 minutes, and the supernatant was discarded to obtain a mixed precipitate.

[0020] The mixed precipitate was washed 2-3 times with deionized water, and then supplemented to the original volume with deionized water, and heated and stirred at 80-90° C. for 5-6 hours to obtain nano-carrier particles.

[0021] Furthermore, when genetic transformation of kidney beans is carried out using a medium for efficient genetic transformation of kidney beans, the method specifically comprises the following steps:

[0022] S1: Sterile bean seeds were placed flat on germination medium for germination. When the germinated embryonic axis reached 2-3 cm, the cotyledons, terminal buds, and radicles were removed, leaving only 1-2 cm of the embryonic axis and cotyledonary nodes to obtain bean explants.

[0023] S2: Activate Agrobacterium GV3101 containing the target plasmid in LB medium until the OD600 reaches 0.6-0.8. Then, collect the activated Agrobacterium suspension by centrifugation and resuspend it in the inoculum until the OD600 reaches 0.4 to obtain a mixed inoculum.

[0024] S3: Immersing the bean explants in the mixed impregnation solution for 10-15 minutes, transferring them to a co-culture medium, culturing them in the dark for 2-3 days, and then transferring them to a washing medium and washing them 3-5 times;

[0025] S4: transferring the bean explants washed with the cleaning medium to a bud induction medium and continuing to culture, replacing the medium every 2-3 weeks, observing the adventitious buds for GFP fluorescence using a variable magnification macromicroscope, and transferring the adventitious buds with GFP fluorescence to an elongation medium and continuing to culture, replacing the medium every 2-3 weeks, until the bean seedlings reach a length of 3-5 cm, thereby obtaining bean sprouts;

[0026] S5: The bean sprouts are transferred to a rooting medium and rooted until at least three roots grow out of the bean sprouts and the root length reaches 3-5 cm, followed by hardening and transplanting.

[0027] Furthermore, the germination medium includes: B5 basal medium, 1 mL / LB5 vitamins, 20 g / L sucrose and 7 g / L agar.

[0028] Furthermore, the cleaning medium includes: MS medium, 20 g / L sucrose, 7 g / L agar, 500 mg / L cephalosporin and 100 mg / L kanamycin.

[0029] Furthermore, the impregnation solution includes: MS liquid culture medium, 0.2% acetosyringone, 0.3% apigenin and 0.1% ferulic acid.

[0030] Furthermore, the elongation medium comprises: MS medium, 0.2 mg / L 6-benzylaminopurine and 500 mg / L cephalosporin.

[0031] Furthermore, the rooting medium includes: MS medium, 20 g / L sucrose, 7 g / L agar and 1 mg / L indoleacetic acid.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] 1. The present invention prepares nanocarrier particles by first dissolving aluminum nitrate nonahydrate and magnesium nitrate hexahydrate, mixing and shearing the mixture with a sodium hydroxide solution, centrifuging and washing the mixture, and then adding deionized water and heating the mixture for reaction. Then, a sol-gel method is used using tetrabutyl titanate as a raw material to support titanium dioxide between the layers of the nanocarrier particles, significantly increasing the interlayer spacing of the nanocarrier particles and providing more space for loading ascorbic acid, thereby achieving the effect of increasing the ascorbic acid loading rate. Moreover, since the composite carrier is used to load ascorbic acid, the ascorbic acid can have a sustained-release property, thereby improving the antioxidant persistence of the prepared co-culture medium and bud induction medium.

[0034] 2. The impregnation solution of the present invention includes MS liquid culture medium, acetosyringone, apigenin and ferulic acid. Apigenin can induce plant cells to produce secondary metabolites, regulate the structure and function of the cell wall, and facilitate the invasion of Agrobacterium. Acetosyringone can not only specifically induce the expression of Vir genes in Agrobacterium to better cut, transfer and integrate T-DNA into the plant matrix, but also enhance the activity of Agrobacterium, enabling it to more effectively adsorb and invade plant cells. When the two are used in combination, apigenin and acetosyringone can work together through different targets to synergistically improve the ability of Agrobacterium to infect bean cells, thereby improving the efficiency of genetic transformation. DETAILED DESCRIPTION

[0035] The following describes in detail a culture medium and a genetic transformation method for efficient genetic transformation of kidney beans provided by the present invention in conjunction with specific examples.

[0036] Example 1

[0037] A method for efficient genetic transformation of kidney beans comprises the following steps:

[0038] (1) Preparation of nanocarrier particles:

[0039] Sodium hydroxide is added to deionized water at a solid-liquid ratio of 1 g: (25-35) mL, and the mixture is stirred and dissolved to obtain a sodium hydroxide solution. Aluminum nitrate nonahydrate and magnesium nitrate hexahydrate are then added to deionized water at a solid-liquid ratio of 1 g: (1.8-2.2) g: (13-15) mL, and the mixture is stirred and dissolved to obtain a mixed solution. Subsequently, the mixed solution is added to the sodium hydroxide solution while stirring, and the mixture is placed in a high-speed shearing machine, sheared for 60-80 seconds, and then centrifuged at a speed of 8000-9000 r / min for 8-10 minutes. The supernatant is discarded to obtain a mixed precipitate. Finally, the mixed precipitate is washed with deionized water for 2-3 times, and then deionized water is added to the original volume, and the mixture is heated and stirred at 80-90° C. for 5-6 hours to obtain nanocarrier particles.

[0040] (2) Preparation of composite antioxidants:

[0041] The nanocarrier particles are dried at 100-110° C. to a constant weight to obtain dry nanocarrier particles for later use. Tetrabutyl titanate is then added to anhydrous ethanol at a volume ratio of 1:(8-10), stirred and dissolved, and then ammonia water is added to adjust the pH to 8-9. The mixture is allowed to stand for aging for 20-24 hours to obtain a titanium dioxide sol. The dry nanocarrier particles are then immersed in the titanium dioxide sol, ultrasonically treated for 20-30 minutes, and stirred for 6-8 hours to obtain a suspension. The suspension is then filtered, and the filter residue is dried at 60-80°C for 8-10 hours, then placed in a muffle furnace and heat treated at 450-550°C for 2-4 hours under nitrogen protection to obtain a composite carrier. Finally, ascorbic acid is dissolved in deionized water to prepare a 0.1 mol / L ascorbic acid aqueous solution, and the composite carrier is added at a solid-liquid ratio of 1 g: (40-50) mL. After ultrasonic loading for 20-30 minutes, the mixture is filtered and dried to obtain a composite antioxidant.

[0042] S1: Sterile bean seeds were placed flat on a germination medium for germination. When the germinated hypocotyl reached 2-3 cm, the cotyledons, terminal buds, and radicles were removed, leaving only 1-2 cm of the hypocotyl and cotyledonary nodes to obtain bean explants. The germination medium consisted of: B5 basal medium, 1 mL / LB5 vitamins, 20 g / L sucrose, and 7 g / L agar.

[0043] S2: Activate Agrobacterium GV3101 containing the target plasmid in LB medium until the OD600 reaches 0.6-0.8, then collect the activated Agrobacterium suspension by centrifugation and resuspend it in an impregnation solution until the OD600 reaches 0.4 to obtain a mixed impregnation solution, wherein the impregnation solution comprises: MS liquid medium, 0.2% acetosyringone, 0.3% apigenin, and 0.1% ferulic acid;

[0044] S3: Immersing the bean explants in the mixed impregnation solution for 10-15 minutes, transferring them to the co-culture medium, culturing them in the dark for 2-3 days, and then transferring them to a washing medium and washing them 3-5 times, wherein the washing medium comprises: MS medium, 20 g / L sucrose, 7 g / L agar, 500 mg / L cephalosporin, and 100 mg / L kanamycin; and the co-culture medium comprises: MS medium, 20 g / L sucrose, 7 g / L agar, 40-50 mg / L acetosyringone, and 0.6-0.8 g / L of the composite antioxidant.

[0045] S4: transferring the bean explants washed with the cleaning medium to the bud induction medium, continuing to culture, replacing the medium every 2-3 weeks, observing the adventitious buds for GFP fluorescence using a variable magnification macromicroscope, and transferring the adventitious buds with GFP fluorescence to an elongation medium for continued culture, replacing the medium every 2-3 weeks, until the bean seedlings reach a length of 3-5 cm, thereby obtaining bean sprouts, wherein the bud induction medium comprises: MS medium, 20 g / L sucrose, 7 g / L agar, 100 mg / L kanamycin, and 0.6-0.8 g / L compound antioxidant, and the elongation medium comprises: MS medium, 0.2 mg / L 6-benzylaminopurine, and 500 mg / L cephalosporin;

[0046] S5: Transferring the bean sprouts to a rooting medium for rooting until the bean sprouts grow at least three roots and the root length reaches 3-5 cm, followed by hardening and transplanting. The rooting medium comprises MS medium, 20 g / L sucrose, 7 g / L agar, and 1 mg / L indoleacetic acid.

[0047] Example 2

[0048] A method for efficient genetic transformation of kidney beans comprises the following steps:

[0049] (1) Preparation of nanocarrier particles:

[0050] Sodium hydroxide is added to deionized water at a solid-liquid ratio of 1 g: (25-35) mL, and the mixture is stirred and dissolved to obtain a sodium hydroxide solution. Aluminum nitrate nonahydrate and magnesium nitrate hexahydrate are then added to deionized water at a solid-liquid ratio of 1 g: (1.8-2.2) g: (13-15) mL, and the mixture is stirred and dissolved to obtain a mixed solution. Subsequently, the mixed solution is added to the sodium hydroxide solution while stirring, and the mixture is placed in a high-speed shearing machine, sheared for 60-80 seconds, and then centrifuged at a speed of 8000-9000 r / min for 8-10 minutes. The supernatant is discarded to obtain a mixed precipitate. Finally, the mixed precipitate is washed with deionized water for 2-3 times, and then deionized water is added to the original volume, and the mixture is heated and stirred at 80-90° C. for 5-6 hours to obtain nanocarrier particles.

[0051] (2) Preparation of composite antioxidants:

[0052] The nanocarrier particles are dried at 100-110° C. to a constant weight to obtain dry nanocarrier particles for later use. Tetrabutyl titanate is then added to anhydrous ethanol at a volume ratio of 1:(8-10), stirred and dissolved, and then ammonia water is added to adjust the pH to 8-9. The mixture is allowed to stand for aging for 20-24 hours to obtain a titanium dioxide sol. The dry nanocarrier particles are then immersed in the titanium dioxide sol, ultrasonically treated for 20-30 minutes, and stirred for 6-8 hours to obtain a suspension. The suspension is then filtered, and the filter residue is dried at 60-80°C for 8-10 hours, then placed in a muffle furnace and heat treated at 450-550°C for 2-4 hours under nitrogen protection to obtain a composite carrier. Finally, ascorbic acid is dissolved in deionized water to prepare a 0.1 mol / L ascorbic acid aqueous solution, and the composite carrier is added at a solid-liquid ratio of 1 g: (40-50) mL. After ultrasonic loading for 20-30 minutes, the mixture is filtered and dried to obtain a composite antioxidant.

[0053] S1: Sterile bean seeds were placed flat on a germination medium for germination. When the germinated hypocotyl reached 2-3 cm, the cotyledons, terminal buds, and radicles were removed, leaving only 1-2 cm of the hypocotyl and cotyledonary nodes to obtain bean explants. The germination medium consisted of: B5 basal medium, 1 mL / LB5 vitamins, 20 g / L sucrose, and 7 g / L agar.

[0054] S2: Activate Agrobacterium GV3101 containing the target plasmid in LB medium until the OD600 reaches 0.6-0.8, then collect the activated Agrobacterium suspension by centrifugation and resuspend it in an impregnation solution until the OD600 reaches 0.4 to obtain a mixed impregnation solution, wherein the impregnation solution comprises: MS liquid medium, 0.2% acetosyringone, 0.3% apigenin, and 0.1% ferulic acid;

[0055] S3: Immersing the bean explants in the mixed impregnation solution for 10-15 minutes, transferring them to the co-culture medium, culturing them in the dark for 2-3 days, and then transferring them to a washing medium and washing them 3-5 times, wherein the washing medium comprises: MS medium, 20 g / L sucrose, 7 g / L agar, 500 mg / L cephalosporin, and 100 mg / L kanamycin; and the co-culture medium comprises: MS medium, 20 g / L sucrose, 7 g / L agar, 40-50 mg / L acetosyringone, and 0.6-0.8 g / L of the composite antioxidant.

[0056] S4: transferring the bean explants washed with the cleaning medium to the bud induction medium, continuing to culture, replacing the medium every 2-3 weeks, observing the adventitious buds for GFP fluorescence using a variable magnification macromicroscope, and transferring the adventitious buds with GFP fluorescence to an elongation medium for continued culture, replacing the medium every 2-3 weeks, until the bean seedlings reach a length of 3-5 cm, thereby obtaining bean sprouts, wherein the bud induction medium comprises: MS medium, 20 g / L sucrose, 7 g / L agar, 100 mg / L kanamycin, and 0.6-0.8 g / L compound antioxidant, and the elongation medium comprises: MS medium, 0.2 mg / L 6-benzylaminopurine, and 500 mg / L cephalosporin;

[0057] S5: Transferring the bean sprouts to a rooting medium for rooting until the bean sprouts grow at least three roots and the root length reaches 3-5 cm, followed by hardening and transplanting. The rooting medium comprises MS medium, 20 g / L sucrose, 7 g / L agar, and 1 mg / L indoleacetic acid.

[0058] Example 3

[0059] A method for efficient genetic transformation of kidney beans comprises the following steps:

[0060] (1) Preparation of nanocarrier particles:

[0061] Sodium hydroxide is added to deionized water at a solid-liquid ratio of 1 g: (25-35) mL, and the mixture is stirred and dissolved to obtain a sodium hydroxide solution. Aluminum nitrate nonahydrate and magnesium nitrate hexahydrate are then added to deionized water at a solid-liquid ratio of 1 g: (1.8-2.2) g: (13-15) mL, and the mixture is stirred and dissolved to obtain a mixed solution. Subsequently, the mixed solution is added to the sodium hydroxide solution while stirring, and the mixture is placed in a high-speed shearing machine, sheared for 60-80 seconds, and then centrifuged at a speed of 8000-9000 r / min for 8-10 minutes. The supernatant is discarded to obtain a mixed precipitate. Finally, the mixed precipitate is washed with deionized water for 2-3 times, and then deionized water is added to the original volume, and the mixture is heated and stirred at 80-90° C. for 5-6 hours to obtain nanocarrier particles.

[0062] (2) Preparation of composite antioxidants:

[0063] The nanocarrier particles are dried at 100-110° C. to a constant weight to obtain dry nanocarrier particles for later use. Tetrabutyl titanate is then added to anhydrous ethanol at a volume ratio of 1:(8-10), stirred and dissolved, and then ammonia water is added to adjust the pH to 8-9. The mixture is allowed to stand for aging for 20-24 hours to obtain a titanium dioxide sol. The dry nanocarrier particles are then immersed in the titanium dioxide sol, ultrasonically treated for 20-30 minutes, and stirred for 6-8 hours to obtain a suspension. The suspension is then filtered, and the filter residue is dried at 60-80°C for 8-10 hours, then placed in a muffle furnace and heat treated at 450-550°C for 2-4 hours under nitrogen protection to obtain a composite carrier. Finally, ascorbic acid is dissolved in deionized water to prepare a 0.1 mol / L ascorbic acid aqueous solution, and the composite carrier is added at a solid-liquid ratio of 1 g: (40-50) mL. After ultrasonic loading for 20-30 minutes, the mixture is filtered and dried to obtain a composite antioxidant.

[0064] S1: Sterile bean seeds were placed flat on a germination medium for germination. When the germinated hypocotyl reached 2-3 cm, the cotyledons, terminal buds, and radicles were removed, leaving only 1-2 cm of the hypocotyl and cotyledonary nodes to obtain bean explants. The germination medium consisted of: B5 basal medium, 1 mL / LB5 vitamins, 20 g / L sucrose, and 7 g / L agar.

[0065] S2: Activate Agrobacterium GV3101 containing the target plasmid in LB medium until the OD600 reaches 0.6-0.8, then collect the activated Agrobacterium suspension by centrifugation and resuspend it in an impregnation solution until the OD600 reaches 0.4 to obtain a mixed impregnation solution, wherein the impregnation solution comprises: MS liquid medium, 0.2% acetosyringone, 0.3% apigenin, and 0.1% ferulic acid;

[0066] S3: Immersing the bean explants in the mixed impregnation solution for 10-15 minutes, transferring them to the co-culture medium, culturing them in the dark for 2-3 days, and then transferring them to a washing medium and washing them 3-5 times, wherein the washing medium comprises: MS medium, 20 g / L sucrose, 7 g / L agar, 500 mg / L cephalosporin, and 100 mg / L kanamycin; and the co-culture medium comprises: MS medium, 20 g / L sucrose, 7 g / L agar, 40-50 mg / L acetosyringone, and 0.6-0.8 g / L of the composite antioxidant.

[0067] S4: transferring the bean explants washed with the cleaning medium to the bud induction medium, continuing to culture, replacing the medium every 2-3 weeks, observing the adventitious buds for GFP fluorescence using a variable magnification macromicroscope, and transferring the adventitious buds with GFP fluorescence to an elongation medium for continued culture, replacing the medium every 2-3 weeks, until the bean seedlings reach a length of 3-5 cm, thereby obtaining bean sprouts, wherein the bud induction medium comprises: MS medium, 20 g / L sucrose, 7 g / L agar, 100 mg / L kanamycin, and 0.6-0.8 g / L compound antioxidant, and the elongation medium comprises: MS medium, 0.2 mg / L 6-benzylaminopurine, and 500 mg / L cephalosporin;

[0068] S5: Transferring the bean sprouts to a rooting medium for rooting until the bean sprouts grow at least three roots and the root length reaches 3-5 cm, followed by hardening and transplanting. The rooting medium comprises MS medium, 20 g / L sucrose, 7 g / L agar, and 1 mg / L indoleacetic acid.

[0069] Comparative Example 1

[0070] The difference between Comparative Example 1 and Example 1 is that the composite carrier in (2) is replaced by an equal amount of nanocarrier particles, that is, ascorbic acid is directly loaded on the nanocarrier particles.

[0071] Comparative Example 2

[0072] The difference between this comparative example 2 and Example 1 is that the apigenin in the impregnation solution of step S2 is replaced by an equal amount of acetosyringone.

[0073] Comparative Example 3

[0074] The difference between this comparative example 3 and Example 1 is that the acetosyringone in the dyeing solution of step S2 is replaced with an equal amount of apigenin.

[0075] Performance testing:

[0076] 1. The loading rate and 72h cumulative release rate of ascorbic acid in the composite antioxidants of Examples 1-3 and Comparative Example 1 were detected and calculated respectively. The results are shown in Table 1.

[0077] Table 1: Comparison of the loading rate and cumulative release rate results of Examples 1-3 and Comparative Example 1

[0078] Load rate (%) 72h cumulative release rate (%) Example 1 95.3 67.8 Example 2 95.5 68.2 Example 3 95.2 67.5 Comparative Example 1 82.7 73.3

[0079] As can be seen from Table 1, when ascorbic acid is directly loaded on nanocarrier particles in Comparative Example 1, the loading rate of ascorbic acid is about 82.7%, which is lower than that in Example 1. This indicates that aluminum nitrate nonahydrate and magnesium nitrate hexahydrate are first dissolved and mixed and sheared with a sodium hydroxide solution, centrifuged and washed, and then deionized water is added and heated to react to prepare nanocarrier particles. Then, a sol-gel method is used using tetrabutyl titanate as a raw material to support titanium dioxide between the layers of the nanocarrier particles, significantly increasing the interlayer spacing of the nanocarrier particles and providing more space for loading ascorbic acid, thereby achieving the effect of increasing the loading rate of ascorbic acid. In addition, in Examples 1-3 and Comparative Example 1, the cumulative release rates of ascorbic acid of the composite antioxidant over 72 h are 67.8%, 68.2%, 67.5% and 73.3%, respectively, indicating that the composite antioxidant has sustained release properties and can improve the antioxidant sustainability of the culture medium.

[0080] 2. The successful integration and expression of the exogenous genes in Examples 1-3 and Comparative Examples 2-3 were confirmed by PCR molecular biology methods. The total number of processed samples and the number of positive transformants were counted, and the genetic transformation rate of kidney bean was calculated. The results are shown in Table 2.

[0081] Table 2: Comparison of genetic conversion rates of Examples 1-3 and Comparative Examples 2-3

[0082] Genetic conversion rate (%) Example 1 25.6 Example 2 25.7 Example 3 25.3 Comparative Example 2 13.2 Comparative Example 3 9.7

[0083] As can be seen from Table 2, when only acetosyringone and apigenin were added to the impregnation solution in Comparative Examples 2 and 3, the genetic transformation rates of kidney bean were approximately 13.2% and 9.7%, respectively, which were lower than those in Examples 1-3. This shows that apigenin and acetosyringone can act together through different targets to synergistically improve the infection ability of Agrobacterium on kidney bean cells, thereby improving the genetic transformation efficiency.

[0084] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A medium for efficient genetic transformation of kidney beans, characterized in that: including a co-culture medium and a bud induction medium; The co-culture medium includes: MS culture medium, 20 g / L sucrose, 7 g / L agar, 40-50 mg / L acetosyringone and 0.6-0.8 g / L composite antioxidant; The bud induction medium includes: MS medium, 20 g / L sucrose, 7 g / L agar, 100 mg / L kanamycin and 0.6-0.8 g / L compound antioxidant.

2. A medium for efficient genetic transformation of kidney beans according to claim 1, characterized in that: The preparation steps of the composite antioxidant are as follows: drying the nanocarrier particles at 100-110° C. to a constant weight to obtain dry nanocarrier particles; Tetrabutyl titanate was added to anhydrous ethanol in a volume ratio of 1:(8-10), and the mixture was fully stirred to dissolve, and then ammonia water was added to adjust the pH to 8-9, and the mixture was allowed to stand for aging for 20-24 hours to obtain a titanium dioxide sol; The dried nano-carrier particles are immersed in the titanium dioxide sol, ultrasonically treated for 20-30 minutes, and stirred for 6-8 hours to obtain a suspension; The suspension is filtered, and the filter residue is dried at 60-80° C. for 8-10 h, and then placed in a muffle furnace, and heat treated at 450-550° C. for 2-4 h under nitrogen protection to obtain a composite carrier; Ascorbic acid was dissolved in deionized water to prepare a 0.1 mol / L ascorbic acid aqueous solution, and then the composite carrier was added at a solid-liquid ratio of 1 g: (40-50) mL. After ultrasonic loading for 20-30 minutes, the composite antioxidant was obtained by filtering and drying.

3. A medium for efficient genetic transformation of kidney beans according to claim 2, characterized in that: The preparation steps of nanocarrier particles are as follows: Add sodium hydroxide into deionized water at a solid-liquid ratio of 1 g: (25-35) mL, stir thoroughly to dissolve, and obtain a sodium hydroxide solution; Add aluminum nitrate nonahydrate and magnesium nitrate hexahydrate into deionized water at a solid-liquid ratio of 1 g: (1.8-2.2) g: (13-15) mL, stir thoroughly to dissolve, and obtain a mixed solution; The mixed solution is added to the sodium hydroxide solution while stirring, and then placed in a high-speed shearing machine. After shearing for 60-80 seconds, the mixture is centrifuged at a rate of 8000-9000 r / min for 8-10 minutes, and the supernatant is discarded to obtain a mixed precipitate; The mixed precipitate is washed 2-3 times with deionized water, and then supplemented to the original volume with deionized water, and heated and stirred at 80-90° C. for 5-6 hours to obtain nano-carrier particles.

4. A medium for efficient genetic transformation of kidney beans according to claim 1, characterized in that: When the culture medium is used for genetic transformation of kidney bean, the method specifically comprises the following steps: S1: Place sterile kidney bean seeds flat on a germination medium for germination. When the germinated hypocotyl reaches 2-3 cm, remove the cotyledons, terminal buds and radicles, leaving only 1-2 cm of the hypocotyl and cotyledon nodes to obtain kidney bean explants. S2: Activate Agrobacterium GV3101 containing the target plasmid in LB medium until OD600 reaches 0.6-0.8, then collect the activated Agrobacterium suspension by centrifugation and resuspend it in the infection solution until OD600 reaches 0.4 to obtain a mixed infection solution; S3: immersing the bean explants in the mixed impregnation solution for 10-15 minutes, transferring them to the co-culture medium, culturing them in the dark for 2-3 days, and then transferring them to the washing medium and washing them 3-5 times; S4: transferring the bean explants washed with the washing medium to a bud induction medium, continuing to culture, changing the medium every 2-3 weeks, observing whether the adventitious buds have GFP fluorescence through a variable magnification macro microscope, and transferring the adventitious buds with GFP fluorescence to an elongation medium for further culture, changing the medium every 2-3 weeks, until the bean seedlings reach a length of 3-5 cm, thereby obtaining bean sprouts; S5: The bean sprouts are transferred to a rooting medium for rooting, and the bean sprouts are hardened and transplanted after they grow at least three roots and the root length reaches 3-5 cm.

5. A medium for efficient genetic transformation of kidney beans and a genetic transformation method according to claim 4, characterized in that: The germination medium includes: B5 basal medium, 1 mL / LB5 vitamins, 20 g / L sucrose and 7 g / L agar.

6. A medium for efficient genetic transformation of kidney beans and a genetic transformation method according to claim 4, characterized in that: The washing medium includes: MS medium, 20 g / L sucrose, 7 g / L agar, 500 mg / L cephalosporin and 100 mg / L kanamycin.

7. A medium for efficient genetic transformation of kidney beans and a genetic transformation method according to claim 4, characterized in that: The staining solution includes: MS liquid culture medium, 0.2% acetosyringone, 0.3% apigenin and 0.1% ferulic acid.

8. A medium for efficient genetic transformation of kidney beans and a genetic transformation method according to claim 4, characterized in that: The elongation medium includes: MS medium, 0.2 mg / L 6-benzylaminopurine and 500 mg / L cephalosporin.

9. A medium for efficient genetic transformation of kidney beans and a genetic transformation method according to claim 4, characterized in that: The rooting medium includes: MS medium, 20 g / L sucrose, 7 g / L agar and 1 mg / L indoleacetic acid.