A genetic transformation method using banana callus as receptor

Abstract

The invention relates to the technical field of tissue culture, and in particular to a genetic transformation method using banana callus as a receptor. Banana callus is mixed with a heavy suspension to obtain infected callus, the infected callus is inoculated on a co-culture medium for co-culture to obtain co-culture callus, the co-culture callus is inoculated on a screening medium for screening culture to obtain newly grown callus, the newly grown callus is inoculated on a proliferation medium for proliferation culture, and genetic transformation using banana callus as a receptor is achieved. The purpose of the invention is to provide a genetic transformation method using banana callus (genotype AAB) as a receptor material, which broadens the application scope of banana genetic transformation compared to the reported banana genetic transformation method, and the genetic transformation efficiency of the invention is high (basically more than 30% positive rate) and good repeatability.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture, in particular to a genetic transformation method using agaraja callus as a receptor. Background Art

[0002] Banana is the world's fourth largest food crop and an important tropical fruit. The research on banana gene function is lagging behind, which has hindered its biotechnology breeding. Biotechnology breeding relies on in-depth research on gene function, and gene function research, homologous or heterologous genetic transformation is the key to gene function research. The genetic transformation efficiency of banana is very low, and the genotype has a great influence on the genetic transformation efficiency, and the repeatability is also poor, which affects the functional research of banana genes.

[0003] When conducting research on the function of banana genes, it was found that due to the lack of a good genetic transformation system, most of the research on the function of banana genes was carried out on other species such as Arabidopsis, rice, and tomato, which has certain limitations. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a genetic transformation method using the callus of Longya banana as a receptor. Due to the different genetic transformation efficiencies of different genotypes, most of the current banana genetic transformation is mainly carried out with Brazilian banana (Fragrant banana, genotype AAA) as a receptor, and mainly focuses on thin slices and male flowers as receptors, with a long cycle and poor repeatability. The purpose of the present invention is to provide a genetic transformation method using the callus of Longya banana Xiangfen No. 1 (genotype AAB) as a receptor material. Compared with the reported banana genetic transformation methods, the application scope of banana genetic transformation is broadened. The genetic transformation efficiency of the present invention is high (basically more than 30% positive rate) and good repeatability. According to existing literature reports, the genetic transformation efficiency of bananas of different genotypes will be different, and the regeneration conditions of bananas of different genotypes or different varieties may be different. Therefore, different regeneration systems and transgenic systems should be adopted for bananas of different genotypes.

[0005] The mechanism of high genetic transformation rate of the present invention is:

[0006] On the one hand, an efficient embryonic regeneration system was used as the receptor system, in which the embryonic callus had good embryonic stability and the ability to regenerate plants; on the other hand, embryonic callus was more easily infected by Agrobacterium and integrated with new genes into the banana genome than other receptors.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a genetic transformation method using agaraja callus as a receptor, comprising the following steps:

[0009] 1) mixing the banana callus with the heavy suspension to obtain infected callus;

[0010] The variety of the longya banana is Xiangfen No. 1;

[0011] The resuspension contains Agrobacterium tumefaciens carrying an overexpression vector of a target gene;

[0012] 2) inoculating the infected callus obtained in step 1) onto a co-culture medium for co-culture to obtain co-cultured callus;

[0013] The co-culture medium is: NB medium + 6-BA 0.1 mg / L + glutamine 200 g / L + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 9 g / L + acetosyringone 100-120 µmol / L, pH value is 5.8;

[0014] 3) inoculating the co-cultured callus obtained in step 2) onto a screening medium for screening and culture to obtain newly grown callus;

[0015] The screening medium is: NB medium + 6-BA 0.1 mg / L + glutamine 200 g / L + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 9 g / L + hygromycin 10-25 mg / L + timentin 100-150 mg / L, pH value is 5.8;

[0016] 4) inoculating the newly grown callus obtained in step 3) onto a proliferation medium for proliferation culture, thereby achieving genetic transformation with the banana callus as a receptor;

[0017] The proliferation medium is: NB medium + 6-BA 0.1 mg / L + glutamine 200 g / L + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 9 g / L, with a pH value of 5.8.

[0018] Preferably, in step 1), the growth time of the banana callus is 15 to 25 days.

[0019] Preferably, the method for preparing the resuspension in step 1) comprises:

[0020] A. mixing and culturing the liquid culture medium with the Agrobacterium tumefaciens carrying the overexpression vector of the target gene to obtain a bacterial liquid;

[0021] The liquid culture medium is LB liquid culture medium containing 100 mg / L kanamycin and 10 mg / L rifampicin;

[0022] The volume ratio of the liquid culture medium to Agrobacterium tumefaciens is 50mL:200µL;

[0023] B. Centrifuge the obtained bacterial solution to obtain bacterial cells;

[0024] The centrifugal conditions include: a rotation speed of 10000 rpm and a time of 2 min;

[0025] C. Mixing the obtained bacterial cells with an equal volume of the suspension to obtain a heavy suspension;

[0026] The suspension comprises: MS culture medium + sucrose 100 g / L + acetosyringone 100-120 µmol / L, and the pH value is 5.0.

[0027] Preferably, the culture conditions include: a temperature of 28° C. and a rotation speed of 220 rpm.

[0028] Preferably, the OD of the bacterial solution is 600 The value is 0.8~1.0.

[0029] Preferably, the overexpression vector is the overexpression vector Pcambia1301-35SN, and the target gene is the FLS1 gene and / or the FLS2 gene.

[0030] Preferably, the mixing time in step 1) is 5 to 10 minutes.

[0031] Preferably, the co-cultivation conditions in step 2) include: dark culturing at a temperature of 28° C. for 2 to 4 days.

[0032] Preferably, the conditions for the screening culture in step 3) include: screening once every 15 to 20 days, for a total of 2 to 5 times, the screening culture temperature is 25 to 30° C., and the culture is cultured in the dark.

[0033] Preferably, the conditions for the proliferation culture in step 4) include: a temperature of 25-30° C. and dark culture.

[0034] Beneficial effects of the present invention:

[0035] Due to the different genetic transformation efficiencies of different genotypes, most of the current banana genetic transformation is mainly carried out with Brazilian banana (Musa acuminata, genotype AAA) as the recipient for genetic transformation, and mainly focuses on thin slices and male flowers as the recipient, with a long cycle and poor repeatability. The purpose of the present invention is to provide a genetic transformation method using the callus tissue of the banana (genotype AAB) as the receptor material, which, compared with the reported banana genetic transformation methods, broadens the application scope of banana genetic transformation, and the genetic transformation efficiency of the present invention is high (basically more than 30% positive rate), and the repeatability is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0037] Figure 1 This is the result of multiple subcultures in M3 medium, parallel 1;

[0038] Figure 2 This is the result of multiple subcultures in M3 medium, parallel 2;

[0039] Figure 3 This is the result of multiple subcultures in M3 medium, parallel 3;

[0040] Figure 4 This is the result of callus death when the concentration of hygromycin in M3 medium is greater than 25 mg / L, parallel 1;

[0041] Figure 5 This is the result of callus death when the concentration of hygromycin in M3 medium is greater than 25 mg / L, parallel 2;

[0042] Figure 6 This is the result of callus death when the concentration of hygromycin in M3 medium is greater than 25 mg / L, parallel 3;

[0043] Figure 7 This is the result of callus death when the concentration of timentin in M3 medium is less than 100 mg / L, parallel 1;

[0044] Figure 8 This is the result of callus death when the concentration of timentin in M3 medium is less than 100 mg / L, parallel 2;

[0045] Fig. 9 This is the result of callus death when the concentration of timentin in M3 medium is less than 100 mg / L, parallel 3;

[0046] Fig.10 This is the result of callus death when the concentration of timentin in M3 medium is less than 100 mg / L, parallel 4;

[0047] Fig.11 This is a gel image of some FLS2-35SN positive test results;

[0048] Fig.12 This is a gel image of some FLS1-35SN positive test results;

[0049] Fig.13 This is a gel image of some of the positive test results of pcambia1301--35SN. DETAILED DESCRIPTION

[0050] The present invention provides a genetic transformation method using agaraja callus as a receptor, comprising the following steps:

[0051] 1) mixing the banana callus with the heavy suspension to obtain infected callus;

[0052] The variety of the longya banana is Xiangfen No. 1;

[0053] The resuspension contains Agrobacterium tumefaciens carrying an overexpression vector of a target gene;

[0054] 2) inoculating the infected callus obtained in step 1) onto a co-culture medium for co-culture to obtain co-cultured callus;

[0055] The co-culture medium is: NB medium + 6-BA 0.1 mg / L + glutamine 200 g / L + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 9 g / L + acetosyringone 100-120 µmol / L, pH value is 5.8;

[0056] 3) inoculating the co-cultured callus obtained in step 2) onto a screening medium for screening and culture to obtain newly grown callus;

[0057] The screening medium is: NB medium + 6-BA 0.1 mg / L + glutamine 200 g / L + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 9 g / L + hygromycin 10-25 mg / L + timentin 100-150 mg / L, pH value is 5.8;

[0058] 4) inoculating the newly grown callus obtained in step 3) onto a proliferation medium for proliferation culture, thereby achieving genetic transformation with the banana callus as a receptor;

[0059] The proliferation medium is: NB medium + 6-BA 0.1 mg / L + glutamine 200 g / L + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 9 g / L, with a pH value of 5.8.

[0060] The invention mixes the longya banana callus with a heavy suspension to obtain the infected callus; the variety of the longya banana is Xiangfen No. 1; and the heavy suspension contains Agrobacterium tumefaciens with an overexpression vector of a target gene.

[0061] In the present invention, the growth time of the callus of the banana is preferably 15 to 25 days, at which time the callus is bright yellow and loose. The present invention has no special limitation on the culture method of the banana callus, and those skilled in the art can use conventional methods.

[0062] In the present invention, the preparation method of the resuspension comprises:

[0063] A. mixing and culturing the liquid culture medium with the Agrobacterium tumefaciens carrying the overexpression vector of the target gene to obtain a bacterial liquid;

[0064] The liquid culture medium is LB liquid culture medium containing 100 mg / L kanamycin and 10 mg / L rifampicin;

[0065] The volume ratio of the liquid culture medium to Agrobacterium tumefaciens is 50mL:200µL;

[0066] B. Centrifuge the obtained bacterial solution to obtain bacterial cells;

[0067] The centrifugal conditions include: a rotation speed of 10000 rom and a time of 2 min;

[0068] C. Mixing the obtained bacterial cells with an equal volume of the suspension to obtain a heavy suspension;

[0069] The suspension comprises: MS culture medium + sucrose 100 g / L + acetosyringone 100-120 µmol / L, and the pH value is 5.0.

[0070] In the present invention, the culture conditions preferably include: a temperature of 28°C and a rotation speed of 220 rpm. 600 The value is preferably 0.8 to 1.0. In the present invention, the overexpression vector is preferably an overexpression vector Pcambia1301-35SN, and the target gene is the FLS1 gene and / or the FLS2 gene.

[0071] The present invention inoculates the obtained infected callus tissue onto a co-culture medium for co-culture to obtain a co-cultured callus tissue; the co-culture medium is: NB medium + 6-BA 0.1mg / L + glutamine 200g / L + activated carbon 0.2g / L + sucrose 30g / L + carrageenan 9g / L + acetosyringone 100-120µmol / L, pH value is 5.8. In the present invention, the co-culture conditions preferably include: dark culture at a temperature of 28°C for 2-4d.

[0072] The present invention inoculates the obtained co-cultured callus onto a screening medium for screening and culture to obtain newly grown callus; the screening medium is: NB medium + 6-BA 0.1 mg / L + glutamine 200 g / L + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 9 g / L + hygromycin 10-25 mg / L + timentin 100-150 mg / L, pH value is 5.8. In the present invention, the conditions of the screening culture preferably include: screening once every 15-20 days, screening 2-5 times in total, the temperature of the screening culture is 25-30°C, and dark culture. In the present invention, when the concentration of hygromycin in the screening medium is greater than 25 mg / L, the callus tissue is completely dead; when the concentration of timentin in the screening medium is less than 100 mg / L, Agrobacterium tumefaciens cannot be effectively killed, thereby causing the death of the callus tissue.

[0073] The present invention inoculates the newly grown callus tissue obtained into a proliferation culture medium for proliferation culture, so as to realize genetic transformation with the callus tissue of the banana as a receptor; the proliferation culture medium is: NB medium + 6-BA 0.1mg / L + glutamine 200g / L + activated carbon 0.2g / L + sucrose 30g / L + carrageenan 9g / L, and the pH value is 5.8. In the present invention, the conditions of the proliferation culture preferably include: a temperature of 25-30°C and dark culture.

[0074] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0075] Example 1

[0076] A genetic transformation method using Aralia ovata callus as a receptor, comprising the following steps:

[0077] 1) Preparation:

[0078] ① Selection of callus receptor material of Longyajiao 'Xiangfen No. 1': The callus growth time of the selected Longyajiao is about 20 days, and the callus at this time is bright yellow and loose.

[0079] ② Preparation of resuspension (M1): MS medium + sucrose 100g / L, pH 5.0, sterilize at 121℃ for 20 minutes, and store at 4℃ for use.

[0080] ③ Preparation of co-culture medium (M2): NB medium + 6-BA 0.1 mg / L + glutamine 200g / L + AC0.2g / L + sucrose 30g / L + carrageenan 9 g / L, pH 5.8, sterilize at 121℃ for 20 minutes, add filter-sterilized acetosyringone (AS) to a final concentration of 100µmol / L when cooled to a level that is not too hot to touch, and place at 4℃ for use.

[0081] ④ Preparation of screening medium (M3): NB medium + 6-BA 0.1mg / L + glutamine 200g / L + AC0.2g / L + sucrose 30g / L + carrageenan 9g / L, pH 5.8, sterilize at 121℃ for 20 minutes, add filter-sterilized hygromycin and timentin when cooled to a final concentration of 10 mg / L and 150mg / L respectively.

[0082] ⑤ Preparation of antibiotic-free callus proliferation medium (M4): NB medium + 6-BA 0.1mg / L + glutamine 200g / L + activated carbon 0.2g / L + sucrose 30g / L + carrageenan 9g / L, pH 5.8, sterilization at 121℃ for 20 minutes.

[0083] 2) Preparation of infection solution: In a 50 ml centrifuge tube, add 30 ml of LB liquid medium containing 100 mg / L kanamycin and 10 mg / L rifampicin, add 200 µl of Agrobacterium tumefaciens GV3101 containing the overexpression vector Pcambia1301-35SN (empty), place in a 28°C shaker at 220 rpm, and shake until the OD reaches 600 is 0.8.

[0084] 3) Resuspend the bacterial solution: set the OD 600 Centrifuge the bacterial suspension at 10000 rpm for 2 minutes at room temperature. Pour out the supernatant after centrifugation in a clean bench, add an equal volume of resuspension solution M1 containing 100 µmol / L acetosyringone (AS) to obtain a resuspension solution, mix thoroughly, and place in ice for more than 1 hour, but not more than 4 hours.

[0085] 4) Infection: Place sterilized filter paper, filter screen (100 mesh), beaker, medicine spoon, 75% ethanol and other consumables in a clean bench for sterilization for more than 30 minutes. Take out the resuspension containing bacteria after the ice bath from the ice, use a medicine spoon to add the callus tissue of Longyajiao 'Xiangfen No. 1' in 1) to the resuspension until the scale line is 40, tighten the lid of the centrifuge tube, and gently invert and mix by hand for 7 minutes. Then filter the resuspension with a 100-mesh filter screen, leaving the callus tissue, and place the callus on the filter paper to absorb the moisture (during this period, you can replace the wet filter paper to speed up the absorption of moisture on the callus, usually 2-5 minutes). The infected callus tissue is evenly distributed in the M2 culture medium (usually 4-6 culture dishes), and co-cultured at 28℃ in the dark for 3 days.

[0086] 5) Washing and screening with M3 screening medium: Prepare sterile water, sterilized filter paper, tissue culture bottles, etc. in advance, and prepare sterile water containing 100 mg / L of Timentin. Scrape all the callus tissue after co-culture with a medicine spoon and place it in a sterilized tissue culture bottle, add about 50 ml of sterile water, gently swirl it with your hands for 1 minute, filter it with a filter sieve, repeat once, then soak it in sterile water containing 100 mg / L of Timentin for 1 minute, filter it with a filter sieve, leaving the callus tissue, place the callus on filter paper to absorb the water (during this period, you can replace the wet filter paper to speed up the absorption of water on the callus, usually 2-5 minutes), and evenly distribute the infected callus tissue to the M3 screening medium (usually 4-6 culture dishes) for screening, the conditions are room temperature and dark culture.

[0087] 6) Subsequent screening and culture: After 20 days, the callus on M3 was subcultured to a new M3 medium. After 4 screenings in the M3 screening medium, many browned or dead callus tissues and newly grown callus tissues were found. The newly grown callus was inoculated into a proliferation medium without antibiotics (antibiotic-free callus proliferation medium (M4)) for proliferation at room temperature and dark culture. Note: Each newly grown callus point was inoculated separately, with a spacing of about 1 cm between different points, and they could not be mixed up, so as to facilitate the subsequent statistics of the positive callus rate.

[0088] 7) Positive callus detection and statistics: The callus that finally survived was numbered, DNA was extracted, and positive identification was performed. Calculation formula: Callus rate (%) = (number of positive callus DNA ÷ total number of extracted DNA) * 100%.

[0089] 8) Results Statistics

[0090] ① Statistics of the positive rate of Pcambia1301-35SN (empty):

[0091] A total of 118 callus blocks survived, of which 66 callus blocks were detected with the target fragment, so the positive callus rate was: (66÷118)*100%=55.9%.

[0092] Example 2

[0093] A genetic transformation method using Aralia ovata callus as a receptor, comprising the following steps:

[0094] 1) Preparation:

[0095] ① Selection of callus receptor material of Longyajiao 'Xiangfen No. 1': The callus growth time of the selected Longyajiao is about 20 days, and the callus at this time is bright yellow and loose.

[0096] ② Preparation of resuspension (M1): MS medium + sucrose 100g / L, pH 5.0, sterilize at 121℃ for 20 minutes, and store at 4℃ for use.

[0097] ③ Preparation of co-culture medium (M2): NB medium + 6-BA 0.1 mg / L + glutamine 200g / L + AC0.2g / L + sucrose 30g / L + carrageenan 9 g / L, pH 5.8, sterilize at 121℃ for 20 minutes, add filter-sterilized acetosyringone (AS) to a final concentration of 110µmol / L when cooled to a level that is not too hot to the touch, and place at 4℃ for later use.

[0098] ④ Preparation of screening medium (M3): NB medium + 6-BA 0.1mg / L + glutamine 200g / L + AC0.2g / L + sucrose 30g / L + carrageenan 9g / L, pH 5.8, sterilize at 121℃ for 20 minutes, add filter-sterilized hygromycin and timentin when cooled to a final concentration of 20 mg / L and 120 mg / L respectively.

[0099] ⑤ Preparation of antibiotic-free callus proliferation medium (M4): NB medium + 6-BA 0.1mg / L + glutamine 200g / L + activated carbon 0.2g / L + sucrose 30g / L + carrageenan 9g / L, pH 5.8, sterilization at 121℃ for 20 minutes.

[0100] 2) Preparation of infection solution: In a 50 ml centrifuge tube, add 30 ml of LB liquid medium containing 100 mg / L kanamycin and 10 mg / L rifampicin, add 200 µl of Agrobacterium tumefaciens GV3101 containing the overexpression vector FLS1-35SN, and place in a 28°C shaker at 220 rpm until the OD reaches 600 is 0.9.

[0101] 3) Resuspend the bacterial solution: set the OD600 The bacterial suspension with a concentration of 0.9 was centrifuged at 10,000 rpm for 2 minutes at room temperature. The supernatant after centrifugation was poured out in a clean bench, and an equal volume of resuspension solution M1 containing 110 µmol / L acetosyringone (AS) was added to obtain a resuspension solution, which was thoroughly mixed and placed in ice for more than 1 hour, but not more than 4 hours.

[0102] 4) Infection: Place sterilized filter paper, filter screen (100 mesh), beaker, medicine spoon, 75% ethanol and other consumables in a clean bench for sterilization for more than 30 minutes. Take out the resuspension containing bacteria after the ice bath from the ice, use a medicine spoon to add the callus tissue of Longyajiao 'Xiangfen No. 1' in 1) to the resuspension until the scale line is 40, tighten the lid of the centrifuge tube, and gently invert and mix by hand for 5 minutes. Then filter the resuspension with a 100-mesh filter screen, leaving the callus tissue, and place the callus on the filter paper to absorb the water (during this period, you can replace the wet filter paper to speed up the absorption of water on the callus, usually 2-5 minutes). The infected callus tissue is evenly distributed in the M2 culture medium (usually 4-6 culture dishes), and co-cultured at 28℃ in the dark for 3 days.

[0103] 5) Washing and screening with M3 screening medium: Prepare sterile water, sterilized filter paper, tissue culture bottles, etc. in advance, and prepare sterile water containing 100 mg / L of Timentin. Scrape all the callus tissue after co-culture with a medicine spoon and place it in a sterilized tissue culture bottle, add about 50 ml of sterile water, gently swirl it with your hands for 1 minute, filter it with a filter sieve, repeat once, then soak it in sterile water containing 100 mg / L of Timentin for 1 minute, filter it with a filter sieve, leaving the callus tissue, place the callus on filter paper to absorb the water (during this period, you can replace the wet filter paper to speed up the absorption of water on the callus, usually 2-5 minutes), and evenly distribute the infected callus tissue to the M3 screening medium (usually 4-6 culture dishes) for screening, the conditions are room temperature and dark culture.

[0104] 6) Subsequent screening and culture: After 20 days, the callus on M3 was subcultured to a new M3 medium. After 4 screenings in the M3 screening medium, many browned or dead callus tissues and newly grown callus tissues were found. The newly grown callus was inoculated into a proliferation medium without antibiotics (antibiotic-free callus proliferation medium (M4)) for proliferation at room temperature and dark culture. Note: Each newly grown callus point was inoculated separately, with a spacing of about 1 cm between different points, and they could not be mixed up, so as to facilitate the subsequent statistics of the positive callus rate.

[0105] 7) Positive callus detection and statistics: The callus that finally survived was numbered, DNA was extracted, and positive identification was performed. Calculation formula: Callus rate (%) = (number of positive callus DNA ÷ total number of extracted DNA) * 100%.

[0106] 8) Results Statistics

[0107] FLS1-35SN positive rate statistics:

[0108] A total of 40 callus blocks survived, of which 18 callus blocks contained target fragments, so the positive callus rate was: (18÷40)*100%=45.0%.

[0109] Example 3

[0110] A genetic transformation method using Aralia ovata callus as a receptor, comprising the following steps:

[0111] 1) Preparation:

[0112] ① Selection of callus receptor material of Longyajiao 'Xiangfen No. 1': The callus growth time of the selected Longyajiao is about 20 days, and the callus at this time is bright yellow and loose.

[0113] ② Preparation of resuspension (M1): MS medium + sucrose 100g / L, pH 5.0, sterilize at 121℃ for 20 minutes, and store at 4℃ for use.

[0114] ③ Preparation of co-culture medium (M2): NB medium + 6-BA 0.1 mg / L + glutamine 200g / L + AC0.2g / L + sucrose 30g / L + carrageenan 9 g / L, pH 5.8, sterilize at 121℃ for 20 minutes, add filter-sterilized acetosyringone (AS) to a final concentration of 120µmol / L when cooled to a level that is not too hot to the touch, and place at 4℃ for use.

[0115] ④ Preparation of screening medium (M3): NB medium + 6-BA 0.1 mg / L + glutamine 200 g / L + AC0.2 g / L + sucrose 30 g / L + carrageenan 9 g / L, pH 5.8, sterilize at 121°C for 20 minutes, add filter-sterilized hygromycin and timentin when cooled to a final concentration of 25 mg / L and 100 mg / L respectively.

[0116] ⑤ Preparation of antibiotic-free callus proliferation medium (M4): NB medium + 6-BA 0.1mg / L + glutamine 200g / L + activated carbon 0.2g / L + sucrose 30g / L + carrageenan 9g / L, pH 5.8, sterilization at 121℃ for 20 minutes.

[0117] 2) Preparation of infection solution: In a 50 ml centrifuge tube, add 30 ml of LB liquid medium containing 100 mg / L kanamycin and 10 mg / L rifampicin, add 200 µl of Agrobacterium tumefaciens GV3101 containing the overexpression vector FLS2-35SN, and place in a 28°C shaker at 220 rpm until the OD reaches 600 is 1.0.

[0118] 3) Resuspend the bacterial solution: set the OD 600 Centrifuge the bacterial solution at 10000 rpm for 2 minutes at room temperature. Pour out the supernatant after centrifugation in a clean bench, add an equal volume of resuspension solution M1 containing 120 µmol / L acetosyringone (AS) to obtain a resuspension solution, mix thoroughly, and place in ice for more than 1 hour, but not more than 4 hours.

[0119] 4) Infection: Place sterilized filter paper, filter screen (100 mesh), beaker, medicine spoon, 75% ethanol and other consumables in a clean bench for sterilization for more than 30 minutes. Take out the resuspension containing bacteria after the ice bath from the ice, add the callus tissue of Longyajiao 'Xiangfen No. 1' in 1) to the resuspension with a medicine spoon until the scale line is 40, tighten the lid of the centrifuge tube, and gently invert and mix by hand for 10 minutes. Then filter the resuspension with a 100-mesh filter screen, leaving the callus tissue, and place the callus on the filter paper to absorb the moisture (during this period, the wet filter paper can be replaced to speed up the absorption of moisture on the callus, usually 2-5 minutes). The infected callus tissue is evenly distributed in the M2 culture medium (usually 4-6 culture dishes), and co-cultured at 28℃ in the dark for 3 days.

[0120] 5) Washing and screening with M3 screening medium: Prepare sterile water, sterilized filter paper, tissue culture bottles, etc. in advance, and prepare sterile water containing 100 mg / L of Timentin. Scrape all the callus tissue after co-culture with a medicine spoon and place it in a sterilized tissue culture bottle, add about 50 ml of sterile water, gently swirl it with your hands for 1 minute, filter it with a filter sieve, repeat once, then soak it in sterile water containing 100 mg / L of Timentin for 1 minute, filter it with a filter sieve, leaving the callus tissue, place the callus on filter paper to absorb the water (during this period, you can replace the wet filter paper to speed up the absorption of water on the callus, usually 2-5 minutes), and evenly distribute the infected callus tissue to the M3 screening medium (usually 4-6 culture dishes) for screening, the conditions are room temperature and dark culture.

[0121] 6) Subsequent screening and culture: After 20 days, the callus on M3 was subcultured to a new M3 medium. After 4 screenings in the M3 screening medium, many browned or dead callus tissues and newly grown callus tissues were found. The newly grown callus was inoculated into a proliferation medium without antibiotics (antibiotic-free callus proliferation medium (M4)) for proliferation at room temperature and dark culture. Note: Each newly grown callus point was inoculated separately, with a spacing of about 1 cm between different points, and they could not be mixed up, so as to facilitate the subsequent statistics of the positive callus rate.

[0122] 7) Positive callus detection and statistics: The callus that finally survived was numbered, DNA was extracted, and positive identification was performed. Calculation formula: Callus rate (%) = (number of positive callus DNA ÷ total number of extracted DNA) * 100%.

[0123] 8) Results Statistics

[0124] FLS2-35SN positive rate statistics:

[0125] A total of 58 callus blocks survived, of which 18 callus blocks contained target fragments, so the positive callus rate was: (18÷58)*100%=31.03%.

[0126] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A genetic transformation method using Aralia ovata callus as a receptor, characterized in that: The following steps are involved: 1) mixing the banana callus with the heavy suspension to obtain infected callus; The variety of the longya banana is Xiangfen No. 1; The resuspension contains Agrobacterium tumefaciens carrying an overexpression vector of a target gene, wherein the overexpression vector is the overexpression vector Pcambia1301-35SN, and the target gene is the FLS1 gene; 2) inoculating the infected callus obtained in step 1) onto a co-culture medium for co-culture to obtain co-cultured callus; The co-culture medium is: NB medium + 6-BA 0.1 mg / L + glutamine 200 g / L + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 9 g / L + acetosyringone 110 µmol / L, pH value is 5.8; 3) inoculating the co-cultured callus obtained in step 2) onto a screening medium for screening and culture to obtain newly grown callus; The screening medium is: NB medium + 6-BA 0.1 mg / L + glutamine 200 g / L + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 9 g / L + hygromycin 20 mg / L + timentin 120 mg / L, pH value is 5.8; 4) inoculating the newly grown callus obtained in step 3) onto a proliferation medium for proliferation culture, thereby achieving genetic transformation with the banana callus as a receptor; The proliferation medium is: NB medium + 6-BA 0.1 mg / L + glutamine 200 g / L + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 9 g / L, pH value is 5.8; In the step 1), the growth time of the callus tissue of the banana plant is 20 days; The resuspension solution in step 1) is: MS medium + sucrose 100 g / L + acetosyringone 110 µmol / L, with a pH value of 5.

0.

2. The genetic transformation method according to claim 1, characterized in that The mixing time in step 1) is 5-10 minutes.

3. The genetic transformation method according to claim 1, characterized in that The co-cultivation conditions in step 2) include: dark cultivation at a temperature of 28° C. for 2-4 days.

4. The genetic transformation method according to claim 1, characterized in that The conditions of step 3) screening culture include: screening once every 15-20 days, for a total of 2-5 times, the screening culture temperature is 25-30° C., and the culture is carried out in the dark.

5. The genetic transformation method according to claim 1, characterized in that The conditions of the proliferation culture in step 4) include: a temperature of 25-30° C. and dark culture.

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