A method for establishing an Agrobacterium-mediated evergreen blueberry transgenic system

Abstract

The present invention provides a method for establishing an Agrobacterium-mediated evergreen blueberry transgenic system, comprising: adding a rhizogenes containing a functional gene to a liquid culture medium to prepare an infection solution; a vacuum infiltration treatment method is: placing an explant in the infection solution for vacuum infiltration treatment, and then placing it on a co-culture medium for co-culture; using the tender shoots of evergreen blueberry seedlings grown under sterile conditions as explants, placing the explants in the infection solution for vacuum infiltration treatment, and then placing them on a co-culture medium for co-culture; washing the explants after co-culture, and then placing them on a regeneration medium for regeneration culture; after 15d to 20d of regeneration culture, the explants gradually grow transgenic callus, and buds appear, and finally the evergreen blueberry transgenic system is obtained. The present invention realizes the transgenic of evergreen blueberry callus for the first time, and obtains transgenic buds, with a conversion rate of 3.27%, and it only takes 2-3 months to obtain transgenic buds.

Description

Technical Field

[0001] The invention relates to the technical field of plant transgenic DNA recombination, and in particular to a method for establishing an evergreen blueberry transgenic system mediated by Agrobacterium. Background Art

[0002] Evergreen blueberry (Latin name: Vaccinium darrowii ), an evergreen shrub of the genus Vaccinium in the family Ericaceae, is a wild blueberry species. Compared with commercial blueberry varieties (with complex ploidy), evergreen blueberries have obvious advantages in genetics: simple genome (diploid), short juvenile period (short research cycle), and important role in the history of blueberry breeding (cross-breeding with northern highbush blueberries to breed southern highbush blueberry varieties). Therefore, evergreen blueberries are widely used as a model blueberry for scientific research, but their plant tissue regeneration is difficult and a transgenic system has not been established.

[0003] In the research reports on blueberry transgenic, polyploid blueberry was mainly used as experimental material for genetic transformation of exogenous genes, and diploid blueberry has not been reported. In 1996, researchers conducted transgenic experiments on the blueberry variety North Country, obtained positive plants containing the GUS gene, and established the genetic transformation system of North Country (GRAHAM J, GREIGK, MCNICOL RJ. Transformation of blueberry without antibiotic selection [J]. Annals of Applied Biology, 1996, 128 (3): 557-564.). In 1998, Cao et al. conducted transgenic research using axillary buds of northern highbush blueberry and southern highbush blueberry as research materials and found that co-culture conditions, strain selection and genotype all affect genetic transformation efficiency (Cao, X., et al. "GUS expression in blueberry (Vacciniumspp.): factors influencing Agrobacterium-mediated gene transfer efficiency." Plant Cell Reports 18 (1998): 266-270.). In 2004, Song Guoqing et al. genetically modified four highbush blueberry varieties and obtained transgenic plants, but the transformation efficiency was generally low (Song, Guo-Qing, and KC Sink. "Agrobacterium tumefaciens-mediated transformation of blueberry(Vaccinium corymbosum L.)." Plant Cell Reports 23 (2004): 475-484.). In 2012, Song Guoqing et al. used the southern highbush blueberry variety 'Legacy' as research material, transferred CBF genes, and clarified the mechanism of CBF regulatory network to improve the cold tolerance of blueberry (Song, Guo-qing, et al. "The Vaccinium corymbosumFLOWERING LOCUS T-like gene (VcFT): a flowering activator reversesphotoperiodic and chilling requirements in blueberry." Plant Cell Reports 32(2013): 1759-1769.).

[0004] Evergreen blueberry is a diploid blueberry with great research advantages. However, unlike other blueberries, tissue regeneration of evergreen blueberry is very difficult. The three main difficulties are: (1) Evergreen blueberry does not grow in tissue culture bottles and has the problem of dead seedlings; (2) Evergreen blueberry explants only grow callus tissue but no regenerated buds; (3) The explants quickly turn brown after contact with Agrobacterium.

[0005] The transgenic system is an important technology for gene function research and one of the main projects of scientific research service companies. With the rapid development of blueberry research, the market demand for blueberry transgenics is gradually increasing. Therefore, it is necessary to provide a method for constructing a transgenic system for evergreen blueberries to facilitate blueberry research and provide transgenic services. Summary of the invention

[0006] The invention provides a method for establishing an evergreen blueberry transgenic system mediated by Agrobacterium tumefaciens. The method realizes the genetic transformation of evergreen blueberry explants, fills the technical gap in the field, and quickly obtains the evergreen blueberry transgenic system in a short time.

[0007] The specific technical solutions are as follows:

[0008] A method for establishing an Agrobacterium-mediated evergreen blueberry transgenic system, comprising:

[0009] (1) Adding Agrobacterium rhizogenes containing a functional gene into a liquid culture medium to prepare an infection solution;

[0010] The formula of the liquid culture medium is: WPM + 2% sucrose + 0.1 mg / L IBA + 0.4 mg / L Cppu + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0011] (2) Using young shoots of evergreen blueberry seedlings grown under sterile conditions as explants, the explants were placed in an infiltration solution for vacuum infiltration and then placed on a co-culture medium for co-cultivation;

[0012] The formula of the co-cultivation medium is: WPM + 2% sucrose + 8.5 g / L agar + 0.05 mg / L IBA + 0.2 mg / L Cppu + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0013] (3) After co-cultivation, the explants were washed with bacteria and placed on a regeneration medium for regeneration culture. After 15 to 20 days of regeneration culture, transgenic callus tissues gradually grew from the explants and buds appeared.

[0014] The formula of the regeneration medium is: WPM + 2% sucrose + 8.5 g / L agar + 0.05 mg / L IBA +0.2 mg / L Cppu + 300 mg / L Carb + 100 mg / L Tim, pH = 5.2 ± 0.02;

[0015] (4) The regeneration medium was replaced every 14 to 16 days for subculture. After 45 to 60 days of the experiment, clusters of transgenic buds grew on the explants, and the evergreen blueberry transgenic system was obtained.

[0016] The present invention carries out a large number of experimental explorations in the early stage of establishing the evergreen blueberry transgenic system. In the early stage of the experiment, only transgenic evergreen blueberry callus tissue can be obtained after multiple genetic transformations, and transgenic evergreen blueberry plants cannot be obtained. Finally, through the combination of "vacuum infiltration treatment + culture medium (especially co-cultivation culture medium)", evergreen blueberry plants capable of regenerating transgenic buds are obtained, thereby establishing a complete transgenic system of evergreen blueberry for the first time.

[0017] Furthermore, in step (1), the Agrobacterium rhizogenes culture medium containing the functional gene is first shaken at 27-28°C until it becomes turbid, centrifuged at 2500-3000 r for 20-25 min, the supernatant is discarded, and then the Agrobacterium rhizogenes cells are added to the liquid culture medium.

[0018] Furthermore, in step (1), the OD of the infection solution 600 It is 0.6~0.7.

[0019] Furthermore, in step (2), the vacuum infiltration treatment method is: vacuum immersing the explant in the infection solution twice, each time for 1 to 2 seconds.

[0020] Furthermore, in step (2), the vacuum infiltration treatment method is:

[0021] (2-1) placing the explant material in a syringe tube, and then adding the infection solution of step (1) into the syringe tube;

[0022] (2-2) Then use a syringe to expel all the air in the tube, plug the nipple of the syringe, and pull the piston of the syringe backwards to keep the infection solution soaking the explant for 1-2 seconds to allow the infection solution to penetrate into the explant material;

[0023] (2-3) Repeat the above syringe vacuum infiltration method once, then drain the infection liquid and clamp out the explant material.

[0024] Furthermore, the mass volume ratio of the explant to the infection solution is 2 g~3 g: 15 mL~20 mL.

[0025] Furthermore, in step (2), the vacuum infiltration infection method is the optimal infection method found by exploration. The number of vacuum treatments and the vacuum immersion time both affect the genetic transformation efficiency. Orthogonal experiments were conducted on the explant materials with vacuum treatments of 1, 2, 3, and 4 times and holding times of 1, 2, 3, and 4 s. The experiment concluded that:

[0026] ① If the vacuum is applied 3 times or more, the explants will die during the later co-culture period regardless of the holding time;

[0027] ② If the vacuum treatment was performed once or twice but the holding time was longer than 2 s, the mortality rate of the explants was high (more than 70%) during the later co-culture period;

[0028] ③ If the vacuum treatment is performed once or twice and the holding time is 1 or 2 s, the mortality rate of the explants during the later co-culture period is low (below 5%); in the later stage, the explants treated with vacuum once and twice are compared, and the conversion rate of the explants treated with vacuum twice is higher. Finally, it is concluded that the optimal vacuum infection method for wax-leaved Vaccinium is to pull out the piston handle to vacuum twice, each time for 1-2 s.

[0029] Furthermore, in step (2), the co-cultivation conditions are: dark cultivation in a constant temperature incubator at 27-28°C for 7-8 days.

[0030] Furthermore, in step (3), the method of washing the bacteria is: first, washing the co-cultured explants once, then washing them three times with sterile water, then soaking them in the washing solution for 10 to 15 minutes, and finally drying the moisture on the surface of the explants;

[0031] The formula of the sterile washing solution is: sterile water + 300 mg / L Carb + 100 mg / L Tim + 2 mL / L Zhipeijing.

[0032] Furthermore, in step (3) and step (4), the regeneration culture conditions are: light culture at 24°C for 16 hours.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The present invention achieved the genetic modification of evergreen blueberry callus for the first time and obtained transgenic buds with a transformation rate of 3.27%.

[0035] (2) The method of the present invention only takes 2-3 months to obtain transgenic buds from evergreen blueberries, a woody plant, which is shorter than the time required for transgenic plants of the genus Vaccinium.

[0036] (3) In the method of the present invention, both young shoots and leaves of evergreen blueberries can be used as explants, and the materials are relatively easy to obtain. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 These are the sprout images under bright and dark fields of fluorescence stereomicroscope (bar = 50 μm);

[0038] Among them, a is a picture of the control group buds with unsuccessful genetic modification under the bright field of a fluorescent stereomicroscope; b is a picture of the positive buds with successful genetic modification under the bright field; c is a picture of the control group buds under the dark field of GFP under a fluorescent stereomicroscope; d is a picture of the positive buds under the dark field of GFP. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific embodiments. The following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto.

[0040] In the following examples, the preparation method of Agrobacterium containing functional genes is as follows: 1 μL of plasmid is transformed into 100 μL of K599 competent Agrobacterium rhizogenes, 600 μL of LB liquid without resistance is added, the mixture is shaken at 28°C for 2-3 h, centrifuged at 6000 r for 1 min to collect the bacteria, 500 μL of supernatant is discarded, the bacterial block is gently blown and resuspended, and coated on an LB plate containing kanamycin and streptomycin antibiotics, and cultured in an incubator at 28°C for 2-3 days, a single clone on the plate is picked for colony detection, and a band with the same size as the plasmid band of the control group is obtained by electrophoresis as a positive single clone, i.e., Agrobacterium rhizogenes containing functional genes.

[0041] In the following examples, "WPM" refers to a medium for plant tissue culture (full name: Lloyd & McCown Woody Plant Basal Medium With Vitamins, brand: Phytotech); "IBA" refers to indolebutyric acid; "Cppu" refers to chlorpyrifos; "GA" refers to gibberellin; "IAA" refers to indoleacetic acid; "KT" refers to kinetin; "ZT" refers to zeatin; "Carb" refers to carbenicillin sodium; and "Tim" refers to timentin.

[0042] Method for determining transformation efficiency: Use a fluorescent protein observation flashlight to observe in a dark room. The evergreen blueberry regenerated buds that show green fluorescence (GFP) under laser irradiation are transgenic buds.

[0043] The transformation efficiency was calculated as follows: number of transgenic buds / total number of explants (the transgenic method of evergreen blueberry was subjected to three biological replicate experiments, and the transformation efficiency of each experiment was obtained and the average value was taken to obtain the final transformation efficiency.)

[0044] Example 1

[0045] A method for establishing an Agrobacterium-mediated evergreen blueberry transgenic system, the specific steps are as follows:

[0046] (1) Use a clean bench to cut well-growing evergreen blueberry shoots from sterile culture bottles as explant materials;

[0047] (2) One day before the explant transgenic infection experiment, the Agrobacterium containing the functional gene was first shaken in a 4 mL medium, and then transferred to a 20 mL conical flask and shaken until the OD600 of the bacterial solution reached 0.6. Centrifuge at 3000 r for 20 min, discard the supernatant, and add an equal amount of liquid culture medium to resuspend and prepare the infection solution;

[0048] The formulation of liquid medium was: WPM + 2% sucrose + 0.1 mg / L IBA + 0.4 mg / L Cppu + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0049] (3) Place the explant material from step (1) into a 20 mL empty syringe, then suck in 20 mL of the infection solution from step (2), expel the air, block the syringe nipple with the left thumb, and pull out the piston handle with the right hand to perform vacuum treatment. Perform vacuum treatment twice, each time for 1-2 seconds, and gently shake the tube wall at the same time to ensure that the explant material is completely immersed in the infection solution;

[0050] (4) The infected material in step (3) is placed on the co-cultivation medium and placed in a constant temperature incubator at 28°C for dark cultivation for 7 days; whether to place filter paper on the co-cultivation medium is determined based on the amount of Agrobacterium grown during the co-cultivation period. If a large amount of Agrobacterium grows, a layer of filter paper can be placed between the medium and the explant material;

[0051] The co-cultivation medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.05 mg / L IBA + 0.2 mg / L Cppu + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0052] (5) The explants co-cultured for 7 days were sterilized. The sterilization steps were as follows: washing once with sterilization solution, washing three times with sterile water, and finally soaking in sterilization solution for 15 min. The surface moisture of the explants was blown dry in a clean bench and then transferred to the regeneration medium for culture. The sterilization solution was prepared as follows: sterile water + 300 mg / L Carb + 100 mg / L Tim + 2 mL / L Zhipeijing.

[0053] Regeneration medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.05 mg / L IBA + 0.2 mg / LCppu + 300 mg / L Carb + 100 mg / L Tim, pH = 5.2 ± 0.02;

[0054] (6) The material treated in step (5) was cultured on a regeneration medium with subculture every 14 days; the regeneration medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.05 mg / L IBA + 0.2 mg / L Cppu + 300 mg / LCarb + 100 mg / L Tim, pH = 5.2 ± 0.02.

[0055] According to statistics, callus with green fluorescence (GFP) label appeared on the explants after 15-20 days, and then developed into buds. After 45-60 days, clusters of transgenic buds (such as Figure 1 as shown).

[0056] Experimental results: This transgenic system only takes 2-3 months from the beginning of infection to obtaining transgenic positive buds, with a conversion rate of up to 3.27%. The experimental data are shown in the following table.

[0057]

[0058] Comparative Example 1

[0059] This comparative example used a similar infection method in the transgenic system of European blueberry callus for transgenic, but because the infection method is not suitable for the regeneration of late buds of evergreen blueberry transgenic callus, it is impossible to obtain evergreen blueberry transgenic positive buds.

[0060] In the end, the experiment failed to produce transgenic positive shoots of evergreen blueberry.

[0061] Except for the different treatment of the implants and the infection method, the other steps or treatments in this comparative example are exactly the same as those in Example 1.

[0062] The specific steps are as follows:

[0063] (1) Cut a number of well-grown evergreen blueberry sprouts from sterile culture bottles on a clean bench, place them on a pre-culture plate and culture them in a constant temperature incubator at 28 degrees for 10 to 14 days in the dark. The pre-culture medium formula is: WPM + 2% sucrose + 0.5 mg / L IAA + 0.5 mg / L KT pH = 5.2 ± 0.02;

[0064] (2) One day before the explant transgenic infection experiment, ClGRF4-GIFThe Agrobacterium was first shaken in 4 mL of small shaker, and then transferred to a 20 mL conical flask and shaken until the bacterial solution OD 600 When the concentration is about 0.6, centrifuge at 3000 r for 20 min, discard the supernatant, add an equal amount of liquid culture medium to resuspend and prepare the infection solution;

[0065] The liquid medium formula was: WPM + 2% sucrose + 0.1 mg / L IBA + 0.4 mg / L Cppu + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0066] (3) Place the pre-cultured explant material in the WPM infection solution. The infection culture conditions are: shake in a shaker at 200 rpm at 28°C for 15-20 min, then subject the material to ultrasonic disruption (amplitude 60%) for 60 s and vacuum infiltration with a vacuum pump for 5 min, repeat the vacuum three times, and finally place in a shaker at 28°C at 200 rpm for 2 h.

[0067] (4) Place the infected material on the co-culture medium and place it in a constant temperature incubator at 28 degrees for dark culture for 7 days; determine whether to place filter paper on the co-culture plate based on the amount of Agrobacterium grown during the co-culture period. If a large number of Agrobacterium grows, place a layer of filter paper between the culture medium and the infected explant material;

[0068] The co-cultivation medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.05 mg / L IBA + 0.2 mg / L Cppu + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0069] (5) The explants co-cultured for 7 days were sterilized by washing with the following steps: washing once with sterile washing solution, washing three times with sterile water, and finally soaking in sterile washing solution for about 15 min. The surface moisture of the explants was blown dry in a clean bench and then transferred to the regeneration medium for culture. The sterile washing solution was prepared as follows: sterile water + 300 mg / L Carb + 100 mg / L Tim + 2 mL / L Zhipeijing.

[0070] Regeneration medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.05 mg / L IBA + 0.2 mg / LCppu + 300 mg / L Carb + 100 mg / L Tim, pH = 5.2 ± 0.02;

[0071] (6) Place the treated materials on the regeneration medium plate and culture them every 14 days; the regeneration medium is: WPM + 2% sucrose + 8.5 g / L agar + 0.05 mg / L IBA + 0.2 mg / L Cppu + 300 mg / LCarb + 100 mg / L Tim, pH = 5.2 ± 0.02.

[0072] The results showed that although a positive callus group with GFP was produced, during the later stage of adventitious bud induction, the positive callus group could not grow obvious buds, and ultimately no positive buds were induced. The experimental data are shown in the following table.

[0073]

[0074] Comparative Example 2

[0075] This comparative example uses different liquid culture media, regeneration culture media and co-culture culture media to conduct transgenic experiments on evergreen blueberries. In the final experiment, only callus tissue grows out of the evergreen blueberry explant material during the culture process, and a small number of positive callus tissues can be obtained. However, because the culture medium system is not suitable for the evergreen blueberry regeneration system, no transgenic positive buds of evergreen blueberries can be obtained.

[0076] Except for the differences in liquid culture medium, regeneration culture medium and co-cultivation culture medium, the remaining steps or treatments in this comparative example are exactly the same as those in Example 1.

[0077] The details are as follows:

[0078] Process 1:

[0079] The formulation of the liquid medium was: WPM + 2% sucrose + 0.2 mg / L IBA + 1 mg / L ZT + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0080] The co-cultivation medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.1 mg / L IBA + 0.5 mg / LZT + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0081] The regeneration medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.1 mg / L IBA + 0.5 mg / LZT + 300 mg / L Carb + 100 mg / L Tim, pH = 5.2 ± 0.02.

[0082] Process 2:

[0083] The formulation of the liquid medium was: WPM + 2% sucrose + 0.2 mg / L IBA + 2 mg / L ZT + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0084] The co-cultivation medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.1 mg / L IBA + 1 mg / LZT + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0085] The regeneration medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.1 mg / L IBA + 1 mg / L ZT + 300 mg / L Carb + 100 mg / L Tim, pH = 5.2 ± 0.02.

[0086] Process 3:

[0087] The formulation of the liquid medium was: WPM + 2% sucrose + 1.4 mg / L IBA + 4 mg / L ZT + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0088] The co-cultivation medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.7 mg / L IBA + 2 mg / LZT + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0089] The regeneration medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.7 mg / L IBA + 2 mg / L ZT + 300 mg / L Carb + 100 mg / L Tim, pH = 5.2 ± 0.02.

[0090] Process 4:

[0091] The formulation of the liquid medium was: WPM + 2% sucrose + 1 mg / L IAA + 1 mg / L ZT + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0092] The co-cultivation medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.5 mg / L IAA + 0.5 mg / LZT + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0093] The regeneration medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.5 mg / L IAA + 0.5 mg / LZT + 300 mg / L Carb + 100 mg / L Tim, pH = 5.2 ± 0.02.

[0094] Process 5:

[0095] The formulation of the liquid medium was: WPM + 2% sucrose + 1 mg / L IBA + 1 mg / L KT + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0096] The co-cultivation medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.5 mg / L IBA + 0.5 mg / L KT + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0097] The regeneration medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.5 mg / L IBA + 0.5 mg / L KT + 300 mg / L Carb + 100 mg / L Tim, pH = 5.2 ± 0.02.

[0098] Process 6:

[0099] The formulation of liquid medium was: WPM + 2% sucrose + 0.2 mg / L NAA + 0.2 mg / L Cppu + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0100] The co-cultivation medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.1 mg / L NAA + 0.1 mg / LCppu + 20 mg / L acetosyringone, pH = 5.2 ± 0.02;

[0101] The regeneration medium was: WPM + 2% sucrose + 8.5 g / L agar + 0.1 mg / L NAA + 0.1 mg / LCppu + 300 mg / L Carb + 100 mg / L Tim, pH = 5.2 ± 0.02.

[0102] The results showed that positive callus tissue was produced in evergreen blueberry in treatments 1 to 6, but the genetic transformation efficiency was not high and no positive buds were induced in the end.

Claims

1. A method for establishing an Agrobacterium-mediated evergreen blueberry transgenic system, characterized in that: include: (1) Adding Agrobacterium rhizogenes containing a functional gene into a liquid culture medium to prepare an infection solution; The formula of the liquid culture medium is: WPM + 2% sucrose + 0.1 mg / L IBA + 0.4 mg / L Cppu + 20 mg / L acetosyringone, pH = 5.2 ± 0.02; (2) Using young shoots of evergreen blueberry seedlings grown under sterile conditions as explants, the explants were placed in an infiltration solution for vacuum infiltration and then placed on a co-culture medium for co-cultivation; The vacuum infiltration treatment method is: vacuum immersing the explant in the infection solution twice, each time for 1 to 2 seconds; The formula of the co-cultivation medium is: WPM + 2% sucrose + 8.5 g / L agar + 0.05 mg / L IBA + 0.2 mg / L Cppu + 20 mg / L acetosyringone, pH = 5.2 ± 0.02; (3) After co-cultivation, the explants were washed with bacteria and placed on regeneration medium for regeneration culture. After 15-20 days of regeneration culture, transgenic callus tissues gradually grew from the explants and buds appeared. The formula of the regeneration medium is: WPM + 2% sucrose + 8.5 g / L agar + 0.05 mg / L IBA + 0.2 mg / L Cppu + 300 mg / L Carb + 100 mg / L Tim, pH = 5.2 ± 0.02; (4) The regeneration medium was replaced every 14 to 16 days for subculture. After 45 to 60 days of the experiment, clusters of transgenic buds grew on the explants, and the evergreen blueberry transgenic system was obtained.

2. The method for establishing the evergreen blueberry transgenic system mediated by Agrobacterium as claimed in claim 1, characterized in that: In step (1), the Agrobacterium rhizogenes culture medium containing the functional gene is first shaken at 27-28°C until it becomes turbid, centrifuged at 2500-3000 r for 20-25 min, the supernatant is discarded, and then the Agrobacterium rhizogenes cells are added to the liquid culture medium.

3. The method for establishing the evergreen blueberry transgenic system mediated by Agrobacterium as claimed in claim 2, characterized in that: In step (1), the OD of the infection solution 600 It is 0.6~0.

7.

4. The method for establishing the evergreen blueberry transgenic system mediated by Agrobacterium as claimed in claim 1, characterized in that: In step (2), the vacuum infiltration treatment method is: (2-1) placing the explant material in a syringe tube, and then adding the infection solution of step (1) into the syringe tube; (2-2) Then use a syringe to expel all the air in the tube, plug the nipple of the syringe, and pull the piston of the syringe backwards to keep the infection solution soaking the explant for 1-2 seconds to allow the infection solution to penetrate into the explant material; (2-3) Repeat the above syringe vacuum infiltration method once, then drain the infection liquid and clamp out the explant material.

5. The method for establishing the evergreen blueberry transgenic system mediated by Agrobacterium as claimed in claim 4, characterized in that: The mass volume ratio of the explant to the infection solution is (2 g~3 g): (15 mL~20 mL).

6. The method for establishing the evergreen blueberry transgenic system mediated by Agrobacterium as claimed in claim 1, characterized in that: In step (2), the co-cultivation conditions are: dark cultivation in a constant temperature incubator at 27-28°C for 7-8 days.

7. The method for establishing the evergreen blueberry transgenic system mediated by Agrobacterium as claimed in claim 1, characterized in that: In step (3), the method of washing bacteria is: first wash the co-cultured explants once with a washing solution, then wash them three times with sterile water, then soak them in the washing solution for 10 to 15 minutes, and finally dry the moisture on the surface of the explants; The formula of the sterile washing solution is: sterile water + 300 mg / L carbenicillin sodium + 100 mg / L timentin + 2 mL / L Zhipeijing.

8. The method for establishing the evergreen blueberry transgenic system mediated by Agrobacterium as claimed in claim 1, characterized in that: In step (3) and step (4), the regeneration culture conditions are: light culture at 24°C for 16 h.

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