A method for establishing a transgenic system of bilberry mediated by Agrobacterium tumefaciens
Through Agrobacterium tumefaciens mediated methods, the European lingonberry genetically modified system was established, which solved the problem of European lingonberry genetically modified, achieved stable genetic transformation and efficient gene introduction, with a conversion rate of 4%.
Patent Information
- Application Number
- CN202211207981.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-30
- Publication Date
- 2025-08-12
- Estimated Expiration
- 2042-09-30
AI Technical Summary
The lack of an effective European lingonberry transgenic system in the existing technology has made it difficult to carry out molecular breeding and genetic research of European lingonberry.
Using Agrobacterium tumefaciens mediated method, the young stems and leaves of European lingonberry were used as explant materials, and infectious culture was used to use specific culture medium and Agrobacterium infectious solution for infectious culture, combined with ultrasonic crushing and vacuum permeation treatment, and then co-culture in co-culture medium. Finally, magenta callus was screened out in the screening medium to achieve stable genetic transformation of European lingonberry.
The genetically modified version of European lingonberry callus was achieved, with a conversion rate of 4%, and the functional gene MYBA1 was successfully transferred, which significantly improved the efficiency and stability of European lingonberry transgenic system.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of forestry transgenic technology, in particular to a method for establishing a transgenic system of European blueberry mediated by Agrobacterium tumefaciens. Background Art
[0002] European bilberry (Vaccinium myrtillus Linn.), also known as European blueberry and bilberry, is a plant of the genus Vaccinium (Vaccinium Spp.) in the family Ericaceae. It is a low shrub native to Northern Europe and is now found in many regions. It contains more than 30 types of anthocyanins, making it the bilberry variety with the most anthocyanin types. It is also one of the richest natural sources of anthocyanins, with a content of up to 37.47%. It has high economic and medicinal value and is listed as a Class 1 herb by the American Herbal Products Association. [1,2] The entire bilberry plant is used in traditional medicine, most commonly to treat eye conditions. [3] , while the berries are mainly eaten fresh or pickled [4] In addition, bilberries are used in dietary supplements. Due to their rich flavor and color, bilberries are often preferred by consumers over blueberries (Vaccinium myrtillus and Vaccinium angustifolium). With the growing demand for bilberries, there is growing interest in improving their cultivation and production.
[0003] Among the genus Vaccinium, the blueberry has the most mature asexual propagation and transgenic technology. The blueberry, with a fruit shape very similar to that of the European bilberry, belongs to the subgenus Vaccinium within the genus Vaccinium. Blueberries have the highest degree of commercialization and cultivation promotion. Existing transgenic methods for Vaccinium plants are all developed using blueberries as the material. Given the value of the bilberry, researchers such as Rowland began transgenic research on the highbush blueberry in 1993, although ultimately unsuccessful. [5] In 1998, Cao et al. used Agrobacterium tumefaciens strain EHA105 and axillary buds of northern highbush blueberry and southern highbush blueberry as research materials to conduct transgenic research. They finally successfully constructed a transgenic system for blueberry and found that the transformation efficiency of Agrobacterium strain EHA105 was higher than that of LBA4404. [6] The co-cultivation days, cultivation years and plant genotype also affect the efficiency of blueberry transgenic [7]In 2004, Song et al. used Agrobacterium tumefaciens-mediated transformation to study blueberry (Vaccinium corymbosum L.) transformation, including four varieties of blueberry: Aurora, Blue crop, Brigitta, and Legacy. The study found that Agrobacterium tumefaciens EHA105 strain was more effective than LBA4404 or GV3101 in terms of genetic modification, and the best genetic modification effect was achieved when the co-culture time was 6 days, which was consistent with the results of the 1998 study. In addition, acetosyringone (AS) also affected the genetic modification efficiency; the corresponding transformation rates of Aurora, Blue crop, Brigitta, and Legacy were 15.3%, 5%, 10%, and 5.6%, respectively. [8] In 2012, scholar Song published new progress on blueberry genetic modification. Using the southern highbush blueberry variety Legacy as research material, he introduced the blueberry C-repeat binding factor (CBF) gene and clarified that the CBF regulatory network can improve the cold tolerance of blueberries. [9] In 2020, scholars Masafumi et al. used two highbush blueberry varieties, 'O'Neal' ('ON') and 'Blue Muffin' ('BM'), grown on an experimental farm in Japan as experimental research materials. With the help of CRISPR / Cas9 technology, they studied the effects of genome editing technology on blueberry transformation. The results showed that genome editing technology can help accelerate blueberry breeding and promote blueberry transformation.
[10] .
[0004] Existing research on genetically modified Vacciniums has primarily focused on blueberries, for which no transgenic system exists. Existing transgenic methods for Vacciniums are limited to highbush blueberries and are not suitable for bilberries. Therefore, there is an urgent need to develop a transgenic system for bilberries to facilitate molecular breeding and genetic research. Summary of the Invention
[0005] The present invention provides a method for establishing a transgenic system of bilberry mediated by Agrobacterium tumefaciens. The method uses young stems and leaves of bilberry as experimental materials, realizes genetic transformation mediated by Agrobacterium tumefaciens on the basis of a bilberry callus regeneration system, and establishes a stable and rapid genetic transformation system of bilberry.
[0006] The specific technical solutions are as follows:
[0007] A method for establishing a transgenic system of bilberry mediated by Agrobacterium tumefaciens, characterized by comprising the following steps:
[0008] (1) Young leaves of well-grown European blueberry seedlings on sterile culture medium were used as explant materials. The explant materials were cut into appropriate sizes and inoculated into callus-inducing culture medium to obtain callus explants. The culture medium formula used was: WPM + 3% sucrose + 1% agar + 0.50 mg / L IBA + 2.0 mg / L ZT, pH = 5.2 ± 0.02;
[0009] (2) After the material has grown in the regeneration medium for 10-14 days, it is used as the infection material and then placed in the prepared Agrobacterium infection solution for infection culture;
[0010] (3) After the infection and culture are completed, the infection solution is poured out, the infected material is dried on filter paper, and placed in a co-culture medium for co-culture; the co-culture medium is: WPM + 3% sucrose + 0.85% agar + 0.70 mg / L IBA + 2.0 mg / L ZT + 20 mg / L AS, pH = 5.2 ± 0.02;
[0011] (4) After the co-cultivation, the transgenic material was washed with sterile water, dried, and placed in a screening medium for screening culture until the transgenic material callus grew larger and showed a magenta color indicated by the reporter gene (betaine synthesis gene); the screening medium was: WPM + 3% sucrose + 0.85% agar + 0.70 mg / L IBA + 2.0 mg / L ZT + 5 mg / L HYG + 400 mg / L Carb, pH = 5.2 ± 0.02;
[0012] The reporter gene used in this invention is a betaine synthesis gene. After expression in callus tissue, the reporter gene can produce betaine in transgenic callus tissue, causing positive transgenic callus to appear magenta. This eliminates the need for additional processing to identify transgenic callus tissue, saving time in establishing a transgenic system.
[0013] Furthermore, in step (1), the young branches were cut into small segments of about 0.8 cm, and the leaves were cut into squares of about 0.5 cm after removing the edges; the explant inoculation method was: the leaves were flattened on the surface of the culture medium with the front side facing down; the callus induction culture conditions were: incubation in the dark at 28°C for 16 hours per day;
[0014] Furthermore, in step (2), the Agrobacterium species is LBA4404 used in the ternary system developed by Chen Qijun's laboratory. It is written here as LBA4404-ternary.
[0015] Furthermore, in step (2), the infection solution is: WPM solution with 1.4 mg / L IBA, 4 mg / L ZT, and 10 mg / L AS added;
[0016] Furthermore, in step (2), the infection conditions are as follows: shaking on a shaker at 28°C for 20-30 min, then ultrasonically disrupting the material (amplitude 60%) for 1 min and vacuum infiltrating the material three times, each for 5 min, and then shaking on a shaker at 28°C for 2 h;
[0017] Furthermore, in step (3), the co-cultivation conditions are: culturing at 28°C in the dark for 8 days;
[0018] Furthermore, in step (4), after the co-cultivation, the transgenic material was washed with sterilized RO water containing 400 mg / L carboxybenzyl for about 10 times, each time for 30-60 seconds; the screening culture conditions were: culture under dark conditions at 28°C, subculture once every two weeks, and after two subcultures, the material was moved to culture under light conditions at 28°C, with 16 hours of light and 8 hours of darkness per day, and a light intensity of 50 μEm -2 s -1 Magenta callus appeared after 40-60 days of culture.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] (1) Transgenic blueberry callus was achieved for the first time. The conversion rate (the ratio of magenta callus to the total number of callus) reached 4%;
[0021] (2) In the present invention, both young branches and leaves of bilberry can be used as explants, and the materials are easy to obtain. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 (Bar = 1 mm) is the magenta callus tissue (the part framed by the oval) in the transgenic material of European blueberry;
[0023] Figure 2 (Bar = 1 mm) To validate this transgenic system, we used it to introduce the functional MYBA1 gene into bilberry. After 1-1.5 months of screening culture (using 50 mg / L kanamycin in the culture medium), the bilberry calli appeared black (enclosed by the oval). qPCR analysis of the resulting black calli revealed significantly higher levels of pigment expression compared to the untransfected calli, demonstrating the successful establishment of the bilberry transgenic system.
[0024] Figure 3 (Bar = 1 mm) shows the establishment process of the Vaccinium tumefaciens-mediated transgenic system in Example 1. The cycle of the entire system is 2-2.5 months. DETAILED DESCRIPTION
[0025] The present invention will be further described below with reference to specific embodiments. The following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto.
[0026] Example 1
[0027] A method for establishing a transgenic system of bilberry mediated by Agrobacterium tumefaciens, comprising the following steps:
[0028] (1) Young leaves of well-grown European blueberry seedlings on sterile culture medium were used as explant materials. The explant materials were cut into appropriate sizes and inoculated into callus-inducing culture medium to obtain callus explants. The culture medium formula used was: WPM + 3% sucrose + 1% agar + 0.50 mg / L IBA + 2.0 mg / L ZT, pH = 5.2 ± 0.02;
[0029] (2) After the material has grown in the regeneration medium for 10-14 days, it is used as the infection material (newly grown buds are removed), and then placed in the Agrobacterium LBA4404-ternary WPM infection solution with an OD600 value of 0.3-0.6 for infection culture, and 1.4 mg / L IBA, 4 mg / L ZT, and 10 mg / L AS are added to the infection solution; the infection culture conditions are: shaking at 28°C for 20-30 minutes, then ultrasonically disrupting the material (amplitude 60%) for 1 minute and vacuum infiltrating the material three times, each for 5 minutes, and then shaking at 28°C for 2 hours; the Agrobacterium infection solution is prepared by conventional methods in the art;
[0030] (3) After the infection and culture are completed, the infection liquid is poured out, the infected material is dried on filter paper, and placed in a co-culture medium for co-culture; the co-culture medium is: WPM + 3% sucrose + 0.85% agar + 0.70 mg / L IBA + 2.0 mg / L ZT + 20 mg / L AS, pH = 5.2 ± 0.02; a sterile filter paper can be placed on the surface of the co-culture medium to prevent large-scale bacterial growth of the material; the co-culture conditions are: culture in the dark at 28°C for 8 days;
[0031] (4) After the co-culture, the transgenic material was washed with sterile RO water containing 400 mg / L carboxybenzyl (Carb) for about 10 times, each time for 30-60 seconds. After drying on sterile filter paper, the material was placed in a screening culture medium for screening culture. The screening culture conditions were: culture at 28°C in the dark, subcultured once every two weeks, and after two subcultures, the material was moved to 28°C under light conditions for culture, with 16 hours of light and 8 hours of darkness per day, and a light intensity of 50 μE m -2 s -1The callus tissue slowly proliferated and grew significantly, and magenta callus tissue appeared after 40-60 days of culture; the screening culture medium was: WPM+3% sucrose+0.85% agar+0.70mg / LIBA+2.0mg / L ZT+5mg / L HYG+400mg / L Carb, pH=5.2±0.02.
[0032] Experimental results show that the cycle of the entire transgenic system is 1.5-2.5 months, and the transgenic efficiency can reach 4%.
[0033] Comparative Example 1
[0034] In this comparative example, another Agrobacterium GV3101 was selected and the transgenic material was screened with the same screening antibiotic hygromycin. It was found that the callus of the transgenic material was easy to brown, the callus was difficult to survive, and no rose-red (magenta) callus appeared.
[0035] The specific steps are as follows (step (1) is the same as step (1) in Example 1):
[0036] (1) Young leaves of well-grown European blueberry seedlings on sterile culture medium were used as explant materials. The explant materials were cut into appropriate sizes and inoculated into callus-inducing culture medium to obtain callus explants. The culture medium formula used was: WPM + 3% sucrose + 1% agar + 0.50 mg / L IBA + 2.0 mg / L ZT, pH = 5.2 ± 0.02;
[0037] (2) After the material is cut, it is used as the infection material and then placed in the prepared Agrobacterium GV3101 MS infection solution for infection culture, and 20 mg / L AS is added to the infection solution; the infection culture conditions are: shaking on a shaker at 28°C for 2 hours; the Agrobacterium infection solution is prepared using conventional methods in the art;
[0038] (3) After the infection and culture are completed, the infection liquid is poured out, the infected material is dried on filter paper, and placed in a co-culture medium for co-culture; the co-culture medium is: WPM + 3% sucrose + 0.85% agar + 0.50 mg / L IBA + 2.0 mg / L ZT + 20 mg / L AS, pH = 5.2 ± 0.02; a sterile filter paper can be placed on the surface of the co-culture medium to prevent large-scale bacterial growth of the material; the co-culture conditions are: culture in the dark at 28°C for 2 days;
[0039] (4) After the co-cultivation, the transgenic material was washed with sterile RO water containing 300 mg / L cephalosporin for about 10 times, each time for 30-60 seconds, and then dried on sterile filter paper and placed in a screening culture medium for screening culture; the screening culture conditions were: 28°C dark conditions, subcultured once every two weeks, and the callus tissue gradually proliferated and grew significantly; the screening culture medium was: WPM + 3% sucrose + 0.85% agar + 0.50 mg / L IBA + 2.0 mg / L ZT + 10 mg / L HYG + 300 mg / LCarb, pH = 5.2 ± 0.02.
[0040] However, callus tissue is prone to browning during the screening process, which greatly reduces the survival rate of transgenic materials during the screening process and seriously affects the transformation efficiency.
[0041] Comparative Example 2
[0042] In this comparative example, another Agrobacterium EHA105 was selected and the transgenic materials were screened with the same screening antibiotic hygromycin concentration. It was found that the transgenic materials grew slowly and the callus tissue was difficult to survive.
[0043] The specific steps are as follows (step (1) is the same as step (1) in Example 1):
[0044] (1) Young leaves of well-grown European blueberry seedlings on sterile culture medium were used as explant materials. The explant materials were cut into appropriate sizes and inoculated into callus-inducing culture medium to obtain callus explants. The culture medium formula used was: WPM + 3% sucrose + 1% agar + 0.70 mg / L IBA + 0.8 mg / L CPPU, pH = 5.2 ± 0.02;
[0045] (2) After the material was cultured in the regeneration medium for 10 days, it was used as the infection material and then placed in the prepared Agrobacterium EHA105 WPM infection solution with an OD600 value of 0.3-0.6 for infection culture. 1.6 mg / L CPPU and 20 mg / L AS were added to the infection solution. The infection culture conditions were: shaking on a shaker at 28°C for 10 minutes, then ultrasonically disrupting for 20 seconds with variable amplitude, and finally shaking on a shaker at 28°C for 2 hours. The Agrobacterium infection solution was prepared using conventional methods in the art.
[0046] (3) After the infection and cultivation is completed, the infection liquid is poured out, the infected material is dried on filter paper, and placed in a co-cultivation medium for co-cultivation; the co-cultivation medium is: WPM + 3% sucrose + 0.85% agar + 0.70 mg / L IBA + 0.8 mg / L CPPU + 20 mg / L AS, pH = 5.2 ± 0.02; a sterile filter paper can be placed on the surface of the co-cultivation medium to prevent large-scale bacterial growth of the material; the co-cultivation conditions are: culturing in the dark at 28°C for 8 days;
[0047] (4) After the co-cultivation, the transgenic material was washed with sterile RO water containing 300 mg / L cephalosporin for about 10 times, each time for 30-60 seconds. After drying on sterile filter paper, the material was placed in a screening culture medium for screening culture. The screening culture conditions were: 28°C dark conditions, subcultured once every two weeks, and after two subcultures, the material was moved to 28°C light conditions for culture, with 16 hours of light and 8 hours of darkness per day, and a light intensity of 50 μE m -2 s -1 The callus tissue gradually proliferated and grew significantly, and some callus tissue showed rose red color; the screening culture medium was: WPM+3% sucrose+0.85% agar+0.70 mg / L IBA+2.0 mg / L ZT+10 mg / L HYG+300 mg / L Carb, pH=5.2±0.02.
[0048] However, the transgenic materials grew relatively slowly during the screening process, and the callus tissue was not easy to survive. Although some individual callus tissues showed rose-red color, the rose-red callus tissue could not proliferate and grow, and even disappeared after a period of time. The false positive rate of transgenic materials was relatively high, which seriously affected the transgenic efficiency.
[0049] References
[0050] [1]CHU W,CHEUNG SCM,LAU R AW,et al.Bilberry(Vaccinium myrtillus L.)[M] / / ND, BENZIE IFF,WACHTEL-GALOR S.Herbal Medicine:Biomolecular andClinical Aspects.Boca Raton(FL).2011.
[0051] [2]HERBALIST R U.Bilberry Fruit Vaccinium myrtillus L.Standards ofAnalysis,Quality Control,and Therapeutics[J].American Herbal Pharmacopoeia TM ,2001.
[0052] [3]Drugs and Lactation Database(LactMed)[Internet].Bethesda(MD):National Library of Medicine(US)[J].Drugs and Lactation Database(LactMed),2021May 17,2006-.Bilberry.
[0053] [4]*A-V F AF N L.Berry Leaves:An Alternative Source of BioactiveNatural Products of Nutritional and Medicinal Value[J].Antioxidants(Basel),2016,5(2).
[0054] [5]OGDEN L J R E L.EFFICIENT SHOOT REGENERATION FROM LEAF SECTIONS OFHIGHBUSH BLUEBERRY SUITABLE FOR USE IN AGROBACTERIUM MEDIATED TRANSFORMATIONS[J].ruit Laboratory Beltsville Agricultural Research Center AgriculturalResearch Service 1993,Beltsville,MD 20705
[0055] [6]A.HOEKEMA P R H,P.J.J.HOOYKAAS,SCHILPEROORT R A.A binary plantvector strategy based on separation of virand T-region of the Agrobacteriumtumefaciens Ti-plasmid[J].Department of Biochemistry,1983,33.
[0056] [7]HAMMERSCHLAG X C Q L L J R F A.GUS expression in blueberry(Vaccinium spp.):factors influencing Agrobacterium-mediated gene transferefficiency[J].Plant Cell Reports,1998,18:266–270.
[0057] [8]SONG G Q,SINK K C.Agrobacterium tumefaciens-mediatedtransformation of blueberry(Vaccinium corymbosum L.)[J].Plant Cell Rep,2004,23(7):475-84.
[0058] [9]SONG G-Q,HANCOCK J F.Recent Advances in Blueberry Transformation[J].International Journal of Fruit Science,2012,12(1-3):316-32.
[0059]
[10] OMORI M,YAMANE H,OSAKABE K,et al.Targeted mutagenesis ofCENTRORADIALIS using CRISPR / Cas9 system through the improvement of genetictransformation efficiency of tetraploid highbush blueberry[J].The Journal ofHorticultural Science and Biotechnology, 2020,96(2):153-61.
Claims
1. A method for establishing a transgenic system of bilberry mediated by Agrobacterium tumefaciens, characterized in that: include: (1) Young leaves of well-grown European blueberry seedlings on sterile culture medium were used as explant materials. The explant materials were cut into appropriate sizes and inoculated into callus-inducing culture medium to obtain callus explants. The culture medium formula used was: WPM + 3% sucrose + 1% agar + 0.50 mg / L IBA + 2.0 mg / L ZT, pH = 5.2 ± 0.02; (2) After the material has grown in the regeneration medium for 10-14 days, it is used as the infection material and then placed in the prepared Agrobacterium infection solution for infection culture; the infection solution is: WPM solution with 1.4 mg / L IBA, 4 mg / L ZT, and 10 mg / L AS added; the infection culture conditions are: shaking at 28°C for 20-30 minutes, then ultrasonically disrupting the material at an amplitude of 60% for 1 minute and vacuum infiltrating the material three times, each for 5 minutes, and then shaking at 28°C for 2 hours; (3) After the infection and cultivation is completed, the infection liquid is poured out, the infected material is dried on filter paper, and placed in the co-cultivation medium for co-cultivation; the co-cultivation medium is: WPM + 3% sucrose + 0.85% agar + 0.70 mg / LIBA + 2.0 mg / L ZT + 20 mg / L AS, pH = 5.2 ± 0.02; (4) After the co-cultivation, the transgenic material was washed with sterile RO water, dried, and placed in a screening medium for screening culture until the callus of the transgenic material grew larger and larger and showed a magenta color presented by the betaine synthesis reporter gene; the screening medium was: WPM + 3% sucrose + 0.85% agar + 0.70 mg / L IBA + 2.0 mg / L ZT + 5 mg / L HYG + 400 mg / L Carb, pH = 5.2 ± 0.
02.
2. The method for establishing a transgenic system of bilberry according to claim 1, characterized in that: In step (3), the co-culture conditions are: culturing at 28°C in the dark for 8 days.
3. The method for establishing a transgenic system of bilberry according to claim 1, wherein: In step (4), after the co-cultivation, the transgenic material was washed with sterilized RO water containing 400 mg / L carboxybenzyl for about 10 times, each time for 30-60 s; the screening culture conditions were: culture at 28°C in the dark, subcultured once every two weeks, and after two subcultures, the material was moved to culture at 28°C under light conditions, with 16 hours of light and 8 hours of darkness per day, and a light intensity of 50 μE m -2 s -1 The callus tissue gradually proliferated and grew significantly, and magenta callus tissue appeared after 40-60 days of culture.