A method for genetic transformation of 'hanfu' apple seed callus and application thereof

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to a genetic transformation method of 'Hanfu' apple seed callus and application. The transformed callus is obtained through induction culture, proliferation culture, immersion culture, co-culture and screening culture of the explant, wherein the explant is 'Hanfu' apple seed; the uninfected 'Hanfu' apple seed callus induced by the method has weak fluorescence signal under ultraviolet excitation light. When the callus is subjected to genetic transformation mediated by Agrobacterium and the pRI101 GFP vector is introduced, the fluorescence signal intensity of the callus is significantly increased, indicating that the 'Hanfu' apple callus can be subjected to genetic transformation containing a GFP label.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a genetic transformation method and application of 'Hanfu' apple seed callus. Background Technology

[0002] In botany, when plant cells are injured, a special cell population—callus—forms on the wound surface. This callus, composed of enlarged and dedifferentiated thin-walled cells, plays a crucial role in plant molecular research and genetic engineering, and is a commonly used experimental material. For genetic transformation, a stable regeneration system is an indispensable prerequisite. However, in the field of apple genetic transformation, numerous challenges exist; transformation is difficult, and the probability of successfully obtaining transformed plants with stable genetic characteristics is low. In contrast, obtaining stable callus tissue for genetic transformation is relatively easier.

[0003] Therefore, the successful cultivation of apple callus and the establishment of a complete apple callus transformation system have enabled the production of genotype-consistent and stable callus tissue. At the same time, a rapid and stable genetic transformation method has been explored. This will provide a new transgenic recipient material and experimental approach for apple molecular biology and genetic engineering research, which has significant research significance and application value.

[0004] Currently, in the field of apple callus research, the materials used are mostly callus tissue induced from 'Wanglin' ​​apple fruits. However, it is worth noting that untransformed 'Wanglin' ​​apple callus tissue exhibits a distinct green fluorescence signal under 488nm ultraviolet excitation. This characteristic makes 'Wanglin' ​​apple callus tissue unsuitable for research involving the transformation of callus tagged with green fluorescent protein (GFP). The inherent fluorescence signal it carries can severely interfere with the observation of experimental results, thus affecting the accuracy and reliability of the research findings. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a genetic transformation method and application for 'Hanfu' apple seed callus.

[0006] A genetic transformation method for 'Hanfu' apple seed callus, wherein the explants are induced culture, proliferation culture, infection culture, co-culture and screening culture to obtain successfully transformed callus, wherein the explants are 'Hanfu' apple seeds;

[0007] The formulation of the induction medium used for the induction culture is 4 g~5 g MS + 0.1 mg~0.4 mg NAA + 1.0 mg~2.0 mg 2,4-D + 0.5 mg~1.0 mg 6-BA + 10 g~30 g sucrose + 7 g~9 g agar, with water added to 1L;

[0008] The proliferation medium used for the proliferation culture was formulated as follows: 4 g~5 g MS + 1.5 mg 6-BA + 0.4 mg~0.8 mg ZT + 10 g~30 g sucrose + 7 g~9 g agar, with water added to a final volume of 1 L.

[0009] The infiltration culture was performed by infiltrating 'Hanfu' apple seed callus tissue with MdPHV-GFP Agrobacterium carrying the target gene vector.

[0010] Preferably, the co-culture medium used in the co-culture is formulated as follows: 4 g~5 g MS + 1.0 mg~2.0 mg 2,4-D + 0.5 mg~1.0 mg 6-BA + 10 g~30 g sucrose + 7 g~9 g agar, with water added to 1 L.

[0011] Preferably, the screening medium used for the screening culture consists of 4 g~5 g MS + 1.0 mg~2.0 mg 2,4-D + 0.5 mg~1.0 mg 6-BA + 20 mg~40 mg kanamycin + 150 mg~250 mg cephalosporin + 10 g~30 g sucrose + 8 g~10 g agar, with water added to a final volume of 1 L.

[0012] Preferably, the induction culture time is 60 to 90 days.

[0013] Preferably, the proliferation culture time is 14 days.

[0014] Preferably, the 'Hanfu' apple seeds are selected 30 to 40 days after flowering.

[0015] Preferably, the 'Hanfu' apple seed sample is a wounded embryo.

[0016] Preferably, the culture medium needs to be adjusted to pH 5.6 using 1 mol / L NaOH solution.

[0017] The application of the described genetic transformation method in genetic transformation.

[0018] Preferably, the genetic transformation is a genetic transformation method containing a GFP tag.

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] This invention provides a genetic transformation method for 'Hanfu' apple seed callus. The callus induced from uninfected 'Hanfu' apple seeds obtained using the culture medium in this method exhibits weak fluorescence under ultraviolet excitation. However, when this callus undergoes Agrobacterium-mediated genetic transformation and is infused with the pRI101 GFP vector, its fluorescence intensity significantly increases. This characteristic indicates that 'Hanfu' apple callus can be transformed using a GFP-tagged genetic transformation method, thereby effectively improving the efficiency of transgenic material identification and providing a more convenient and accurate means for related research.

[0021] The genetic transformation method provided by this invention further reduces the fluorescence signal of callus induced by 'Hanfu' apple seeds under ultraviolet excitation light, so that the fluorescence signal intensity of the callus after Agrobacterium-mediated genetic transformation increases more significantly, making it easier to identify transgenic materials. Attached Figure Description

[0022] Figure 1 The images show the proliferation of 'Hanfu' apple seed callus and 'Wanglin' ​​apple callus within 14 days. In the images, A represents day 1 after inoculation of 'Wanglin' ​​apple callus, B represents day 7 after inoculation of 'Wanglin' ​​apple callus, C represents day 14 after inoculation of 'Wanglin' ​​apple callus, D represents day 1 after inoculation of 'Hanfu' apple seed callus, E represents day 7 after inoculation of 'Hanfu' apple seed callus, and F represents day 14 after inoculation of 'Hanfu' apple seed callus.

[0023] Figure 2 Comparison of fluorescence characteristics of different callus tissues under ultraviolet irradiation, where A is an image of 'Hanfu' apple seed callus tissue and B is an image of 'Wanglin' ​​apple callus tissue.

[0024] Figure 3 Comparison of fluorescence characteristics of 'Hanfu' apple seed callus under ultraviolet irradiation. In this study, A represents 'Hanfu' apple seed callus, and B represents overexpression. GFP The 'cold-rich' apple seed callus tissue of the gene.

[0025] Figure 4 Comparison of fluorescence characteristics of 'Wanglin' ​​apple callus under ultraviolet irradiation, where A represents 'Wanglin' ​​apple callus and B represents overexpression. GFP The 'Wang Lin' apple callus tissue of the gene.

[0026] Figure 5 A bar graph showing the proliferation coefficients of 'Hanfu' apple seed callus and 'Wanglin' ​​apple callus within 14 days. Detailed Implementation

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0028] Materials used in the embodiments

[0029] Plant material: 'Hanfu' apple seeds harvested in the current year from the fruit tree germplasm resource nursery.

[0030] Agrobacterium strain: EHA105.

[0031] Plasmid: pRI101GFP is the pRI101GFP reported in the literature Yue Ma, Hao Xue, Feng Zhang, Qiu Jiang, Shuang Yang, Pengtao Yue, Feng Wang, Yuanyan Zhang, Linguang Li, Ping He, Zhihong Zhang. (2021) The miR156 / SPL module regulates apple salt stress tolerance by activating MdWRKY100 expression. Plant Biotechnology Journal. 19, 2, 311-323.

[0032] Primer synthesis was completed at Shanghai Sangon Biotech Co., Ltd.

[0033] The reagents used in the examples were MS medium, sucrose and agar, all purchased from Cooler Master Technology Co., Ltd., and PhantaMax Super-Fidelity DNA Polymerase purchased from Novizan Nanjing Biotechnology Co., Ltd.

[0034] The YEP liquid culture medium formula is: 10 g yeast extract, 10 g tryptone, 5 g NaCl, and water to a final volume of 1 L.

[0035] The YEP solid plate formulation is as follows: 10 g yeast extract, 10 g tryptone, 5 g NaCl, 100 mg kanamycin, 100 mg rifampin, 15 g agar, and water to a final volume of 1 L.

[0036] Example 1

[0037] Freeze-thaw transformation of Agrobacterium competent cells

[0038] (1) Take 1 μL (100 ng / μL) of pRI101GFP Escherichia coli plasmid and mix it into 50 μL of EHA105 Agrobacterium competent cells. Gently pipette and mix well to obtain a mixed solution.

[0039] (2) The mixed solutions were placed on ice for 5 minutes, then in liquid nitrogen for 5 minutes, and finally in a 28°C water bath for 5 minutes, and then placed on ice for 5 minutes for thermal shock conversion.

[0040] (3) After the transformation is complete, add 700 μL of YEP liquid medium to the centrifuge tube and culture at 28℃ for 2 h with shaking at 180 rpm.

[0041] (4) After the culture is completed, centrifuge the bacterial cells at 5000 rpm for 2 minutes. Discard the supernatant, keep the precipitate, mix it by suction and spread it on YEP solid plates.

[0042] (5) Incubate at 28℃ upside down for 48 h. PCR verification was performed on the colonies that grew, and positive clones were picked and added to YEP liquid medium (with the corresponding antibiotics) and cultured at 28℃ and 180 rpm to obtain activated Agrobacterium strain EHA105pRI101-GFP, i.e., positive colonies. After the solution became turbid, it was mixed with 50% glycerol at a volume ratio of 1:1 and frozen at 80℃ for later use.

[0043] Example 2

[0044] To detect the positive colonies obtained in Example 1 (5), colony PCR detection was performed.

[0045] Primers were designed based on the apple whole genome database and the sequence information of the target gene GFP, wherein the primers include an upstream primer F and a downstream primer R, and the specific sequences are shown below:

[0046] Upstream primer F: 5' GACGCACAATCCCACTATCC3';

[0047] Downstream primer R: 5'CACGCTGAACTTGTGGCCGTTCAC3'.

[0048] RNA was extracted from the leaves of 'Hanfu' variegated plant, and cDNA was obtained by reverse transcription. This cDNA served as a template for PCR amplification. PCR amplification was performed using the primers described above. The reaction system was as follows:

[0049] Table 1 PCR reaction system

[0050]

[0051] The PCR products were electrophoresed on a 1% agarose gel to observe gene amplification, and the amplification products were observed using a gel imaging system.

[0052] In this embodiment, the specific band had a molecular weight of approximately 500 bp, indicating that Agrobacterium colonies with the EHA105pRI101-GFP plasmid inserted were obtained for subsequent genetic transformation. The desired positive colonies were then obtained.

[0053] Example 3

[0054] Induced proliferation and genetic transformation of 'Hanfu' apple seed callus

[0055] The method for inducing proliferation and genetic transformation of 'Hanfu' apple seed callus provided in this embodiment of the invention includes the following steps:

[0056] This embodiment uses GFP Using the target gene, a genetic transformation system for 'Hanfu' apple seed callus was established, which specifically includes the following steps:

[0057] (1) Obtaining genetic transformation receptors.

[0058] The collected 'Hanfu' apple seed samples were disinfected and sterilized by shaking them in a disinfectant solution containing 2% sodium hypochlorite for 20-30 minutes, followed by rinsing them 3-5 times with sterile water. The rinsed seeds were then peeled in a laminar flow hood and placed on sterilized gauze. The peeled seeds were then collected and placed in a disinfectant solution containing 0.5% sodium hypochlorite for 1 minute, followed by rinsing them 2-3 times with sterile water. The seeds were then blotted dry in sterile filter paper and set aside for later use.

[0059] (2) Induction and proliferation of callus tissue from 'Hanfu' apple seeds.

[0060] After sterilization, 'Hanfu' apple seeds were inoculated onto induction medium and cultured in the dark at 25℃ for 60-90 days. Once callus tissue was obtained, it was transferred to proliferation medium for further proliferation culture under a 16 / 8 h photoperiod and at 25℃. The induction medium consisted of 4.43 g / L MS + 0.4 mg / L NAA + 1.5 mg / L 2,4-D + 0.5 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar, while the proliferation medium consisted of 4.43 g / L MS + 1.5 mg / L 6-BA + 0.4 mg / L ZT + 30 g / L sucrose + 8 g / L agar.

[0061] (3) Immersion test

[0062] 1) Agrobacterium culture

[0063] The activated Agrobacterium strain EHA105pRI101-GFP from Example 1 was added to YEP liquid medium containing kanamycin and rifampin and cultured. The culture was shaken at 180 rpm for 1216 h at 28°C until the Agrobacterium concentration reached OD600 = 0.4~0.6, at which point it was ready for use as the Agrobacterium culture solution.

[0064] 2) Preparation of Agrobacterium tumefaciens inoculum

[0065] Pour the Agrobacterium tumefaciens bacterial suspension into a sterile centrifuge tube, centrifuge at 5000g for 5 minutes at 4℃, collect the Agrobacterium tumefaciens cells, and discard the supernatant; resuspend the Agrobacterium tumefaciens with ddH2O, and determine the concentration of the Agrobacterium tumefaciens bacterial suspension, i.e., OD600 = 0.4~0.6, using a UV spectrophotometer;

[0066] 3) Callus inoculation, co-culture, and screening culture

[0067] The Agrobacterium tumefaciens infection solution prepared in step 2) was co-cultured with 'Hanfu' apple seed callus in a constant temperature shaker at 28°C and 100 rpm in the dark for 30 min to obtain the infected 'Hanfu' apple seed callus.

[0068] After the above-obtained 'Hanfu' apple seed callus was placed on filter paper to absorb the staining solution, it was placed in a co-culture medium and co-cultured in the dark for 1 day.

[0069] In this process, a sterile filter paper needs to be placed on the co-culture medium, and then the infected callus is spread out on the co-culture medium. The co-culture medium is 4.43 g / L MS + 1.5 mg / L 2,4-D + 0.4 mg / L 6-BA + 30 g / L sucrose + 9 g / L agar.

[0070] 4) Screening and Cultivation

[0071] The 'Hanfu' apple seed callus obtained in step 3) was cultured in the dark at 25°C for 30 days on solid MS screening medium (4.43 g / L MS + 1.5 mg / L 2,4-D + 0.4 mg / L 6-BA + 30 mg / L Kana + 200 mg / L Cef + 30 g / L sucrose + 9 g / L agar). After screening, the callus was irradiated with a handheld fluorescence irradiator for fluorescence observation.

[0072] Comparative Example 1

[0073] The difference between Comparative Example 1 and Example 3 is that the callus tissue in steps 3) and 4) is 'Wang Lin' apple callus tissue. Steps 3) and 4) are as follows:

[0074] 3) Callus inoculation, co-culture, and screening culture

[0075] The Agrobacterium tumefaciens infection solution prepared in step 2) was co-cultured with 'Wanglin' ​​apple callus in a constant temperature shaker at 28°C and 100 rpm in the dark for 30 min to obtain infected 'Wanglin' ​​apple callus.

[0076] After the above-obtained 'Wang Lin' apple callus was placed on filter paper to absorb the staining solution, it was placed in a co-culture medium and co-cultured in the dark for 1 day.

[0077] In this process, a sterile filter paper needs to be placed on the co-culture medium, and then the infected callus is spread out on the co-culture medium. The co-culture medium is 4.43 g / L MS + 1.5 mg / L 2,4-D + 0.4 mg / L 6-BA + 30 g / L sucrose + 9 g / L agar.

[0078] 4) Screening and Cultivation

[0079] The 'Wang Lin' apple callus obtained in step 3) was placed in solid MS selection medium (4.43 g / L MS + 1.5 mg / L 2,4-D + 0.4 mg / L 6-BA + 30 mg / L Kana + 200 mg / L Cef + 30 g / L sucrose + 9 g / L agar) and cultured in the dark at 25℃ for 30 days. After screening, the callus was irradiated with a handheld fluorescence irradiator for fluorescence observation.

[0080] The results showed that successfully infected callus tissues from 'Hanfu' apple seeds and 'Wanglin' ​​apples were green. The fluorescently labeled callus tissues were placed individually in solid MS selection medium and cultured for three generations at 34 weeks each to obtain homozygous resistant callus tissues from 'Hanfu' apple seeds and 'Wanglin' ​​apples, respectively. The culture conditions were: dark culture at 25℃.

[0081] Figure 2 Images show unstained callus tissues of 'Hanfu' apple seeds and 'Wanglin' ​​apples. Fluorescent labeling revealed that the 'Hanfu' apple seed callus exhibited weaker fluorescence than the 'Wanglin' ​​apple callus.

[0082] Figure 3 Images show unstained and stained 'Hanfu' apple seed callus. Fluorescent labeling revealed a clear fluorescence in the stained 'Hanfu' apple seed callus, with a significant contrast between the two.

[0083] Figure 4Images show unstained and stained 'Wang Lin' apple callus. Fluorescent labeling revealed that the stained 'Wang Lin' apple callus exhibited fluorescence compared to the unstained one, but the contrast was not significant.

[0084] Table 2. Callus induction rate of 'Hanfu' apple seeds under different induction media

[0085]

[0086] Note: The analysis was performed using Ducan's multiple range test. Different lowercase letters were significantly different at the p < 0.05 level.

[0087] Induction rate = Number of calluses that produce callus / Total number of inoculated calluses

[0088] The experimental data were first summarized using Microsoft Excel 2010, and then statistically analyzed using IMS SPSS Statistics 22. Significance analysis was performed at the P ≤ 0.05 level.

[0089] Table 3. Statistical table of callus proliferation of 'Hanfu' apple seeds under different proliferation culture media.

[0090]

[0091] Note: The ideal condition is when the callus is yellowish-white, loose and moist, and grows quickly; the next best condition is when the callus is milky white or slightly brown, harder and grows relatively quickly; and the worst condition is when the callus is brownish, harder, and grows slowly.

[0092] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0093] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0094] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A genetic transformation method for GFP-tagged callus tissue from 'Hanfu' apple seeds, wherein successfully transformed callus tissue is obtained from explants through induction culture, proliferation culture, infection culture, co-culture, and selection culture, characterized in that... The explants were 'Hanfu' apple seeds; The formulation of the induction medium used for the induction culture is 4 g~5g MS + 0.1 mg~0.4 mg NAA + 1.0 mg~2.0 mg 2,4-D + 0.5 mg~1.0 mg 6-BA + 10 g~30 g sucrose + 7 g~9 g agar, with water added to 1L; The proliferation medium used for the proliferation culture was formulated as follows: 4 g~5 g MS + 1.5 mg 6-BA + 0.4 mg~0.8 mg ZT + 10 g~30 g sucrose + 7 g~9 g agar, with water added to a final volume of 1 L. The infiltration culture was performed by infecting 'Hanfu' apple seed callus tissue with Agrobacterium carrying the target gene vector pRI101-GFP. The 'Hanfu' apple seeds were selected 30 to 40 days after flowering, and the 'Hanfu' apple seed samples were embryos with wounds.

2. The genetic transformation method according to claim 1, characterized in that, The co-culture medium used in the co-culture was formulated as follows: 4 g~5 g MS + 1.0 mg~2.0 mg 2,4-D + 0.5 mg~1.0 mg 6-BA + 10 g~30 g sucrose + 7 g~9 g agar, with water added to a final volume of 1 L.

3. The genetic transformation method according to claim 1, characterized in that, The screening medium used for the screening culture consisted of 4 g~5 g MS + 1.0 mg~2.0 mg 2,4-D + 0.5 mg~1.0 mg 6-BA + 20 mg~40 mg kanamycin + 150 mg~250 mg cephalosporin + 10 g~30 g sucrose + 8 g~10 g agar, with water added to a final volume of 1 L.

4. The genetic transformation method according to claim 1, characterized in that, The induction culture time is 60 to 90 days.

5. The genetic transformation method according to claim 1, characterized in that, The proliferation culture time is 14 days.

6. The genetic transformation method according to claim 1, characterized in that, The culture medium needs to be adjusted to pH 5.6 using 1 mol / L NaOH solution.

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