Application of walnut YX4 as receptor material in agrobacterium-mediated genetic transformation

Abstract

The invention discloses an application of walnut YX4 as a receptor material in agrobacterium tumefaciens-mediated genetic transformation. The invention relates to the field of genetic transformation, and particularly discloses a method for carrying out genetic transformation by taking Juglans regia YX4 CGMCC (China General Microbiological Culture Collection Center) No.46084 as an acceptor material. According to the invention, genotype walnuts with high somatic embryo induction rate, strong regeneration capacity and high genetic transformation efficiency are screened as genetic transformation receptors, and an efficient genetic transformation system and method are optimized and established. By adopting the transformation method disclosed by the invention, the transformation efficiency (P = 0.008) of agrobacterium infection is remarkably improved. The screened excellent receptor material of the YX-4 genotype is named as YX4, and is preserved in the China General Microbiological Culture Collection Center (CGMCC). The screening of excellent receptor materials and the optimization of a genetic transformation system have important practical significance for establishing a walnut rapid propagation system, and the commercialization process of walnut breeding is promoted.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of walnut YX4 as a receptor material in Agrobacterium-mediated genetic transformation. Background Art

[0002] The walnut genus encompasses 20 species (studied by Gleeson in 1982). Among them, walnut (Juglans regia), as one of the most important economic tree species, is cultivated all over the world. Walnut kernels are rich in unsaturated fatty acids, proteins, minerals, and vitamins, and are of great significance to human health. China leads the world in walnut production and cultivation, and is also one of the main global centers of walnut genetic diversity, providing valuable germplasm resources for walnut breeding work (Bernard et al., 2018; Chen et al., 2014). In the early days, in order to more effectively utilize these germplasm resources, traditional breeding methods were used to cultivate genetically stable excellent varieties, but the efficiency of this method was relatively low. Recent studies have shown that transgenic technology can break through the limitations of traditional breeding techniques and greatly accelerate the process of germplasm improvement.

[0003] Agrobacterium-mediated genetic transformation is one of the widely used methods for developing transgenic plants in many species, such as oak (Vidal et al. 2010), willow (Liu et al. 2015), cotton (Chen et al. 2014), ash (Palla and Pijut 2015), and chestnut (Sun et al. 2020). Somatic embryos (SEs) in plants are of single-cell origin and can serve as excellent experimental materials for genetic transformation (Polito et al. 1989). Since the first report of walnut somatic embryogenesis (SEs) in 1985, research on the use of walnut SEs for genetic transformation has been carried out (Tang et al. 2000). First, McGranahan et al. improved the Agrobacterium infection process in terms of antibiotic gene selection (McGranahan et al. 1988). The application of GFP fluorescent protein not only saved manpower and material resources but also improved the detection efficiency (Escobar et al. 2000). Later, a new non-invasive monitoring gene DsRED was found to be able to be used for visual detection of material exchange in transgenic walnuts (Zhang et al. 2015; Liu et al. 2017). In addition, research on functional genes has also been successively reported. Overexpression of the crystal gene in walnuts (Dandekar et al. 1998) can increase their resistance to insects; overexpression of the JrWOX11 gene (Chang et al. 2022) can significantly improve the rooting ability of walnuts, which has laid a foundation for the development of a series of walnut varieties with excellent traits. Although great progress has been made in walnut genetic transformation research in recent years, problems such as poor regeneration ability of receptor materials, long growth cycles, and low transformation efficiency of existing genetic transformation systems are particularly prominent.

[0004] In view of this, screening excellent receptor materials and developing new and efficient genetic transformation systems are of great practical significance for establishing a rapid propagation system for walnuts and lay a foundation for studying walnut gene functions. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to improve the Agrobacterium-mediated genetic transformation efficiency of walnuts. The technical problems to be solved are not limited to the described technical topics, and those skilled in the art can clearly understand other technical topics not mentioned herein through the following description.

[0006] To solve the above technical problems, the present invention first provides the application of walnut (Juglans regia) YX4 as a receptor material in Agrobacterium-mediated genetic transformation. The preservation number of the walnut (Juglans regia) YX4 in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 46084.

[0007] The present invention also provides an Agrobacterium-mediated walnut genetic transformation method, which includes using the walnut (Juglans regia) YX4 as a receptor material for genetic transformation.

[0008] Furthermore, the method may include the following steps:

[0009] A1) Pre-culturing the somatic embryos of the walnut (Juglans regia) YX4 to obtain pre-cultured somatic embryos;

[0010] A2) Infecting the pre-cultured somatic embryos with a recombinant Agrobacterium solution containing a target gene;

[0011] A3) Inducing and culturing the identified positive transgenic somatic embryos to obtain regenerated plants;

[0012] The pre-culturing conditions in step A1) include: pre-culturing the somatic embryos in a medium and subculturing once every 6 - 8 days.

[0013] Furthermore, the pre-culturing conditions in step A1) include: pre-culturing the somatic embryos in a medium, subculturing once every 7 days, and culturing for 28 - 32 days.

[0014] Furthermore, the OD 600 value of the recombinant Agrobacterium solution in step A2) can be 0.6 - 0.8.

[0015] Furthermore, the diameter of the somatic embryos in step A1) can be 3 mm to 9 mm.

[0016] The 3 mm to 9 mm described herein can be 3, 4, 5, 6, 7, 8, or 9 mm.

[0017] Furthermore, the diameter of the somatic embryos can be 6 mm.

[0018] In the above method, before step A1), it may further include the step of inducing the regeneration of somatic embryos from young embryos of the walnut (Juglans regia) YX4 with a diameter of 3 mm to 9 mm using an induction medium.

[0019] In the above method, the infection time in step A2) can be 5 - 30 minutes.

[0020] Further, the infection time in step A2) can be 5, 10, 15, 20, 25 or 30 minutes.

[0021] In the above method, the infection time in step A2) can be 15 minutes.

[0022] In the above method, the pre-culture medium in step A1) can be the DKW medium containing 30 g / L of sucrose and 3.0 g / L of phytagel.

[0023] Further, the pH value of the pre-culture medium can be 5 - 6, and preferably can be 5.6.

[0024] In the above method, the induction medium can be the DKW medium containing 30 g / L of sucrose, 2.0 mg / L of 6-BA (6-benzylaminopurine) and 3.0 g / L of phytagel.

[0025] Further, the pH value of the induction medium can be 5 - 6, and preferably can be 5.6.

[0026] Further, the method for identifying positive in A3) is known in the art. For example, the transformed transgenic plants or materials (including transgenic progeny materials) can be identified by techniques such as PCR detection, Southern hybridization, immunoblotting, Northern hybridization, enzyme-linked immunosorbent assay (ELISA), functional identification (testing for the presence of selectable marker genes and target genes) and / or in situ hybridization, etc.

[0027] The Agrobacterium-mediated walnut genetic transformation method described herein specifically may include the following steps:

[0028] (1) Pre-culture the cell embryos of walnut body YX4 with a diameter of about 6 mm in the medium, subculture every 7 days, and culture for about 30 days to obtain pre-cultured somatic embryos;

[0029] (2) Infect the pre-cultured somatic embryos with the recombinant Agrobacterium solution containing the target gene, and the OD 600 value of the recombinant Agrobacterium solution is 0.6, and the infection time is 5 - 30 minutes;

[0030] (3) Induce and culture the positive transgenic somatic embryos obtained through identification to obtain regenerated plants.

[0031] The Agrobacterium-mediated walnut genetic transformation method described herein specifically may also include the following steps:

[0032] (1) Pre-culture somatic embryos of walnut YX4 with a diameter of about 6 mm in a medium, subculture every 7 days for about 30 days to obtain pre-cultured somatic embryos;

[0033] (2) Infect the pre-cultured somatic embryos with a recombinant Agrobacterium liquid containing the target gene, the OD 600 value of the recombinant Agrobacterium liquid is 0.8, and the infection time is 5 - 30 minutes;

[0034] (3) Induce and culture the identified positive transgenic somatic embryos to obtain regenerated plants.

[0035] In the above method, the preparation method of the recombinant Agrobacterium liquid containing the target gene in step A2) is: introduce the recombinant vector containing the target gene into Agrobacterium, obtain positive recombinant bacteria through screening and identification, and resuspend the positive recombinant bacteria with a liquid DKW basal medium containing acetosyringone to obtain a recombinant Agrobacterium liquid (i.e., the infection liquid).

[0036] Furthermore, the introduction method includes but is not limited to introducing the recombinant vector containing the target gene into Agrobacterium by chemical transformation methods (such as Ca ion-induced transformation method, polyethylene glycol-mediated transformation method, or metal cation-mediated transformation method, etc.) or physical transformation methods (such as electroporation transformation method).

[0037] In one or more embodiments of the present invention, the introduction method is the electroshock method (i.e., the electroporation transformation method).

[0038] The target gene described herein can be a nucleic acid molecule encoding a target protein (any protein of interest), and it can be the gene that can bring the expected phenotypic traits and needs to be cloned and transformed into the recipient (walnut). The target gene can be from the organism itself or from different organisms. The methods for obtaining the target gene are well-known to those skilled in the art. For example, the complex organism genome can be digested by enzymatic digestion and other steps, first construct a genomic library, and then isolate the DNA fragment carrying the target gene from the library; or use the reverse transcription method to start from mRNA and reverse transcribe to obtain cDNA as the target gene; it is also possible to artificially synthesize the target gene by enzymatic or chemical synthesis methods; it is also possible to directly amplify the target gene in vitro from the donor organism genome or the existing target gene clone using PCR technology, etc.

[0039] The so-called reporter gene generally refers to a group of genes encoding proteins or enzymes that are easily detectable. The coding sequence of the reporter gene is fused with the gene expression regulatory sequence, or fused with other target genes, and expressed under the control of the regulatory sequence. The expression regulation of the target gene is "reported" by detecting the expression product of the reporter gene. The reporter gene may include β-glucuronidase (GUS) gene, luciferase gene, chloramphenicol acetyltransferase (CAT) gene, β-galactosidase (β-gal) gene, secreted human placental alkaline phosphatase (SEAP) gene, yellow fluorescent protein (YFP) gene, green fluorescent protein (GFP) gene, etc.

[0040] In one or more embodiments of the present invention, the target gene is the GUS gene or the YFP gene, but the present invention is not limited to this gene, and it can also be other target genes, such as other reporter genes, disease-resistant genes, insect-resistant genes, stress-resistant genes, high-yield genes, genes related to improving quality, genes related to protein content, genes related to oil content, development-related genes, or long-chain unsaturated fatty acid dehydrogenase genes, etc., but not limited thereto.

[0041] Through extensive and in-depth research, the inventors of this application screened 30 walnut germplasms with representative and different characteristics from thousands of walnut varieties as candidate materials, and further screened the best receptor materials from them. Finally, a genotype walnut with a high somatic embryo induction rate, strong regeneration ability, and high genetic transformation efficiency was selected as the walnut genetic transformation receptor, and an efficient genetic transformation system and method were optimized and established. The transformation efficiency of Agrobacterium infection was significantly improved by using the transformation method of the present invention (P = 0.008). The excellent receptor material of the screened YX-4 genotype was named YX4, and it has been deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC), and the deposit number is CGMCC No. 46084. The screening of excellent receptor materials and the optimization of the genetic transformation system in the present invention have important practical significance for establishing a rapid propagation system for walnuts, and contribute to promoting the commercialization process of walnut breeding.

[0042] Deposit description

[0043] Proposed taxonomic name: Juglans regia

[0044] Biological material (strain) referred to: YX4

[0045] Deposit unit: General Microbiological Center of the China Committee for Culture Collection of Microorganisms

[0046] Abbreviation of deposit unit: CGMCC

[0047] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0048] Deposited date: July 18, 2024

[0049] Registration number in the preservation center: CGMCC No.46084 Description of the drawings

[0050] Figure 1 Is the somatic embryo induction rate of immature embryos with different genotypes

[0051] Figure 2 Is the somatic embryo proliferation rate of different genotypes

[0052] Figure 3 Is the somatic embryo plant conversion rate of different genotypes

[0053] Figure 4 Is the somatic embryo transformation efficiency of different genotypes

[0054] Figure 5 Is the effect of the diameter of the immature embryo of Juglans regia YX4 on the induction regeneration rate. Among them: a: Phenotypic characteristics of receptor materials with different sizes; b: Induction regeneration rate of immature embryos of Juglans regia with different diameters

[0055] Figure 6 Is the phenotypic map of somatic embryos with a diameter of 6 mm cultured for 30 days at different subculture frequencies. Among them: a: Subcultured once every 7 days; b: Subcultured once every 10 days; c: Subcultured once every 14 days

[0056] Figure 7 Is the genetic transformation of the YFP gene in somatic embryos of Juglans regia YX4. Among them: a: DNA detection of the YFP gene in transgenic somatic embryos, M: 200 bp molecular weight standard, P: Plasmid positive control, N: Negative control (non-transgenic sample), 1-22: Transgenic somatic embryos; b: Semi-quantitative RT-PCR detection of the YFP gene in transgenic somatic embryos; c: Overexpression level of YFP in somatic embryos; d: Fluorescence observation of Juglans somatic embryos under a fluorescence microscope; e: Positive rate of transgenic YFP somatic embryos in E1, E2 and E3 generations

[0057] Figure 8 Is the genetic transformation of the GUS gene in somatic embryos of Juglans regia YX4. Among them: a: DNA detection of the GUS gene in transgenic somatic embryos, M: 200 bp molecular weight standard, P: Plasmid positive control, N: Negative control (non-transgenic sample), 1-22: Transgenic somatic embryos; b: Staining reaction of Juglans somatic embryos transfected with the GUS gene; c: Positive rate of transgenic somatic embryos transfected with the GUS gene in E1, E2 and E3 generations Specific implementation manners

[0058] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0059] In the experimental methods in the following embodiments, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0060] The plasmid pMDC164-DR5::GUS in the following embodiments uses pMDC164 as the backbone vector. The DR5 fragment and the pMDC164 vector (containing the GUS reporter gene) are double-digested with KpnI and BamHI, and the DR5 promoter is directionally inserted into the multiple cloning site upstream of the GUS gene through T4 ligase. It is transformed into Escherichia coli DH5α (provided by Beijing Huayueyang Biotechnology Co., Ltd., product number NRR01060). Positive clones are screened through kanamycin resistance, and the correctness of the inserted sequence is verified by colony PCR and sequencing to complete the construction of the recombinant vector, which is named pMDC164-DR5::GUS.

[0061] The plasmid pMDC164 (containing the GUS reporter gene) was kindly provided by Professor Liubin Liu of Fujian Agriculture and Forestry University and is described in the following literature: Liu B, Wang L, Zhang J, et al. WUSCHEL-related Homeobox genes in Populustomentosa: diversified expression patterns and a functional similarity in adventitious root formation[J]. BMC genomics, 2014, 15: 1-14. The public can obtain it from the applicant to repeat the experiments of this application.

[0062] The YFP reporter gene-labeled plasmid pBS-35S::YFP empty vector in the following embodiments was purchased from NewProBio, and the product number is V009083.

[0063] The DKW medium in the following embodiments was purchased from phytotech company, and the product number is D2470.

[0064] SE and SEs in the following embodiments have the same meaning, both representing somatic embryos (abbreviated as somatic embryos), and can be used interchangeably.

[0065] Example 1. Screening of the best genotype somatic embryo receptor for walnut genetic transformation

[0066] 1. Comparison of somatic embryo induction rates of immature embryos with different genotypes

[0067] To establish an efficient genetic transformation system, we first screened excellent receptor materials. Thirty germplasms with different characteristics were selected as candidate test materials from the main walnut-producing areas across the country, such as Xinjiang, Tibet, Henan, Hebei, Beijing, and Liaoning. At the optimal fruit development stage (50 days after the full bloom of female flowers), 50 immature embryos of each variety were taken respectively, and the induction rates of immature embryos of different germplasms were compared under the same induction conditions.

[0068] Induction and regeneration of walnut somatic embryos: Using the immature embryos of walnuts as materials, sampling of walnut young fruits was carried out at different time periods starting from 8 weeks after pollination. After surface disinfection, the immature embryos were induced to regenerate and cultured at 24 °C with somatic embryo (SE) induction medium (SEIM), and the induction and regeneration rates of walnut immature embryos were counted.

[0069] Configuration method of somatic embryo induction medium (SEIM): Based on DKW medium, sucrose with a final concentration of 30 g / L, 6-BA (6-benzylaminopurine) with a concentration of 2.0 mg / L, and phytagel with a concentration of 3.0 g / L were added, and the pH was adjusted to 5.6.

[0070] The results showed that seven lines such as YX-1 and YX-7 were direct somatic embryo formation types. Globular embryos appeared 25 days after induction, and cotyledonary embryos with complete structures were formed around 35 days, showing strong somatic embryo induction ability. Callus was produced on the surface of the immature embryos of the second type (YX-10, YX-13) and the third type (such as YX-17, YX-19) around 25 days of induction, and complete cotyledonary somatic embryos were formed until around 45 days. The callus persisted after the formation of somatic embryos and showed browning and necrosis. The overall induction state of the fourth type of immature embryos was poor, and the formation of somatic embryos was restricted.

[0071] The comparison results of somatic embryo induction rates of four types of immature embryos are as Figure 1 shown. The induction rates of immature embryos of most varieties in type I were significantly higher than those of the other three types. The induction rates of some varieties were close to 40%, especially for YX-4, whose induction rate of immature embryos reached more than 50%. In contrast, the induction rates of the second and third types of immature embryos were the second, but there were also significant differences in the induction rates among different varieties of walnuts within these two types. Overall, the immature embryos of walnuts in type I showed a high induction efficiency in a relatively short induction time through the direct formation of somatic embryos, demonstrating good induction response ability. In subsequent experiments, we took the somatic embryos of type I as the research object and carried out a comparative analysis on the process of cultivating them into regenerated lines.

[0072] 2. Comparison of somatic embryo regeneration abilities of walnuts with different genotypes

[0073] To further screen for ideal recipient somatic embryo materials, we conducted a comparative analysis of the regeneration ability (proliferation rate and germination rate of somatic embryos) during the regeneration process of seven different genotypes of somatic embryos with high germination rates that were screened out. The results showed that there were significant differences in the proliferation rates of somatic embryos among the seven genotypes ( Figure 2 ): Among them, the proliferation rate of YX-4 was the highest, approaching 80%, significantly higher than that of other genotypes (p < 0.05). The proliferation rate of the YX-2 genotype was the second highest, approximately 70%. The proliferation rates of the YX-1, YX-3, YX-5, and YX-7 genotypes were relatively low, and the proliferation efficiency was only about 40%.

[0074] To obtain complete plant lines, we further analyzed the ability of different somatic embryos to germinate into plant lines ( Figure 3 ), and the conversion rate of YX-4 was the highest (85.3%), significantly higher than that of somatic embryos of other genotypes. The germination rates of YX-5, YX-2, and YX-3 were the second highest, 79.3%, 70.7%, and 62.6% respectively, while the germination rate of YX-6 was the lowest, and the germination conversion rate was only about 46.7%.

[0075] 3. Comparison of transformation efficiencies of somatic embryos of different genotypes

[0076] We used the pCAMBIA1300 vector containing the reporter gene Vens as a marker to verify and analyze the genetic transformation results. First, fluorescence detection showed that strong fluorescence signals were observed in three randomly selected transgenic somatic embryos (35S:Vens-1, 35S:Vens-2, 35S:Vens-3) in the green channel, while no fluorescence signal was observed in the wild-type (WT) control, indicating that the target gene was successfully expressed. Further statistical analysis of the positive rates of genetic transformation of different somatic embryos showed that the positive rate of the YX-4 line was 76.4%, significantly higher than the positive rates of somatic embryos of the other genotypes ( Figure 4 ).

[0077] In summary, in terms of factors such as the induction rate, proliferation rate, conversion rate, and positive rate of genetic transformation, the somatic embryos of the YX-4 genotype showed significantly better comprehensive performance than those of other genotypes and were suitable as excellent recipient materials for establishing a walnut genetic transformation system.

[0078] The excellent recipient material of the YX-4 genotype finally screened was named YX4. Juglans regia YX4 was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC) on July 18, 2024, and the deposit number is CGMCC No. 46084. Juglans regia YX4 CGMCC No. 46084 is also simply referred to as walnut YX4 in this article.

[0079] Example 2: Application of Walnut YX4 as a Receptor Material

[0080] In this example, an efficient genetic transformation system was established using walnut YX4 as the receptor material. The specific method is as follows:

[0081] 1. Effect of Young Embryo Diameter on Induction Regeneration Rate

[0082] Using the young embryos of walnut YX4 as materials, samples of walnut young fruits were taken at different time periods starting from 8 weeks after self-pollination. After surface disinfection, young embryos with four diameter sizes of 3 mm, 6 mm, 9 mm, and 12 mm were used for induction regeneration. They were cultured at 24 °C with somatic embryo (SE) induction medium (SEIM), and the induction regeneration rates of walnut young embryos with different diameter sizes were counted.

[0083] Configuration method of somatic embryo induction medium (SEIM): Based on DKW medium, add sucrose with a final concentration of 30 g / L, 6-BA (6-benzylaminopurine) with a concentration of 2.0 mg / L, and plant gel with a concentration of 3.0 g / L, and adjust the pH to 5.6.

[0084] The results are as Figure 5 shown. The induction rates of the four diameter-sized young embryos from high to low are: 6 mm > 3 mm > 9 mm > 12 mm, and there are significant differences among them. Therefore, walnut young embryos with a diameter of 6 mm are more suitable as explants for induction regeneration.

[0085] 2. Genetic Transformation of Walnut Somatic Embryos

[0086] (1) Preparation of Agrobacterium Infection Solution

[0087] The plasmids pMDC164-DR5::GUS and pBS-35S::YFP containing reporter genes were respectively integrated into the GV3101 Agrobacterium strain by electroporation. Single colonies were placed in LB liquid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin and cultured at 28 °C and 200 rpm. Samples were taken when OD 600 was 0.4, 0.6, and 0.8. After centrifuging at 5000 rpm for 10 minutes and discarding the supernatant, the cells were resuspended with liquid DKW basal medium containing 100 μM acetosyringone to obtain a resuspended bacterial solution (i.e., the infection solution), which was then used to infect walnut somatic embryos (SEs).

[0088] (2) Pre-culture and Transformation of Somatic Embryos

[0089] Select somatic embryos with a complete structure and the same appearance, 6 mm in size (induced and regenerated from young embryos with a diameter of 6 mm as explants), and transfer them to a solid basic DKW medium (using DKW medium as the basal medium, supplemented with sucrose at a final concentration of 30 g / L and phytagel at 3.0 g / L, and adjusted to pH 5.6) for pre-culture of SEs. The pre-culture conditions are as follows: subculture every 7 days, 10 days, and 14 days respectively. After culturing for about 30 days, transfer them into the prepared Agrobacterium liquid (the infection solution obtained in step (1)), and let them stand at room temperature for 10 minutes, 15 minutes, and 20 minutes respectively (Table 1). Then remove the infection solution, transfer the SEs to clean filter paper, fully absorb the bacterial liquid, and inoculate them onto a solid DKW basic medium containing 100 μM acetosyringone (using DKW medium as the basal medium, supplemented with sucrose at a final concentration of 30 g / L, phytagel at 3.0 g / L, and 100 μM acetosyringone, and adjusted to pH 5.6). After dark culturing at 25°C for 48 h, transfer the SEs to a DKW selection medium containing 200 mg / L ticarcillin and 80 mg / L hygromycin (using DKW medium as the basal medium, supplemented with sucrose at a final concentration of 30 g / L, 200 mg / L ticarcillin, 80 mg / L hygromycin, and phytagel at 3.0 g / L, and adjusted to pH 5.6) for screening and counting the positive rate of newly generated SEs.

[0090] The orthogonal experiment on the main factors affecting the genetic transformation efficiency of walnut SEs is shown in Table 1.

[0091] Table 1. Factors and levels of the genetic transformation effect experiment of walnut somatic embryos

[0092]

[0093]

[0094] 2. Detection of transgenic walnut somatic embryos

[0095] Genomic DNA was extracted from resistant and untransformed SEs using a plant genomic DNA kit (Tiangen, China, catalog number: DP350). The quality of the purified total DNA was observed by 1.0% (w / v) agarose gel electrophoresis, and its concentration was determined using a NanoDrop One (Thermo Scientific, USA). The final concentration of the isolated total DNA was diluted to 100 ng / μL and stored at -20 °C for later use. The insertion of the exogenous YFP gene was detected using the 35S-forward primer (5′-ctatccttcgcaagacccttcctct-3′) and the YFP-reverse primer (5′-tggcggacttgaagaagtcgtg-3′), and the insertion of the exogenous GUS gene was detected using the GUS-forward primer (5′-gaccacgcattaatggactggatt-3′) and the GUS-reverse primer (5′-GTACCTTCTCTGCCGTTTCCAAAT-3′). The PCR program was: 94 °C for 2 min, 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, 72 °C for 5 min, for 35 cycles.

[0096] The expression of the YFP gene was observed using a fluorescence microscope, and the expression levels were detected by semi - quantitative RT - PCR and qRT - PCR. The total RNA of SEs was isolated using the Plant RNA kit (product of Tiangen, catalog number: DP419), and 1 μg of total RNA was synthesized into cDNA using an RT kit (product of TaKaRa, catalog number RR047A). The program for qRT - PCR amplification of the YFP gene was: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. Amplification was carried out in the standard mode using a Roche Light Cycle 480Ⅱ real - time PCR system (Roche, Switzerland) and a TB Green Premix Ex Taq kit (product of TaKaRa, catalog number CN830S). All reactions were carried out in a 20 - μL solution, using 2 μL of 10 - fold diluted cDNA as the template and the walnut GAPDH gene as the internal reference. The following primers were used to detect the GAPDH and YFP genes: GAPDH - forward (5'-ATGATGTCAAGGAGAAGGACTC - 3') and GAPDH - reverse (5'-CACAATGAT CTCAGCTCCG - 3'), qYFP - forward (5'-ATCATGGCCGACAAGCAGAA - 3') and qYFP - reverse (5'-AACTC CAGCAGGACCATGTG - 3'). The program for semi - quantitative RT - PCR detection of the YFP gene was: 94°C for 2 min, 94°C for 30 s, 58°C for 30 s, 72°C for 10 s, 72°C for 5 min, for 28 cycles. The reaction volume was 20 μL, and the primers were the same as those for qRT - PCR amplification. The YFP fluorescence of SEs was detected using a fluorescence stereomicroscope (Carl LSM 510, Zeiss, Germany). The excitation wavelength and emission filter set were 514 nm (Ex) / BP520 - 555 nm (Em).

[0097] To detect the expression efficiency of the GUS gene, transgenic walnut SEs containing the GUS gene were analyzed using a GUS staining kit (product of Solarbio, catalog number G3060) under the control of the DR5 promoter. The SEs were placed in the GUS staining solution, and the color change was observed after incubation at 37°C for 8 hours.

[0098] 3. Statistical analysis of the transgenic success rate of walnut somatic embryos

[0099] SE survival rate (%) = (number of living SEs / number of infected SEs) × 100%, SE positive rate (%) = (number of positive SEs / total number of tested SEs) × 100%. Using the pre-culture of SEs, the concentration of Agrobacterium, and the inoculation time of the transformation system as the influencing factors of Agrobacterium transformation, 3 levels were set for each factor, and experiments were statistically analyzed according to the L9(34) orthogonal table (Table 1). A total of 9 treatments were set, and 50 SEs were tested for each treatment. All experiments were carried out with at least three independent replicates, and each replicate consisted of 50 samples.

[0100] 4. Experimental results

[0101] 4-1. Main factors affecting the genetic transformation efficiency of walnut SEs

[0102] Select SEs with consistent phenotypes and explore the effects of pre-culture, Agrobacterium concentration, and infection time (inoculation time) on genetic transformation efficiency (Table 1). The orthogonal experiment showed that among the 9 combinations, the survival rate of SEs in the A1B2C2 group was 77.78% after resistance screening, which was significantly higher than that of other groups. The treatment method was as follows: Subculture SEs with a diameter of about 6 mm every 7 days, and infect with Agrobacterium (OD 600 = 0.6) for 15 minutes after 30 days. The survival rates of other combinations from high to low were: A1B3C3, A1B1C1, A2B2C3, A2B1C2, A2B3C1, A3B1C3, A3B3C2, A3B2C1 (Table 2).

[0103] Table 2. Effects of three factors on the genetic transformation of walnut somatic embryos

[0104]

[0105] Through range analysis, among the 3 experimental factors, the pre-culture of SEs had the most significant effect on the survival rate of genetic transformation. Subculturing every 7 days, the SEs after 30 days showed relatively healthy white before infection ( Figure 6 in a), while the SEs subcultured every 10 days ( Figure 6 in b) and every 14 days ( Figure 6 in c) had a darker yellow color; after Agrobacterium infection, the average survival rate of SEs subcultured every 7 days was 72.22%, while the average survival rates of those subcultured every 10 days and 14 days were only 53.71% and 14.18% respectively (Table 3). The concentration of Agrobacterium had a greater impact on the survival rate of SEs. When OD 600 was 0.6 and 0.8, the average survival rates of SEs were 50.37% and 53.34% respectively, while when OD 600When it was 0.4, the average survival rate of SEs was only 37.04% (Table 3). The infection time in the transformation system had the least effect on the survival rate of SEs. When SEs were infected for 10, 15, and 20 minutes, the average survival rates were 46.30%, 51.85%, and 42.59% respectively (Table 3). The results of variance analysis (Table 4) showed that the pre-culture of SEs had a significant effect on the survival rate of genetic transformation (P = 0.008). However, the concentration of Agrobacterium and the infection time in the transformation system had no significant effect on the survival rate of SEs (P = 0.084 and 0.241).

[0106] Table 3. Interval analysis of genetic transformation of walnut somatic embryos

[0107]

[0108]

[0109] Note: and respectively represent the average survival rates of each factor at different levels.

[0110] Table 4. Variance analysis of genetic transformation of walnut somatic embryos

[0111] Source of Variance Sum of Squares Degree of Freedom Mean Square F Value P Value A 5151.466 2 2575.733 124.460 0.008 B 130.330 2 65.169 3.149 0.241 C 452.269 2 226.134 10.927 0.084 Error 41.391 2 21.668 Total 5734.065

[0112] 4-2. Genetic transformation of YFP gene in walnut SEs

[0113] SEs were randomly selected to extract DNA for PCR verification. Specific YFP bands were generated in both the positive control and transgenic lines, while no band appeared in the negative control ( Figure 7 in a), so we speculated that YFP had been transferred into SEs. After statistics, the positive rates of SEs in E1-E3 generations were 20%, 40%, and 60% respectively ( Figure 7 in e). To further confirm the YFP expression level in positive SEs, detection was carried out by qRT-PCR and semi-quantitative RT-PCR, and it was found that the expression of YFP gene was detected in transgenic SEs, while the expression of YFP gene was not detected in wild type ( Figure 7 in b, c). At the same time, longitudinal sections of SEs in E3 generation were made and detected by fluorescence microscope, and strong yellow fluorescence signals were found in large areas in SEs ( Figure 7 in d), indicating that the YFP gene had been successfully integrated into the genome of SEs using this genetic transformation system.

[0114] 4-3. Genetic transformation of GUS gene in walnut SEs

[0115] SEs were randomly selected to extract DNA for PCR verification. Specific GUS bands were produced in both the positive control and transgenic lines, while no band appeared in the negative control ( Figure 8 as shown in a)). Therefore, we speculated that GUS had been transferred into SEs. Statistically, the positive rates of SEs in generations E1 - E3 were 50%, 80% and 100% respectively ( Figure 8 as shown in c)). In addition, SEs were rinsed and stained with a GUS staining kit. The blue SEs were GUS - positive, and the blue signals of SEs in generations E1 and E2 were stronger, indicating that the vector containing the GUS reporter gene had been successfully transformed into SEs ( Figure 8 as shown in b)).

[0116] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including changes made with conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Application of walnut (Juglans regia) YX4 as a receptor material in Agrobacterium-mediated genetic transformation, wherein the deposit number of the walnut (Juglans regia) YX4 in the General Microbiological Center of the China Microbiological Culture Collection Administration is CGMCC No.46084.

2. The method for genetic transformation of walnut mediated by Agrobacterium tumefaciens is characterized in that: The method comprises genetic transformation using the walnut (Juglans regia) YX4 described in claim 1 as a recipient material.

3. The method according to claim 2, characterized in that The method comprises the following steps: A1) pre-culturing the somatic embryos of walnut (Juglans regia) YX4 according to claim 1 to obtain pre-cultured somatic embryos; A2) infecting the pre-cultured somatic embryos with a recombinant Agrobacterium solution containing the target gene; A3) inducing and culturing the positive transgenic somatic embryos obtained through identification to obtain regenerated plants; The pre-culture conditions in step A1) include: pre-culturing the somatic embryos in a culture medium and subculturing once every 6-8 days.

4. The method according to claim 3, characterized in that The pre-culture conditions in step A1) include: pre-culturing the somatic embryos in a culture medium, subculturing once every 7 days, and culturing for 28-32 days.

5. The method according to claim 3 or 4, characterized in that: The OD of the recombinant Agrobacterium solution in step A2) 600 The value is 0.6-0.

8.

6. The method according to any one of claims 3 to 5, characterized in that: The diameter of the somatic embryos in step A1) is 3 mm to 9 mm.

7. The method according to claim 6, characterized in that The diameter of the somatic embryos is 6 mm.

8. The method according to any one of claims 3 to 7, characterized in that: Prior to step A1), the method further comprises the step of inducing the regeneration of the immature embryos of walnut (Juglans regia) YX4 of claim 1 with a diameter of 3 mm to 9 mm into walnut somatic embryos using an induction medium.

9. The method according to any one of claims 3 to 8, characterized in that: The infection time in step A2) is 5-30 minutes.

10. The method according to any one of claims 3 to 9, characterized in that: The pre-culture medium in step A1) is a DKW medium containing 30 g / L sucrose and 3.0 g / L phytagel.

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