Method for improving apple leaf disc regeneration and genetic transformation efficiency

Abstract

The invention belongs to the technical field of genetic transformation, and particularly relates to a method for improving apple leaf disc regeneration and genetic transformation efficiency. And culturing the apple leaf disc in the apple leaf disc regeneration culture medium to obtain a leaf disc regeneration adventitious bud. The amino acid sequence of the MdREF1 small peptide is as shown in SEQ ID NO. 1. The apple homologous peptide MdREF1 is identified in an NCBI (National Center of Biotechnology Information) database on the basis of AtPROPEP1-8 and AtPep1-8 sequences of arabidopsis thaliana. The synthetic peptide of the apple MdREF1 is added into a basic regeneration culture medium, so that the leaf disc callus growth amount and the adventitious bud differentiation number can be remarkably increased. Wherein by adding 50nmol / L of MdREF1 peptide, the regeneration frequency can be improved by 1.74 times, the average regeneration bud number can be increased by 2.16 times, and the conversion efficiency can be improved by 3.14 times.

Description

Technical Field

[0001] This invention belongs to the field of genetic transformation technology, specifically relating to a method for improving the regeneration and genetic transformation efficiency of apple leaf discs. Background Technology

[0002] Apples are perennial woody plants with highly heterozygous varieties and complex genetic bases, requiring long breeding cycles for conventional methods. However, advancements in plant molecular biology have opened a new avenue for the genetic improvement of apple varieties and rootstocks. This allows for the modification and refinement of individual traits without altering the superior traits of the parent plants, enabling the development of new varieties that meet expectations in a short period. Agrobacterium-mediated transformation utilizes the natural transformation mechanism of Agrobacterium to integrate target genes into the crop genome. Due to its high efficiency and stability, it has become one of the most commonly used genetic engineering methods. However, limited by low genetic transformation efficiency, research on the formation mechanisms of apple functional genomics and important agronomic traits lags far behind that of major food crops and other fruits.

[0003] As research into developmental regulators deepens, scientists have discovered that these regulators play crucial roles at various stages of plant growth and development, and demonstrate significant potential in improving the efficiency of plant genetic transformation. In 2024, Chinese scientists identified for the first time the primary wound signaling molecule that induces plant regeneration—the regeneration factor REF1 (REGENERATIONFACTOR1). REF1 is a small peptide that participates in the plant regeneration process by regulating the local wound response and regeneration capacity. Studies have shown that when plant cells are damaged, REF1, as the initial wound signaling molecule, is recognized by the leucine-rich receptor kinases PEPR1 / 2, thereby activating the expression of the downstream key regulator WIND1 and initiating tissue repair and organ regeneration processes. As a local wound signal, REF1 promotes the regeneration capacity of plants and has been successfully applied in various difficult-to-transform crops such as wild tomatoes, wheat, and soybeans, demonstrating a conserved regeneration model.

[0004] Rosaceae fruit trees have low conversion efficiency, compared to pears ( Pyrus communis ),Peach( Prunus persica ),cherry( Prunus avium ) and Li ( Prunus domestica Compared to other fruit trees in the Rosaceae family, such as apples, Malusdomestica Apple was one of the first tree species to be successfully genetically transformed, but the transformation efficiency remains relatively low. Currently, the conventional method for establishing an apple regeneration system involves using detached leaves from sterile apple seedlings as explants, inoculating them onto MS basal medium supplemented with cytokinins (such as TDZ or 6-BA) and auxins (such as NAA or IBA), and then inducing adventitious bud differentiation through a period of dark culture. However, this traditional method suffers from low genetic transformation efficiency. Summary of the Invention

[0005] To address the low genetic transformation efficiency of conventional methods in existing apple regeneration systems, this invention comprehensively identifies, synthesizes, and verifies the role of the apple regeneration-promoting MdREF1 peptide in improving apple leaf-disc regeneration and transformation efficiency. The results show that the MdREF1 peptide effectively enhances apple leaf-disc regeneration and genetic transformation efficiency, providing a method for improving these processes. To achieve the above objectives, this invention employs the following technical solution.

[0006] This invention provides a method for improving the regeneration and genetic transformation efficiency of apple leaf discs, comprising the following steps: Apple leaf discs were cultured in apple leaf disc regeneration medium to obtain adventitious buds regenerated from the leaf discs.

[0007] Each 11L of apple leaf disc regeneration medium is prepared from the following final concentration of material: 4 g / L~5 g / L MS medium, 6 g / L~10 g / L agar, 28 g / L~32 g / L sucrose, 1 mg / L~3 mg / L cytokinin TDZ, 0.1 mg / L~2 mg / L auxin NAA and 10 nmol / L~100 nmol / L MdREF1 peptide, with the balance being water.

[0008] The amino acid sequence of the MdREF1 peptide is shown in SEQ ID NO.1: IARARPKNHNKPPLSSGKGGQIN.

[0009] The method provided by this invention includes the steps of inoculating apple leaf discs onto an apple leaf disc regeneration medium and then culturing the apple leaf discs. Each 1L of apple leaf disc regeneration medium is prepared from the following materials at the following final concentrations: 4g / L~5g / L MS medium, 6g / L~10g / L agar, 28g / L~32g / L sucrose, 1mg / L~3mg / L cytokinin TDZ, 0.1mg / L~2mg / L auxin NAA, and 10nmol / L~100nmol / L MdREF1 peptide, with the remainder being water. The core innovation lies in the introduction of the MdREF1 peptide, which acts as a highly efficient regeneration signaling molecule, synergistically promoting cell division and differentiation potential in the apple leaf disc regeneration medium, in order to actively promote cell division and differentiation potential in the leaf discs. Simultaneously, by using a concentration gradient screening method to determine the optimal concentration of the peptide, the stable and efficient expression of this core active substance is ensured. This invention directly activates the totipotency expression of apple leaf disc cells at the molecular regulatory level by exogenously adding the MdREF1 peptide, fundamentally overcoming the variety-dependent problem caused by differences in the expression levels of endogenous regeneration signaling substances among different genotypes. Specifically, the MdREF1 peptide, as a key regeneration-promoting signaling molecule, can stably act on leaf disc tissues of different varieties within a concentration range of 10 nmol / L to 100 nmol / L, bypassing the concentration adaptation barrier caused by variety-specific receptor differences and varying signal transduction efficiencies in traditional hormone regulation pathways, significantly improving the universality of culture medium formulations. Simultaneously, the MdREF1 peptide specifically promotes callus formation and efficient differentiation of bud primordia, directly increasing the frequency and quality of adventitious bud regeneration in leaf discs, shortening the regeneration cycle, and thus proportionally increasing the number of selectable transformable cell clusters. Ultimately, this achieves an overall improvement in genetic transformation efficiency, thereby addressing the low genetic transformation efficiency of conventional methods in existing apple regeneration systems.

[0010] Furthermore, the cultivation conditions are as follows: Temperature 23℃~27℃, light cycle 14h~18h, light / dark cycle 6h~10h, dark culture for 20~25 days, then culture under light for 15~30 days.

[0011] Furthermore, when preparing the apple leaf disc regeneration medium, MS medium, agar, sucrose, cytokinin TDZ and auxin NAA are first prepared into a basic regeneration medium. Then, MdREF1 small peptide is dissolved in sterile water, filtered to remove bacteria, and added to the basic regeneration medium.

[0012] Furthermore, sterilization through filtration refers to filtration using a microporous membrane.

[0013] Furthermore, the pore size of the microporous filter membrane is 0.2μm~0.45μm.

[0014] Furthermore, the MdREF1 peptide is added after the basic regeneration medium has been sterilized and the temperature has been lowered to 40°C~60°C.

[0015] Furthermore, the method also includes converting the target gene into apple leaf discs before cultivation.

[0016] Furthermore, the apple leaf disc is taken from the leaves of tissue-cultured seedlings of apple clone 'GL-3'.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for improving the regeneration and genetic transformation efficiency of apple leaf discs. The method includes inoculating apple leaf discs onto an apple leaf disc regeneration medium, followed by culturing the discs. Each 1L of apple leaf disc regeneration medium is prepared from the following materials at the following final concentrations: 4g / L~5g / L MS medium, 6g / L~10g / L agar, 28g / L~32g / L sucrose, 1mg / L~3mg / L cytokinin TDZ, 0.1mg / L~2mg / L auxin NAA, and 10nmol / L~100nmol / L MdREF1 peptide, with the remainder being water. The core innovation lies in the introduction of the MdREF1 peptide, which acts as a highly efficient regeneration signaling molecule, synergistically promoting cell division and differentiation potential in the leaf disc regeneration medium, in turn, actively enhancing the cell division and differentiation potential of the leaf disc. Furthermore, by using a concentration gradient screening method to determine the optimal concentration of the peptide, the stable and efficient expression of this core active substance is ensured. This invention directly activates the totipotency expression of apple leaf disc cells at the molecular regulatory level by exogenously adding the MdREF1 peptide, fundamentally overcoming the variety-dependent problem caused by differences in the expression levels of endogenous regeneration signaling substances among different genotypes. Specifically, the MdREF1 peptide, as a key regeneration-promoting signaling molecule, can stably act on leaf disc tissues of different varieties within a concentration range of 10 nmol / L to 100 nmol / L, bypassing the concentration adaptation barrier caused by variety-specific receptor differences and varying signal transduction efficiencies in traditional hormone regulation pathways, significantly improving the universality of culture medium formulations. Simultaneously, the MdREF1 peptide specifically promotes callus formation and efficient differentiation of bud primordia, directly increasing the frequency and quality of adventitious bud regeneration in leaf discs, shortening the regeneration cycle, and thus proportionally increasing the number of selectable transformable cell clusters. Ultimately, this achieves an overall improvement in genetic transformation efficiency, thereby addressing the low genetic transformation efficiency of conventional methods in existing apple regeneration systems.

[0018] The method provided by this invention also has the potential to broaden the scope of its application. The MdREF1 peptide originates from a conserved damage response signaling pathway in plants, and this signaling mechanism has high homology in Rosaceae plants. Therefore, the core scheme established in this invention—"adding specific regeneration peptides to enhance in vitro regeneration efficiency"—is not limited to apples, but also provides a novel and referable technical path for optimizing the genetic transformation systems of other important Rosaceae crops (such as pears, peaches, strawberries, and roses), possessing significant methodological extension value. Attached Figure Description

[0019] Figure 1 This is the phylogenetic tree of apples and Arabidopsis thaliana PROPEPs in this invention.

[0020] Figure 2 This invention relates to multiple sequence alignment and Web-Logo analysis of apple and Arabidopsis thaliana Peps; wherein: A is a multiple sequence alignment of apple and Arabidopsis thaliana Peps; B represents the analysis of the web-logo of Apple and Arabidopsis thaliana Peps.

[0021] Figure 3 In this invention, the error bars representing the relative expression levels of apple MdPROPEP1 and MdPROPEP2 at different time points indicate the standard deviation of three biological replicates. Different letters represent the different levels of expression at different time points. P <0.05).

[0022] Figure 4 To illustrate the promoting effect of MdREF1 peptide on the leaf disc regeneration capacity of apple 'GL-3' in this invention, 10 nmol / L, 20 nmol / L, 50 nmol / L, 100 nmol / L, 150 nmol / L, and 200 nmol / L represent different concentrations of MdREF1 peptide (regeneration peptide MdREF1). A represents CK, and 0 contains MdREF1 peptide. B is a small MdREF1 peptide with a final concentration of 10 nmol / L; C represents the MdREF1 peptide with a final concentration of 20 nmol / L; D is a small MdREF1 peptide with a final concentration of 50 nmol / L; E represents a MdREF1 peptide with a final concentration of 100 nmol / L; F represents the MdREF1 peptide with a final concentration of 150 nmol / L; G is a small MdREF1 peptide with a final concentration of 200 nmol / L; The multiple samples in each of the figures are parallel samples.

[0023] Figure 5 This invention demonstrates the promoting effect of the MdREF1 peptide on the genetic transformation efficiency of apple 'GL-3' leaf discs; wherein: A shows the regeneration of the exogenously applied MdREF1 peptide during genetic transformation, displaying a representative image of the leaf disc; multiple samples are parallel samples. B represents the regeneration efficiency of exogenously applied MdREF1 peptide (error bars indicate the standard deviation of three biological replicates; different letters indicate the regeneration efficiency at different stages). P <0.05); C represents the conversion efficiency of exogenously applied MdREF1 peptide (error bars indicate the standard deviation of three biological replicates; different letters indicate the conversion efficiency at different stages). P <0.05); D represents the PCR detection results: lane 5 represents DNA molecular weight Marker 2000, lanes 1-4 and 6-9 represent transformed plants, and lane 10 represents the negative control. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0025] This invention discloses a method for identifying, verifying the expression level, synthesizing, adding and applying the apple regeneration-promoting small peptide MdREF1 (MdREF1 small peptide), and analyzing the results.

[0026] 1) The steps for finding the apple regeneration-promoting peptide MdREF1 in the above method are as follows: Apples in the NCBI database, using Arabidopsis thaliana AtPROPEP1-8 and Arabidopsis thaliana AtPep1-8 as the source sequences respectively. Malus domestica The BLASTP function was used in the protein library to search for homologous Pep sequences in apples. The found apple Pep sequences possess a stable SSG-x2-G-x2-N motif.

[0027] The protein sequence numbers of Arabidopsis thaliana AtPROPEP1-8 are AtPROPEP1 (AT5G64900), AtPROPEP2 (AT5G64890), AtPROPEP3 (AT5G64905), AtPROPEP4 (AT5G09980), AtPROPEP5 (AT5G09990), AtPROPEP6 (AT2G22000), AtPROPEP7 (AT5G09978), and AtPROPEP8 (AT5G09976).

[0028] The protein sequences of Arabidopsis thaliana AtPep1-8 correspond to the conserved 23rd amino acid sequence at the end of AtPROPEP1-8.

[0029] The apple Pep sequence, also known as the apple MdREF1 protein sequence or apple regeneration-promoting small peptide MdREF1, is the 23rd amino acid sequence at the end of XP028949464.

[0030] 2) The apple variety used in this invention is 'GL-3', which is disclosed in the literature: "Hongyan Dai, Wenran Li, Guofen Han, Yi Yang, Yue Ma, He Li, Zhihong Zhang, Development of a seedling clone with high regeneration capacity and susceptibility to Agrobacterium in apple, Scientia Horticulturae, Volume 164, 2013, Pages 202-208, ISSN 0304-4238, https: / / doi.org / 10.1016 / j.scienta.2013.09.033".

[0031] 3) The steps for verifying the expression level of the apple regeneration-promoting peptide MdREF1 in the above method are as follows: Select tender leaves from the top of 35-day-old 'GL-3' tissue culture seedlings. Make three transverse cuts perpendicular to the leaf veins, and inoculate the leaves with the underside facing upwards onto the basal regeneration medium. Samples were taken from the wound sites on the regenerated leaf discs every two days. Sampling continued until day 21, with day 1 serving as a control. RNA was extracted from samples at different wound stages of 'GL-3' apple leaves to synthesize cDNA for quantitative real-time PCR.

[0032] The method for obtaining 'GL-3' tissue culture seedlings with a seedling age of 35 days is as follows: Select healthy, uncontaminated, sterile tissue culture seedlings, cut off the top stem tip about 1.8 cm long under sterile conditions as explants, inoculate them into fresh subculture proliferation medium, and culture them for about 35 days under standard tissue culture conditions (temperature 25℃±2℃, photoperiod 16h light / 8h dark).

[0033] The basic regeneration medium is made from materials at the following final concentrations: 4.43 g / L MS medium, 8 g / L agar, 30 g / L sucrose, 2 mg / L cytokinin TDZ and 0.5 mg / L auxin NAA, with the remainder being water.

[0034] Subculture proliferation medium is prepared from materials at the following final concentrations: 4.43 g / L MS medium, 8 g / L agar, 30 g / L sucrose, 0.3 mg / L cytokinin 6-BA and 0.2 mg / L auxin IAA, with the remainder being water.

[0035] The steps for performing quantitative real-time PCR are as follows: After a period of propagation and culture, the resistant shoots obtained by transformation were used to establish a clone. Using leaves of sterile test-tube seedlings as material, DNA was extracted from transformed and non-transformed plants using the CTAB method. Transformed plants were detected by PCR. Specific primers were designed based on the target gene on the vector, and a negative control was set up. The results were determined by agarose gel electrophoresis.

[0036] 4) The (conserved) amino acid sequence MdREF1 with regeneration-promoting effects identified in the above method was synthesized by Shanghai ChuTai Biotechnology Co., Ltd. The conserved amino acid sequence MdREF1 with regeneration-promoting effects is also known as apple regeneration-promoting peptide MdREF1, regeneration-promoting peptide MdREF1, or MdREF1 peptide.

[0037] The (conserved) amino acid sequence of the apple regeneration-promoting peptide MdREF1 is shown in SEQ ID NO.1: IARARPKNHNKPPLSSGKGGQIN.

[0038] 5) The synthesized regeneration-promoting peptide MdREF1 is in powder form and needs to be dissolved before use.

[0039] Example 1: 1. Obtaining the peptide solution: The preparation of the peptide solution was carried out under aseptic conditions in a laminar flow hood. The specific steps are as follows:

[0040] MdREF1 small peptide powder (synthesized by Shanghai Chupeptide Biotechnology Co., Ltd.) was dissolved in sterile water and shaken to mix thoroughly. The resulting solution was then filtered through a microporous membrane with a pore size of 0.45 μm to remove bacteria, thus obtaining a sterile small peptide stock solution, referred to as peptide solution.

[0041] 2. Application of peptide solutions in promoting leaf disc regeneration: Preparation of (apple) leaf disc regeneration medium: Add MS basal salt to a final concentration of 4.43 g / L, MS sucrose to a final concentration of 30 g / L, cytokinin TDZ to a final concentration of 2 mg / L, auxin NAA to a final concentration of 0.5 mg / L, and agar to a final concentration of 8 g / L to obtain the basal regeneration medium. Sterilize the basal regeneration medium at 121℃ for 21 minutes. When the temperature drops to 50℃, add pre-dissolved peptide solution to achieve a final concentration of 50 nM / L. Then, dispense the solution into disposable plastic petri dishes to obtain the regeneration culture plate containing the regenerating peptide MdREF1. The medium containing the regenerating peptide MdREF1 is the apple leaf disc regeneration medium or (apple) leaf disc regeneration medium.

[0042] Select the top tender leaves of 35-day-old 'GL-3' apple tissue culture seedlings, make three horizontal cuts perpendicular to the leaf veins without cutting all the way through, and then inoculate the leaves with the underside facing up onto the apple leaf disc regeneration medium. The 35-day-old 'GL-3' apple tissue culture seedlings are those that are 35 days old.

[0043] The above methods can increase the formation of callus and the number of adventitious buds.

[0044] 3. Application of peptide solutions in promoting leaf disc genetic transformation: The selection medium for apple leaf disc genetic transformation was prepared as follows: MS basal salts (final concentration 4.43 g / L), MS sucrose (final concentration 30 g / L), cytokinin TDZ (final concentration 2 mg / L), auxin NAA (final concentration 0.5 mg / L), Cef (final concentration 0.5 mg / L), Kna (final concentration 0.5 mg / L), and agar (final concentration 8 g / L) were added. The mixture was sterilized at 121°C for 21 minutes. After cooling to 50°C, a pre-dissolved peptide solution was added to achieve a final concentration of 50 nM / L. The solution was then aliquoted into disposable plastic petri dishes to obtain the selection medium containing the regenerating peptide MdREF1. This selection medium containing the regenerating peptide MdREF1 is either the selection medium supplemented with the regenerating peptide MdREF1 (100 nmol / L) or the selection medium used in apple leaf disc genetic transformation.

[0045] Select tender leaves from the top of 'GL-3' apple tissue culture seedlings that have grown for 35 days. Make three horizontal cuts perpendicular to the leaf veins but do not cut all the way through. Infect the leaves with bacterial solution for 7 minutes, transfer them to sterile filter paper to remove excess bacterial solution, and then inoculate them into co-culture medium and extension medium for 2 days in sequence. Finally, transfer them to selection medium supplemented with regenerative peptide MdREF1 (100 nmol / L) for further culture.

[0046] The bacterial culture refers to Agrobacterium K599 bacterial culture, which is obtained by shaking Agrobacterium K599 containing 2300 vectors until OD. 600The concentration was 0.7. After high-speed centrifugation to collect the bacterial cells, a resuspension, i.e., bacterial solution, was obtained. Agrobacterium K599 containing the 2300 vector refers to the Agrobacterium K599 engineered strain carrying the pCAMBIA2300 binary expression vector for genetic transformation, which was purchased from Beijing Coollab Technology Co., Ltd.

[0047] The co-culture medium was prepared from the following final concentrations: 4.43 g / L MS medium, 8 g / L agar, 30 g / L sucrose, 2 mg / L cytokinin TDZ, 0.5 mg / L auxin NAA, 1 mg / L acetylsuccione AS and 1 mg / L betaine Bet, with the remainder being water.

[0048] The extension medium was prepared from the following final concentrations of materials: 4.43 g / L MS medium, 8 g / L agar, 30 g / L sucrose, 2 mg / L cytokinin TDZ, 0.5 mg / L auxin NAA and 1 mg / L cephalosporin Cef, with the remainder being water.

[0049] The above methods can increase the formation of callus, the number of adventitious buds, and the number of resistant buds.

[0050] In all aspects described in this invention, the leaf discs must first be cultured in the dark for 21 days, followed by light cultivation; the light cultivation lasts for 16 hours, followed by 8 hours in the dark, with a light intensity of 2000 lx and a temperature of 25℃ ± 2℃. The method provided by this invention can significantly improve the regeneration efficiency and genetic transformation efficiency of apples.

[0051] Table 1 shows the apple homologous protein sequences obtained using the BLASTP function in the GDR (https: / / www.rosaceae.org / ) and NCBI database (https: / / www.ncbi.nlm.nih.gov / ) based on Arabidopsis PEPs and PROPEPs as query source sequences. The phylogenetic trees of apple and Arabidopsis PROPEPs are shown below. Figure 1 .

[0052] Table 1. Apple PEP protein sequence information obtained from identification. Depend on Figure 1Phylogenetic analysis of apple and Arabidopsis PROPEPs revealed that the two apple PROPEP protein sequences exhibited high homology and differed from AtPROPEP. Specifically, MdPROPEP1 and MdPROPEP2 clustered with AtPROPEP5-6, indicating strong homology and potential similar functions. This aligns with the evolutionary relationship between tomato SlPROPEP6 (Solyc04g072310), which has been validated for its regeneration-promoting effects, and Arabidopsis. Multiple sequence alignment and Web-Logo analysis of the conserved C-terminal PEPs of eight Arabidopsis and two apple PROPEP protein sequences showed that all sequences contained the SxG key motif sequence. MdPep1 had a complete 23-amino acid C-terminus, consistent with AtPeps, while MdPep2 had 21 amino acids, lacking two amino acids (e.g., ...). Figure 2 As shown in the figure, it may have some impact on the overall activity.

[0053] This invention analyzed the expression characteristics of MdPROPEP1 and MdPROPEP2 genes at different time points during the callus growth process of apple 'GL-3' tissue culture seedlings at different stages using real-time quantitative PCR. The specific steps are as follows: RNA was extracted from 'GL-3' apple leaves at different injury stages using the CTAB method. 1 μg of RNA was reverse transcribed into cDNA using the PrimeScript RT kit and the gDNA Eraser kit (TaKaRa). Primers were designed and their specificity determined using SnapGene. Real-time quantitative PCR (RT-qPCR) was performed using a CFX96 Touch instrument. The amplification program included: 95°C for 30 seconds; 40 cycles (95°C for 10 seconds, 60°C for 30 seconds); melting curve analysis was performed using 2... -△△CT The method calculates the relative expression level.

[0054] MdActin (MDP0000752428) was used as an internal reference gene.

[0055] The designed primer sequences are as follows: The nucleotide sequence of MdPROPEP1F is shown in SEQ ID NO.2: 5'-TCAGGGAGAACAAGCACGAC-3'.

[0056] The nucleotide sequence of MdPROPEP1R is shown in SEQ ID NO.3: 5'-GACCACCTTTACCGGAGCTT-3'.

[0057] The nucleotide sequence of MdPROPEP2 F is shown in SEQ ID NO.4: 5'-TCAGGGAGAACAAGCACGAC-3'.

[0058] The nucleotide sequence of MdPROPEP2 R is shown in SEQ ID NO.5: 5'-TCAGCAGTATTGTTTTCCGGA-3'.

[0059] The primer sequences for the internal reference gene are as follows: The nucleotide sequence of MdActin F is shown in SEQ ID NO.6: 5'-TCCGGAGCTTAAAGGTGGTT-3'.

[0060] The nucleotide sequence of MdActin R is shown in SEQ ID NO.7: 5'-TGCTCACTATGCCGTGCTCA-3'.

[0061] The results are as follows Figure 3 As shown, real-time quantitative PCR technology was used to analyze the healing process of cut surfaces on apple 'GL-3' tissue culture seedlings at different stages. MdPROPEP1 and MdPROPEP2 The expression dynamics of the genes were analyzed. The results showed that there were significant differences in the expression patterns of the two genes. MdPROPEP1 The expression of [specific marker] showed a gradually increasing trend, reaching its peak on day 21. In comparison, MdPROPEP2 The expression level only showed a slight upregulation from day 11 to day 16, and the overall expression level was significantly lower than that during the entire process. MdPROPEP1 Based on the analysis of callus development stages, day 16 marks the active stage of wound healing, day 19 is the critical period for callus formation, and day 21 is the tissue remodeling stage. MdPROPEP1 The expression level was significantly higher than MdPROPEP2 From the above, we can see that... MdPROPEP1 It plays a leading role in signal transduction during the callus proliferation phase.

[0062] The effects of different concentrations of MdREF1 peptide on the regeneration ability of apple 'GL-3' leaves are shown in Table 2: Table 2. Effects of different concentrations of MdREF1 peptide on the regeneration capacity of 'GL-3' leaves. Except for the control group (CK), the regeneration frequency of all treatment groups was 100%, indicating that the addition of MdREF1 peptide did not affect the leaf regeneration frequency but significantly altered the number of regenerated buds. Different concentrations of MdREF1 peptide had different effects on the regeneration ability of Gala leaves, exhibiting a concentration-dependent effect: 50 nmol / L was the optimal concentration, at which the average number of regenerated buds reached a peak of 13.27, 2.09 times that of CK, effectively doubling the average number of regenerated buds, while maintaining a stable regeneration frequency of 100%. The effect gradually decreased after the concentration exceeded 50 nmol / L, and even at 200 nmol / L, the effect dropped to 9.55, still higher than CK. Specific results are as follows... Figure 4 As shown: 50 nmol / L is the optimal concentration, at which the average number of regenerated buds reaches a peak of 13.27, doubling the average number of regenerated buds, and the regeneration frequency remains stable at 100%.

[0063] The effect of adding MdREF1 peptide on the genetic transformation and regeneration capacity of apple 'GL-3' is as follows: Figure 5 As shown: Compared with culture medium without MdREF1 peptide, culture medium with added MdREF1 peptide significantly improved the regeneration frequency and average number of regenerated shoots during the apple transformation process. Statistical data showed that applying 50 nmol / L MdREF1 increased the average regeneration frequency of 'GL-3' from 42.34% to 73.67%, an increase of approximately 1.74 times; the average number of regenerated shoots increased from 1.22 to 2.64, an increase of approximately 2.16 times; and the transformation efficiency increased from 25.00% to 78.50%, an increase of 3.14 times.

[0064] As shown above, the apple homologous regeneration factor MdREF1 (MdREF1 peptide) can improve the regeneration and transformation efficiency of apple leaf discs. Synthesizing and dissolving apple MdREF1 peptides, and then applying them externally to the apple genetic transformation selection medium, with the addition of 50 nmol / L of the regeneration peptide MdREF1, significantly increased the genetic transformation efficiency of apple 'GL-3' by more than three times. Furthermore, this effect is conserved and applicable to other apple varieties. Therefore, REF1 holds promise as an effective strategy for overcoming the challenges of genetic transformation in the Rosaceae family.

[0065] It should be noted that when numerical ranges are involved in 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, this invention describes preferred embodiments.

[0066] 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, all of which fall within the scope of the invention.

Claims

1. A method for improving the efficiency of apple leaf disc regeneration and genetic transformation, characterized in that, Includes the following steps: Apple leaf discs were cultured in apple leaf disc regeneration medium to obtain adventitious buds regenerated from the leaf discs; Each 1L of apple leaf disc regeneration medium is prepared from the following final concentration of material: 4 g / L~5 g / L MS medium, 6 g / L~10 g / L agar, 28 g / L~32 g / L sucrose, 1 mg / L~3 mg / L cytokinin TDZ, 0.1 mg / L~2 mg / L auxin NAA and 10 nmol / L~100 nmol / L MdREF1 peptide, balance water; The amino acid sequence of the MdREF1 peptide is shown in SEQ ID NO.

1.

2. The method of claim 1, wherein, The cultivation conditions are as follows: Temperature 23℃~27℃, light cycle 14h~18h, light / dark cycle 6h~10h, dark culture for 20~25 days, then culture under light for 15~30 days.

3. The method of claim 1, wherein, When preparing the apple leaf disc regeneration medium, MS medium, agar, sucrose, cytokinin TDZ and auxin NAA are first prepared into a basic regeneration medium. Then, MdREF1 peptide is dissolved in sterile water, filtered to remove bacteria, and added to the basic regeneration medium.

4. The method of claim 3, wherein, The process of removing bacteria through filtration refers to filtration using a microporous membrane.

5. The method of claim 4, wherein, The microporous filter membrane has a pore size of 0.2μm to 0.45μm.

6. The method of claim 3, wherein, The MdREF1 peptide was added after the basic regeneration medium was sterilized and the temperature was lowered to 40℃~60℃.

7. The method of claim 1, wherein, The method also includes converting the target gene into apple leaf discs before culturing.

8. The method of claim 7, wherein, The apple leaf discs were taken from the leaves of tissue-cultured seedlings of the apple clone 'GL-3'.

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