WO2024088372A1 - 一种利用发根农杆菌高效快速获得苹果稳定转基因植株的方法 - Google Patents

一种利用发根农杆菌高效快速获得苹果稳定转基因植株的方法 Download PDF

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WO2024088372A1
WO2024088372A1 PCT/CN2023/127030 CN2023127030W WO2024088372A1 WO 2024088372 A1 WO2024088372 A1 WO 2024088372A1 CN 2023127030 W CN2023127030 W CN 2023127030W WO 2024088372 A1 WO2024088372 A1 WO 2024088372A1
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leaves
culture
apple
transgenic
agrobacterium rhizogenes
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PCT/CN2023/127030
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English (en)
French (fr)
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李鹏民
王玉珠
王娱乐
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西北农林科技大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/74—Rosaceae, e.g. strawberry, apple, almonds, pear, rose, blackberries or raspberries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/40—Afforestation or reforestation

Definitions

  • the present application belongs to the technical field of plant genetic engineering, and specifically relates to a method for efficiently and quickly obtaining stable transgenic apple plants by using leaves of apple plants as receptors and Agrobacterium rhizogenes.
  • Agrobacterium tumefaciens There are two main types of Agrobacterium: Agrobacterium tumefaciens and Agrobacterium rhizogenes.
  • Agrobacterium plasmid is a natural system that can achieve DNA transfer and integration. The most commonly used plasmids in plasmid vector systems are: Ti plasmid and Ri plasmid. Ti plasmid exists in Agrobacterium tumefaciens, and Ri plasmid exists in Agrobacterium rhizogenis. Ti plasmid and Ri plasmid have many similarities in structure and function and have basically the same characteristics. The Ti plasmid is approximately between 160 and 240 kB.
  • the Ti plasmid is integrated into the plant receptor genome through the T-DNA region and can be expressed in plant cells, leading to the occurrence of crown galls. It can also be passed to offspring through meiosis.
  • the T-DNA is between 12 and 24 kb in length, with a border sequence containing a 25 bp repeating sequence at each end.
  • the T-DNA between the left and right border sequences can be transferred and integrated into the host cell genome.
  • Agrobacterium rhizogenes infection can induce the production of hairy roots (Hair Root), which is caused by the Ri (Root Inducing) plasmid contained in Agrobacterium rhizogenes.
  • the Ri plasmid is a root inducing plasmid (Root Inducing Plasmid); the size of the Ri plasmid is 200-800 kb. It contains genes responsible for autonomous root growth and opine synthesis. In terms of structure, it has a toxic region (Vir region), a T-DNA region that is transferred into the plant cell nucleus, and an opine synthesis functional region inside it.
  • the Ri plasmid is transferred to the infected plant through T-DNA, causing it to generate hairy roots.
  • the T-DNA on the Ri plasmid is discontinuously distributed, which is related to the formation of hairy roots with TL-DNA, TR-DNA and genes on TL-DNA, and also affects the morphology of the aboveground part of the regenerated plant and some physiological traits (Wu E et al. 2018).
  • the main method is to use Agrobacterium tumefaciens containing the target gene for mediation, and finally obtain transgenic regenerated plants.
  • Agrobacterium tumefaciens containing the target gene for mediation
  • the leaf disc method is currently mainly used, using Agrobacterium tumefaciens to infect the leaves of apple explants and co-cultivate for a short period of time, and then inducing apple transgenic strains under different conditions in the later stage (Fang Xinwu et al. 2014).
  • transgenic hairy roots that have been obtained to further obtain transgenic strains that can be stably inherited
  • the hairy roots are first dedifferentiated and induced to form callus tissue, and then redifferentiated and induced to obtain transgenic regenerated plants (Li Xiangyun 2010; Wang Yan 2009; Zhang Chengcheng 2011).
  • the conditions for in vitro tissue culture of hairy roots must be further explored, and the steps are cumbersome and the time period is long. Therefore, it is particularly important to develop a method for efficiently and quickly obtaining transgenic plants that can be stably inherited using Agrobacterium rhizogenes.
  • the application overcomes the traditional prejudice against Agrobacterium rhizogenes and pioneered the use of apple leaves as receptors.
  • Agrobacterium rhizogenes as a medium, stably inherited transgenic adventitious buds are obtained through adventitious bud induction culture. This is the first time that a stably inherited transgenic apple strain is obtained efficiently and quickly using the "Agrobacterium rhizogenes + leaf infection + induction culture of adventitious buds" method.
  • Agrobacterium rhizogenes is used to mediate the infection of apple leaves that are in good condition and easy to infect after subculture. After co-culture and differentiation culture, a large number of adventitious buds differentiated from leaves can be quickly obtained. After continued cultivation, the adventitious buds are taken and the transgenic apple regeneration plants are determined by PCR molecular detection. Compared with the previously reported apple transgenic method mediated by Agrobacterium rhizogenes, the method protected by this application obtains adventitious buds that can be stably inherited, rather than hairy roots. After the adventitious buds are induced to root in the later stage, transgenic plants with stable inheritance can be obtained. It has many advantages such as simplicity, high efficiency, and short cultivation and cycle.
  • the present application first provides a method for inducing and culturing adventitious buds of apple or apple, comprising the following steps:
  • the receptors are apple leaves;
  • the leaves in 1) are tender leaves
  • the preparation step 1) is specifically as follows: selecting apple tender branches to establish apple plant explants, subculturing the explants 3-4 times, and taking tissue culture seedling leaves below the top tender leaves and above the bottom old leaves as transgenic receptors.
  • the vector construction in step 2) is to construct an expression vector containing the target gene and/or marker gene;
  • the marker gene is a fluorescent marker gene
  • the expression vector includes but is not limited to pCAMBIA1301 vector or RNAi silencing PK7WIWG2D-PGT1::GFP vector;
  • the construction step is specifically as follows: using plasmid pCAMBIA1301 as the original vector, replacing the hygromycin phosphotransferase gene and the glycosidase gene GUS with the target gene and the fluorescent marker gene to obtain a recombinant expression vector.
  • the Agrobacterium is Agrobacterium rhizogenes
  • the Agrobacterium rhizogenes includes but is not limited to Agrobacterium rhizogenes K599, Agrobacterium rhizogenes 8196, Agrobacterium rhizogenes R1601 or Agrobacterium rhizogenes C58C1.
  • the transformation in step 3) is to transform the recombinant expression vector into Agrobacterium rhizogenes, and then infect the cells in a MES-KOH resuspension containing acetosyringone after dark culture;
  • the step 3) transformation step is specifically as follows: transforming the recombinant expression vector into Agrobacterium rhizogenes, culturing on a LB solid plate containing kanamycin and streptomycin at 19-30°C in the dark for 13 days, picking a single colony in liquid LB containing kanamycin and streptomycin, shaking at 25-28°C and 150-220rpm until the OD600 value reaches the range of 0.8-1.5, centrifuging at 5000-7000rpm at room temperature to remove the supernatant, and resuspending the cells in a MES-KOH resuspension containing acetosyringone for infection.
  • step 4 the impregnation in step 4) is carried out by methods including but not limited to traditional scratching or vacuum penetration;
  • the infection is carried out by vacuum infiltration, and Agrobacterium rhizogenes is introduced into apple leaf cells, so that the T-DNA of the target gene carried by the Agrobacterium is inserted into the plant genome;
  • the vacuum infiltration method for infection comprises placing the leaves of the tissue culture seedlings in an infection solution, vacuum treating the leaves at a pressure of 0.04-0.1 MPa, and then transferring the leaves to a co-culture medium containing acetosyringone and betaine for culture;
  • the vacuum infiltration method for infection steps is: placing the tissue culture seedling leaves in the infection solution, the vacuum infiltration time is 1-60min, and the infiltration treatment is performed at a vacuum degree of 0.04-0.1Mpa. After the vacuum infiltration treatment, the bacterial solution on the surface of the leaves is absorbed with sterile filter paper, and the leaves are transferred to a co-culture medium containing acetosyringone and betaine, and the back of the leaves is facing up and the front is close to the culture medium for cultivation. The culture medium is placed at a temperature of 25 ⁇ 5°C and cultured in the dark for 1-3 days.
  • MS basal medium supplemented with a certain concentration of thidiazuron TDZ and/or 6-benzyladenine, and ⁇ -naphthylacetic acid NAA
  • the specific components are: MS+0-5 mg/L thidiazuron TDZ+0.05-3 mg/L ⁇ -n
  • the step 5) inducing the adventitious buds to obtain adventitious buds but not hairy roots; the step 5) inducing the adventitious buds to obtain adventitious buds is to transfer the co-cultivated apple leaves to an adventitious bud differentiation medium and culture them in the light to obtain adventitious buds;
  • the specific steps of step 5) adventitious bud induction culture are: transferring the co-cultivated apple leaves to a differentiation medium, conducting adventitious bud induction culture under the conditions of a light intensity of 500-5000 lux, a photoperiod of 12-18/12-6h, and a temperature of 25 ⁇ 5°C, and culturing for 2-8 weeks to obtain adventitious buds.
  • the present application also provides a method for constructing a stable genetic transgenic system of apple and apple, the method comprising any of the above method steps, and further comprising the following steps:
  • step 6 the screening and identification of transgenic adventitious buds in step 6) is based on fluorescent marker screening and identification;
  • the screening and identification steps of the transgenic adventitious buds in step 6) are specifically as follows: using ultraviolet light to perform preliminary fluorescent screening on the adventitious buds differentiated from leaves on the differentiation medium, placing the preliminarily screened adventitious buds on a subculture medium containing cephalosporin for culturing, and after 2-4 weeks, performing fluorescent identification again on the preliminarily screened adventitious buds, and placing the identified fluorescently marked adventitious buds on a subculture medium containing cephalosporin for continued culturing.
  • step 7) rooting and transplanting the transgenic plants are: transferring the differentiated adventitious buds into a rooting medium, performing rooting culture under the conditions of a light intensity of 500-5000 lux, a photoperiod of 12-18 h light/12-6 h dark, and a temperature of 25 ⁇ 5° C. to obtain regenerated plants, and transplanting the rooted regenerated plants into soil to obtain complete transgenic plants with stable inheritance;
  • the above method may further include the following steps:
  • the DNA level of the fluorescently labeled regenerated plants obtained above was detected by PCR method to confirm that the target gene had been integrated into the apple genome DNA, and finally the transgenic plants were confirmed.
  • the present application uses Agrobacterium rhizogenes to infect leaves, and directly obtains adventitious buds through co-culture and differentiation culture, rather than the hairy roots described by the predecessors.
  • the transgenic adventitious buds obtained can be subcultured and rooted to obtain a complete transgenic plant with stable genetic ability.
  • This method has the advantages of simplified operation process, greatly reduced difficulty, and greatly improved transformation efficiency.
  • the existing method of using Agrobacterium tumefaciens to infect apple leaves has a low probability of obtaining transgenic adventitious buds that can be stably inherited, and the operation steps are cumbersome.
  • the Agrobacterium tumefaciens infects the leaf, it is necessary to culture it in the dark for 4 weeks and then culture it in the light for 2-6 weeks to obtain transgenic adventitious buds.
  • the infected apple leaves are directly transferred to the differentiation medium and exposed to the light for 2-6 weeks after dark culture to obtain transgenic adventitious buds. Therefore, the genetic transformation method of the present application has more streamlined steps, and the entire experimental cycle is shortened by about half, while greatly reducing the input of manpower, material resources, financial resources and other resources.
  • This application overcomes the traditional technical concept, pioneeringly uses apple leaves as receptors, and uses Agrobacterium rhizogenes to mediate, to obtain transgenic adventitious buds with stable inheritance.
  • Agrobacterium rhizogenes to mediate, to obtain transgenic adventitious buds with stable inheritance.
  • GL-3 apple transgenic mentioned in this application as an example, if you want to obtain a transgenic strain using the traditional "leaf disc method" (mediated by Agrobacterium tumefaciens), you must wound the apple leaves in a disposable culture dish containing an infection solution, so it can only be repeated in small quantities and multiple times, with high technical requirements, cumbersome experimental steps, long time, and a small number of transgenic adventitious buds obtained, and low efficiency.
  • a large number of leaves can be immersed in a tissue culture bottle (or a larger container) containing a bacterial solution at one time, without the need to perform a wounding operation on the leaves, and Agrobacterium infection can be carried out by vacuum infiltration treatment.
  • the vacuum infiltration treatment method since the vacuum drying vessel has a large volume and can accommodate multiple tissue culture bottles (or a larger container), it is possible to achieve synchronous infection of multiple different transgenics. The steps are simple and convenient, saving time, and a large number of transgenic adventitious buds that can be stably inherited are obtained in one experiment, with a high probability and a greatly improved transformation efficiency.
  • FIG1 is a diagram showing the growth state of the GL-3 apple tissue culture seedlings used in the present application, which is suitable for infection;
  • FIG2 is a vector map of the plasmid pCAMBIA1301-PGT2-Egfp of the present application.
  • FIG3 is a fluorescence imaging diagram of the plasmid pCAMBIA1301-PGT2-Egfp of the present application verified by transient expression in tobacco; in the figure, G: strain K599 containing pCAMBIA1301-PGT2-Egfp plasmid; ck: strain K599 containing pCAMBIA1301 plasmid;
  • FIG4 is a diagram showing the state of the infected leaves being transferred to the co-cultivation medium of the present application
  • FIG5 is a diagram showing the infected leaves of the present application after being cultured in a differentiation medium exposed to light for 4 weeks;
  • FIG6 is a state diagram of some adventitious buds differentiated from infected leaves of the present application, which were transferred to subculture medium and cultured for 4 weeks after preliminary fluorescence screening under ultraviolet light;
  • FIG7 is a fluorescence imaging image of some adventitious buds obtained in the present application identified by the PlantView100 plant living imaging system, the left image is a composite image, and the right image is a bright field;
  • FIG8 shows the PCR test results of some transgenic plants obtained in the present application; in the figure, M: DL2000 DNA Marker; CK: untransformed plants; 1-11: transformed plants;
  • FIG9 is a fluorescence imaging diagram of some transgenic strains obtained in the present application identified by the PlantView100 plant living imaging system, the left diagram is a composite image, and the right diagram is a bright field;
  • FIG10 is a fluorescence imaging diagram of some transgenic strains obtained in Example 2 of the present application identified by the PlantView100 plant living imaging system, the left image is a bright field, and the right image is a composite image;
  • Figure 11 is the PCR test results of some transgenic plants obtained in Example 2 of the present application; in the figure, M: DL2000 DNA Marker; CK: untransformed plants; 1, 2: some transformed plants obtained
  • FIG12 is a fluorescent image of transgenic adventitious buds obtained from GL-3 leaves mediated by C58C1 Agrobacterium rhizogenes strain in Example 3 of the present application, which were identified by the PlantView100 plant living imaging system, the left image is a composite image, and the right image is a bright field image;
  • FIG13 is a fluorescent image of transgenic adventitious buds obtained from GL-3 leaves mediated by C58C1 Agrobacterium rhizogenes strain in Example 3 of the present application, which were identified by the PlantView100 plant living imaging system, the left image is a composite image, and the right image is a bright field image;
  • FIG14 is a fluorescent imaging image of transgenic adventitious buds obtained from GL-3 leaves mediated by C58C1 Agrobacterium rhizogenes strain in Example 3 of the present application, which was identified by the PlantView100 plant living imaging system, the left image is a composite image, and the right image is a bright field image;
  • Figure 15 is a map of the PK7WIWG2D-PGT1::GFP vector used in Example 4 of this application;
  • FIG. 16 is a phenotypic comparison diagram of some transgenic strains obtained by Example 4 in the present application and the wild type.
  • FIG17 is the PCR detection result of the PGT1-RNAi::GFP apple transgenic line obtained in Example 4 of the present application; in the figure, M: DL2000 DNA Marker; CK: untransformed plant; F1-1, F1-2: transformed plants;
  • FIG18 is a fluorescent imaging image of transgenic adventitious buds obtained by infecting GL-3 leaves using other wounding methods in Example 5 of the present application, which were identified by the PlantView100 plant living imaging system, the left image is a composite image, and the right image is a bright field image;
  • FIG19 is an operation flow chart of the apple transgenic technology mediated by traditional Agrobacterium tumefaciens - "leaf disc method" in Example 6 of the present application;
  • FIG20 is an operation flow chart of the apple transgenic technology mediated by Agrobacterium rhizogenes in this application;
  • Figure 21 is a fluorescence imaging comparison diagram of the ability of transgenic adventitious buds obtained by using Agrobacterium tumefaciens and Agrobacterium rhizogenes in Example 6 of the present application to be identified by the PlantView100 plant living imaging system
  • Figure A is a composite image obtained by the PlantView100 plant living imaging system for adventitious buds obtained by using Agrobacterium tumefaciens
  • Figure B is the corresponding bright field
  • Figure C is a composite image obtained by the PlantView100 plant living imaging system for adventitious buds obtained by using Agrobacterium rhizogenes
  • Figure D is the corresponding bright field.
  • the terms “comprises”, “comprising”, “having”, “containing” or “involving” are inclusive or open-ended and do not exclude other unrecited elements or method steps.
  • the term “consisting of” is considered a preferred embodiment of the term “comprising”. If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.
  • the methods in this application include methods for inducing and cultivating adventitious buds of apple and apple, and methods for constructing a stable genetic transgenic system of apple and apple. These methods are based on overcoming the traditional prejudice against the use of Agrobacterium rhizogenes, pioneering the use of apple leaves as receptors, and using Agrobacterium rhizogenes to mediate adventitious bud induction and cultivate to obtain stable genetically modified adventitious buds, that is, for the first time, using the method of "Agrobacterium rhizogenes + leaf infection + induction and cultivation of adventitious buds” to efficiently and quickly obtain a stable and inherited apple transgenic strain. Therefore, in theory, any basic steps including the above-mentioned "Agrobacterium rhizogenes + leaf infection + induction and cultivation of adventitious buds" are within the scope of protection of this application.
  • the examples take the establishment of PGT2 gene markers related to the presence or absence of traits of the apple plant Tripterygium wilfordii and its assisted breeding technology as an example to explain in detail the technical content of the present application. This example is only used to explain the method ideas of the present application and does not limit the protection content.
  • Those skilled in the art can expect that the selection of apple varieties, root-generating Agrobacterium species, vector types, target genes and marker genes, etc., will generally not affect the ability of the core basic method of the present application to induce a stable genetic transgenic system; similarly, the selection of some basic culture media or the setting of condition parameters are not limited. For example, when differentiation culture is required, the art can select corresponding specific differentiation culture media and condition parameters for differentiation culture.
  • Example 1 Using GL-3 apple material as the starting material, establishing an apple stable transformation system using Agrobacterium rhizogenes K599
  • This example uses GL-3 apple material as the starting material.
  • Selection of GL-3 apple tissue culture seedling leaves Select tender branches from March and April of the same year to establish explants of GL-3 apple plants.
  • the established explants are placed in culture medium for culture. Subculture is performed every 4 weeks for 3-4 times.
  • the subculture medium for continued growth of the plants is: basic culture medium MS (Murashige and Skoog), with the addition of exogenous hormones 6-BA, i.e., 6-benzyladenine, and IAA, i.e., 3-indoleacetic acid.
  • the concentration of 6-BA is 1 mg/L
  • the concentration of IAA is 0.5 mg/L.
  • Plasmid pCAMBIA1301 was used as the original vector. Plasmid pCAMBIA1301 contained the hygromycin phosphotransferase gene HPT and the glucuronidase gene GUS of the CaMV 35S promoter. The vector was modified by using the restriction site XhoI for single restriction digestion to replace the hygromycin phosphotransferase gene with the target gene PGT2. Secondly, to facilitate the subsequent transgenic plant screening, NcoI and BstEII were used for double restriction digestion to replace the glucuronidase gene GUS with Egfp enhanced green fluorescent protein to obtain the vector pCAMBIA1301-PGT2-Egfp. The map of the vector after modification is shown in Figure 2.
  • Agrobacterium rhizogenes strain K599 was selected, and the vector pCAMBIA1301-PGT2-Egfp was transformed into the K599 strain.
  • the transformed K599 strain containing the target gene and the Egfp green fluorescent protein plasmid was verified by transient expression in tobacco, as shown in Figure 3.
  • Agrobacterium containing the target plasmid was selected, and inoculated on a solid LB plate (containing 50 mg/L kanamycin (Kan) and 50 mg/L streptomycin (Stre)), and cultured in the dark at 28°C for 48 hours.
  • the bacterial solution was placed in a 50 ml centrifuge tube, and centrifuged at room temperature and 5500 rpm for 5 minutes to remove the supernatant.
  • the resuspended liquid is transferred to an appropriate container for infection.
  • the Agrobacterium infection liquid is allowed to stand at room temperature for more than half an hour before use for infection.
  • MES-KOH resuspension MES, i.e., MES monohydrate, and MgCl2 , i.e., magnesium chloride, are added.
  • concentration of MES is 10mM
  • concentration of MgCl2 is 10mM
  • pH is 5.6.
  • Infection and co-cultivation Put the prepared apple leaves into the Agrobacterium infection solution, soak the apple leaves in the heavy suspension solution by vacuum infiltration, vacuumize, and soak for 15 minutes at a pressure of 0.09Mpa. Use sterile filter paper to absorb the bacterial solution on the surface of the leaves in the clean bench, transfer the leaves infected with Agrobacterium to the co-cultivation medium, and culture them in the dark at 23 ⁇ 2°C for 3 days. The state of the leaves transferred to the co-cultivation medium is shown in Figure 4. If the leaves are water-stained, it means that the infection is successful.
  • Co-culture medium basic medium MS (Murashige and Skoog), added with exogenous hormones TDZ (thidiazuron) and NAA ( ⁇ -naphthylacetic acid), TDZ concentration is 2 mg/L, NAA concentration is 0.5 mg/L, then add 30 g/L sucrose and 7.5 g/L agar powder, adjust the pH to 5.8, add Ace (acetosyringone) and BT (betaine) to the medium after sterilization, Ace concentration is 0.1 mM, BT concentration is 1 mM.
  • Differentiation medium basic medium MS (Murashige and Skoog), with the addition of exogenous hormones TDZ, i.e., thidiazuron, and NAA, i.e., ⁇ -naphthylacetic acid.
  • the concentration of TDZ was 2 mg/L, and the concentration of NAA was 0.5 mg/L.
  • 30 g/L of sucrose and 7.5 g/L of agar powder were added, and the pH was adjusted to 5.8.
  • the antibiotic Cef i.e., cephalosporin
  • the concentration of Cef used is 250 mg/L.
  • the adventitious buds obtained from the preliminary screening were fluorescently identified again by a multi-spectral dynamic fluorescence imaging system, and the adventitious buds with fluorescent labels after identification were placed in a subculture medium containing cephalosporin and continued to be cultured at 25°C, a light intensity of 2400 lux, and a photoperiod of 16/8h.
  • Subculture medium see Figure 6: basic medium MS (Murashige and Skoog), added with exogenous hormones 6-BA, i.e. 6-benzyladenine and IAA, i.e.
  • 3-indoleacetic acid 6-BA used at a concentration of 1 mg/L
  • IAA used at a concentration of 0.5 mg/L
  • Rooting culture When the regenerated plants grown from differentiation culture grow to 4-6 cm, they are transferred to the rooting medium and cultured in the dark at 25°C for 2 weeks. After 2 weeks, they are cultured for about 2 weeks under the conditions of 2400 lux, 16/8h photoperiod, and 25°C. The regenerated plants differentiate into roots and are planted in the soil to obtain transgenic plants with stable inheritance.
  • the rooting medium is: basic medium MS (Murashige and Skoog), with exogenous hormones IAA (3-indoleacetic acid) and IBA (3-indolebutyric acid) added. The concentration of IAA is 0.5 mg/L, and the concentration of IBA is 1 mg/L. Then, 30 g/L of sucrose and 7.5 g/L of agar powder are added, and the pH is adjusted to 5.8.
  • DNA of fluorescently labeled regenerated plants was extracted and subjected to PCR detection.
  • Two primers for PCR detection of resistant seedlings were designed based on the vector sequence and PGT2 gene sequence, namely primer 1: 5’-CTCGAGATGGAGGCGACAGCTATAGTTTTATATCC-3’, primer 2: 5’-GATCTGGATTTTAGTACTGGATTTTGGTTTTAGGA–3’.
  • the PCR program was as follows: pre-denaturation at 94°C for 5 min; then denaturation at 94°C for 30 s, annealing at 56°C for 15 s, extension at 72°C for 20 s, and 35 cycles; extension at 72°C for 10 min.
  • Example 2 Using Royal Gala apple material as the starting material, establishing an apple stable transformation system using Agrobacterium rhizogenes K599
  • Example 2 The Agrobacterium rhizogenes strains, vectors, infection experiment procedures, fluorescence screening and identification, rooting culture, and transgenic plant identification methods and steps used in the experiment are the same as those in Example 1.
  • Leaf co-culture after infection The co-culture time and external conditions were the same as those of the co-culture after infection with GL-3 material. The following method was based on Yao (1995).
  • the co-culture medium after Royal Gala infection was the basic medium MS (Murashige and Skoog), with the addition of exogenous hormones 6-BA (6-benzyladenine) and NAA ( ⁇ -naphthylacetic acid), with the concentration of 6-BA being 5 mg/L and the concentration of NAA being 0.2 mg/L.
  • 30 g/L of sucrose and 7.5 g/L of agar powder were added, and the pH was adjusted to 5.8.
  • 0.5 mg/L of vitamin B5, Ace (acetosyringone) and BT (betaine) were added to the medium, with the concentration of Ace being 0.1 mM and the concentration of BT being 1 mM.
  • Example 3 Establishment of a stable apple transformation system mediated by different Agrobacterium rhizogenes
  • Transformation and culture to obtain a strain for transformation the pCAMBIA1301-PGT2-Egfp expression vector was used as a vector, and the target vector plasmid was transferred into Agrobacterium rhizogenes competent cells 8196, R1601, and C58C1 using the heat shock method.
  • the cells were spread and inoculated on LB solid culture medium for culture, and cultured in the dark at 28° C. for 48 h. A single colony was picked and placed in 50 ml of liquid LB, and shaken at 28° C. and 200 rpm to an OD600 value of 1.5.
  • the bacterial liquid was centrifuged and resuspended in 1.5 times the volume of MES-KOH resuspension liquid.
  • the resuspended liquid was transferred to an appropriate container for infection.
  • the Agrobacterium infection liquid was allowed to stand at room temperature for more than half an hour before use for infection.
  • Example 3 The operation procedures of the apple leaf experiment mediated by three different rhizogenes Agrobacterium strains were the same as those in Example 1, and the steps of culturing apple leaves after infection and inducing adventitious buds were the same as those in Example 1.
  • Example 4 Establishment of a stable apple transformation system based on different vectors
  • Vector construction Create an RNAi silencing PK7WIWG2D-PGT1::GFP vector using pDONR222 as an intermediate vector.
  • the vector map is shown in FIG15 .
  • Example 3 The methods of Agrobacterium rhizogenes transformation, infection and co-cultivation, differentiation culture, fluorescence screening and identification, and rooting culture were the same as those in Example 1, and finally some transgenic regenerated strains were obtained, as shown in FIG16 .
  • Example 5 Establishment of a stable apple transformation system under different infection treatments
  • Example 2 Different from the infection treatment method - vacuum infiltration treatment in Example 1, this example adopts a different wound treatment method.
  • a sterilized blade is used to make 3-4 1 cm wounds on the back of the apple leaves (other wound treatments for the recipient plants are also acceptable), and the leaves with wounds are immersed in the infection solution for 5-30 minutes. After 5-30 minutes, the leaves are taken out and the liquid on the surface of the leaves is dried with sterile filter paper.
  • the leaves are placed on a co-culture medium for culture, and the subsequent differentiation culture, fluorescence screening and identification, rooting culture, and transgenic plant identification are the same as in Example 1.
  • the strain used for leaf disc infection is GV3101 Agrobacterium tumefaciens strain; the strain used for rooting is K599 Agrobacterium rhizogenes strain.
  • the plasmids are both vectors pCAMBIA1301-PGT2-Egfp.
  • Leaf disc method refer to Hongyan Dai (2013), take appropriate amount of leaves and place them in bacterial solution, use sterile blade to make 3-4 1cm wounds on the back of leaves, immerse the wounded leaves in infection solution for 8 minutes, take them out after 8 minutes, use sterile filter paper to absorb the liquid on the surface of leaves, and place the leaves on co-culture medium.
  • the leaves on co-culture are transferred to extended culture medium for dark culture for 4 weeks, then taken out and cultured in light for 4-6 weeks.
  • the steps of wound operation process are shown in Figure 19.
  • the temperature of the whole culture process is 22-25°C, the light intensity is 2400lux, and the number of transgenic adventitious buds obtained by infection is counted after 2-6 weeks of light exposure.
  • Rooting method The steps are as in Example 1, taking 1.5 hours, 3 days of dark culture treatment, after dark culture treatment, transfer to differentiation medium and expose to light for 2 weeks to differentiate callus and adventitious roots, and 4 weeks to obtain transgenic adventitious buds. The specific steps of the operation are shown in Figure 20. After 2-8 weeks of light culture, the number of transgenic adventitious buds is counted; the external conditions of the experiment, the number of each batch and the number of repeated tests are consistent with the leaf disc method.
  • FIG. 21 The fluorescence imaging comparison diagram identified by the PlantView100 plant living imaging system is shown in Figure 21.
  • Figure A is a composite image of adventitious buds obtained by using Agrobacterium tumefaciens mediation and obtained by the PlantView100 plant living imaging system, and Figure B is the corresponding bright field.
  • Figure C is a composite image of adventitious buds obtained by using Agrobacterium rhizogenes mediation and obtained by the PlantView100 plant living imaging system, and Figure D is the corresponding bright field.
  • the adventitious buds obtained by using Agrobacterium tumefaciens mediation are solitary adventitious buds, and some adventitious buds obtained by using Agrobacterium rhizogenes mediation are clustered adventitious buds.

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Abstract

一种利用发根农杆菌高效快速获得苹果稳定转基因植株的方法，该方法以苹果叶片为受体，利用发根农杆菌介导获得不定芽而非毛状根，再基于不定芽获得可稳定遗转的苹果转基因植株。

Description

一种利用发根农杆菌高效快速获得苹果稳定转基因植株的方法
相关申请的交叉引用
本申请要求于2022年10月28日提交中国专利局的申请号为2022113420212、名称为“一种利用发根农杆菌高效快速获得苹果稳定转基因植株的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请属于植物基因工程技术领域，具体涉及一种以苹果植株的叶片作为受体，利用发根农杆菌高效快速获得苹果稳定转基因植株的方法。
背景技术
农杆菌主要有两种：根癌农杆菌和发根农杆菌。农杆菌质粒是一种能实现DNA转移和整合的天然系统，质粒载体系统中最常用的质粒有:Ti质粒和Ri质粒。Ti质粒存在于根癌农杆菌(Agrobacterium tumefaciens)中，Ri质粒存在于发根农杆菌(Agrobacterium rhizogenis)中。Ti质粒和Ri质粒在结构和功能上有许多相似之处，具有基本一致的特性。Ti质粒大约在160～240kB之间，Ti质粒是通过T-DNA区转移整合入植物受体基因组并能在植物细胞中表达从而导致冠瘿瘤的发生，且可通过减数分裂传递给子代的区域，T-DNA长度为12-24kb之间，两端各有一个含25bp重复序列的边界序列，在整合过程中左右边界序列之间的T-DNA可以转移并整合到宿主细胞基因组中，研究发现只有边界序列对DNA的转移是必需的，而边界序列之间的T-DNA并不参与转化过程，因而可以用外源基因将其替换，从而将外源基因导入到宿主的基因组中(白永廷等1984；王裕鹏2018)；发根农杆菌感染能诱导产生毛状根(Hair Root),这是由发根农杆菌含有的Ri(Root Inducing)质粒引起,Ri质粒为根诱导质粒(Root Inducing Plasmid)；Ri质粒的大小为200～800kb,它含有负责发根自主性生长和冠瘿碱合成的基因,在结构上有致毒区(Vir区),转移进入植物细胞核的T-DNA区及其内部的冠瘿碱合成功能区等。Ri质粒通过T-DNA转移到被侵染植物中,使其生成毛状根农杆碱型Ri质粒上的T-DNA呈不连续分布,与TL-DNA、TR-DNA及TL-DNA上的基因与毛状根的形成有关,同时也影响着再生植株地上部分形态和一些生理性状(吴萼等2018)。
目前，对于大多数植物农杆菌介导的植物遗传转化，主要是采用含有目的基因的根癌农杆菌进行介导，最终获得转基因再生植株。以苹果转基因为例，目前主要采用的是叶盘法，利用根癌农杆菌侵染苹果外植体的叶片，并短期共培养，后期经过不同条件的诱导获得苹果转基因株系(方信武等2014)。在植物转基因体系中，还存在一种方式，使用发根农杆菌介导植株获得转基因毛状根，主要是用植物茎秆作为受体，利用发根农杆菌介导获得转基因毛状根。但该方法存在一定局限性，仅获得的毛状根是转基因材料，而叶片及茎等其他组织仍为非转基因材料，因此无法获得稳定遗传的转基因植株(曹庆丰2012；丁雪2017；肖璇2014)。如果想利用已经获得的转基因毛状根进一步获得可稳定遗传的转基因株系，则需要对毛状根进行离体组织培养，毛状根先脱分化诱导形成愈伤组织,再经过再分化诱导获得转基因再生植株(李想韵2010；王艳2009；张程程2011)。但是，使用这种方法获得可稳定遗传的转基因再生植株的要对毛状根离体组织培养的条件进行进一步摸索，其步骤繁琐，时间周期长。因此，开发出一种利用发根农杆菌介导高效快速获得可稳定遗传的转基因植株的方法，显得尤为重要。
有鉴于此，提出本申请。
发明内容
申请针对现有技术存在的问题，本申请克服对于发根农杆菌的传统偏见，开创性的使用苹果叶片作为受体，利用发根农杆菌介导，通过不定芽诱导培养获得稳定遗传的转基因不定芽，即首次利用“发根农杆菌+叶片侵染+诱导培养不定芽”的方式高效快速获得可稳定遗传的苹果转基因株系。
具体而言，利用发根农杆菌介导，以经过继代培养获得的状态较好、易侵染的苹果叶片作为受体进行侵染，侵染后的叶片经共培养和分化培养，可快速获得大量叶片分化的不定芽，不定芽经过继续培养，取其叶片，通过PCR分子检测，确定转基因苹果再生植株。与之前已报道的发根农杆菌介导的苹果转基因方法相比，本申请保护的方法获得的是可稳定遗传的不定芽，而非毛状根，不定芽经过后期诱导生根后即可获得稳定遗传的转基因植株。具有简便、高效、培和周期短等诸多优势。
本申请具体提供如下技术方案如下：
本申请首先提供量一种苹果或苹果属的不定芽诱导培养方法，包括如下步骤：
1)转基因受体制备：所述受体为苹果叶片；
2)表达载体构建；
3)农杆菌的转化；
4)叶片侵染及共培养；
5)不定芽诱导培养。
进一步的，所述1)中的叶片为嫩叶片；
优选的，所述步骤1)制备步骤具体为：选苹果嫩枝建立苹果植株外植体，外植体继代3-4次，取顶端嫩叶以下底端老叶以上的组培苗叶片作为转基因的受体。
进一步的，所述步骤2)中的载体构建为构建包含目的基因和/或标记基因的表达载体；
优选的，所述标记基因为荧光标记基因；
优选的，所述表达载体包括但不仅限于pCAMBIA1301载体或RNAi沉默PK7WIWG2D-PGT1::GFP载体；
在一些特定方式中，所述构建步骤具体为：采用质粒pCAMBIA1301为原始载体，将潮霉素磷酸转移酶基因和糖苷酸酶基因GUS替换为目的基因和荧光标记基因，得到重组表达载体。
进一步的，所述步骤3)中，所述农杆菌为发根农杆菌；
优选的，所述发根农杆菌包括但不限于发根农杆菌K599、发根农杆菌8196、发根农杆菌R1601或发根农杆菌C58C1。
在一些特定方式中，所述步骤3)中的转化为将重组表达载体转化到发根农杆菌中，暗培养后至含有乙酰丁香酮的MES-KOH重悬液中进行侵染；
优选的，所述MES-KOH重悬液为：8-12mM MES一水合物,5-15mM氯化镁,0.05-0.3mM乙酰丁香酮，pH＝5.3-5.6；
在一些更特定的方式中，所述步骤3)转化步骤具体为：将重组表达载体转化到发根农杆菌中，在含有卡那霉素和链霉素的LB固体平板上，19-30℃暗培养13d，挑取单菌落于含有卡那霉素和链霉素的液体LB中，25-28℃，150-220rpm摇至OD600值达0.8-1.5范围，室温5000-7000rpm离心去上清，将菌体重悬至含有乙酰丁香酮的MES-KOH重悬液中侵染。
进一步的，所述步骤4)中所述浸染采用包括但不限于传统划伤或真空渗透方式进行侵染；
优选的，所述侵染采用真空渗透方式进行侵染，将发根农杆菌导入苹果叶片细胞，使农杆菌携带的目的基因的T-DNA插入植物基因组；
在一些特定方式中，所述真空渗透方式进行侵染步骤为：将组培苗叶片置于侵染液中，0.04-0.1Mpa压力下真空处理，之后将叶片转移至含有乙酰丁香酮和甜菜碱的共培养培养基上培养；
在一些更特定的方式中，所述真空渗透方式进行侵染步骤为：将组培苗叶片置于侵染液中，真空渗透时间1-60min，真空度0.04-0.1Mpa下进行渗透处理，真空渗透处理后，用无菌滤纸将叶片表面菌液吸干，将叶片转移至含有乙酰丁香酮和甜菜碱的共培养培养基上，叶片背面朝上正面紧贴培养基进行培养，培养基置于温度25±5℃的条件下，黑暗培养1-3d。
优选的，所述共培养基为MS基础培养基中添加一定浓度的噻苯隆TDZ和/或6-苄基腺嘌呤，和α-萘乙酸NAA；更优选的，组分具体为：MS+0-5mg/L的噻苯隆TDZ+0.05-3mg/L的α-萘乙酸NAA+25-35g/L蔗糖或35-45g/L山梨醇+6-8g/L的琼脂粉或2-3g/L植物凝胶，0.05-0.3mM的乙酰丁香酮，0.8-1.2mM的甜菜碱，pH＝5.6-6.0。
进一步的，所述步骤5)不定芽诱导培养获得不定芽，不获得毛状根；所述步骤5)不定芽诱导培养为，将经共培养的苹果叶片转移至不定芽分化培养基，见光培养获得不定芽；
优选的，所述不定芽分化培养基为MS基础培养基中添加一定浓度的噻苯隆TDZ和/或6-苄基腺嘌呤，和α-萘乙酸NAA；更优选的，组分具体为：MS+0-5mg/L的噻苯隆TDZ+0.05-3mg/L的α-萘乙酸NAA+25-35g/L蔗糖或35-45g/L山梨醇+6-8g/L的琼脂粉或2-3g/L植物凝胶，pH＝5.6-6.0；
在一些特定方式中，所述步骤5)不定芽诱导培养的具体步骤为：将经共培养的苹果叶片转移至分化培养基，在光强500-5000lux，光周期12-18/12-6h，温度25±5℃的条件下进行不定芽诱导培养，培养2-8周获得不定芽。
本申请还提供一种苹果及苹果属稳定遗传转基因体系的构建方法，所述方法包括上述任一所述方法步骤，并进一步包括如下步骤：
6)转基因不定芽的筛选和鉴定；
7)转基因植株生根与移栽。
进一步的，所述步骤6)的转基因不定芽的筛选与鉴定为基于荧光标记的筛选和鉴定；
优选的，所述步骤6)的转基因不定芽的筛选和鉴定步骤具体为：使用紫外灯对分化培养基上叶片分化的不定芽进行初步荧光筛选，将初步筛选出的不定芽置于含头孢霉素的继代培养基上培养，2-4周后对初步筛选出的不定芽进行再次荧光鉴定，将鉴定后的具有荧光标记的不定芽置于含头孢霉素的继代培养基中继续培养。
进一步的，所述步骤7)转基因植株生根与移栽的具体步骤为：把分化培养出的不定芽移入生根培养基，在光强500-5000lux，光周期12-18h光照/12-6h黑暗，温度25±5℃的条件下进行生根培养，获得再生植株，将带根的再生植株移栽至土壤中，获得稳定遗传的完整转基因植株；
优选的，所述生根培养基为：MS+0.1-2.0mg/L 3-吲哚乙酸+0.02-2mg/L 3-吲哚丁酸+25-35g/L蔗糖或35-45g/L山梨醇+6-8g/L琼脂粉或2-3g/L植物凝胶，pH＝5.6-6.0。
进一步的，上述方法还可进一步包括如下步骤：
8)转基因植株的分子水平鉴定：
用PCR方法对上述获得的具有荧光标记的再生植株进行DNA水平检测，确定目的基因已经整合到苹果基因组DNA中，最终确定转基因植株。
与现有技术相比，本申请至少具有如下明显技术优势：
1)现有的利用发根农杆菌侵染仅能够直接获得转基因毛状根，茎秆、叶片等其他组织并不能被发根农杆菌侵染，仍不是转基因组织，其材料不能稳定遗传，想要获得可稳定遗传的转基因株系还需对转基因毛状根进行进一步离体组织培养，诱导根系脱分化生成愈伤，愈伤再分化生成不定芽(此过程极为困难)，然后不定芽再经过继代培养及生根才能获得具有稳定遗传能力的完整转基因植株。技术流程繁琐且时间周期长、效率低。而本申请利用发根农杆菌侵染叶片，经共培养和分化培养直接获得不定芽，而非前人所述的毛状根，其获得的转基因不定芽通过继代培养及生根后可以获得具有稳定遗传能力的完整转基因植株。使用本申请方法，可以越过转基因毛状根及随后的毛状根脱分化和分化过程，直接获得不定芽，进而获得完整转基因植株，该方法具有操作流程简化，难度大大降低，转化效率大副提升等优势。
2)而现有的利用根癌农杆菌侵染苹果叶片的方法，获得可稳定遗传的转基因不定芽的概率低，操作步骤繁琐。比如，在叶片侵染后的培养阶段，根癌农杆菌侵染叶片后需要暗培养4周后再见光培养2-6周才能获得转基因不定芽。但按照本申请的方法，侵染后的苹果叶片暗培养3d后直接转移至分化培养基中见光2-6周即可获得转基因不定芽。因此本申请的遗传转化方法步骤更加精简，实验整个周期缩短一半左右的同时大幅减少了人力、物力、财力等资源投入。
3)本申请克服传统技术观念，开创性的使用苹果叶片作为受体，利用发根农杆菌介导，获得稳定遗传的转基因不定芽。以本申请中提到的GL-3苹果转基因为例，使用传统“叶盘法”(根癌农杆菌介导)想要获得转基因株系，须对苹果叶片在盛有侵染液的一次性培养皿中进行创伤侵染，因此只能少量多次重复进行，技术要求高，实验步骤繁琐，时间长，获得转基因不定芽的数量少、效率低。但是使用本申请利用发根农杆菌介导获得转基因植株的方法，可将大量叶片一次性浸入含有菌液的组培瓶(或更大的容器)中，无需对叶片进行造伤操作，通过真空渗透处理的方式，进行农杆菌侵染。使用真空渗透的处理方式，由于真空干燥器皿容积大，可容下多个组培瓶(或更大的容器)，所以可以实现多种不同转基因的同步侵染。步骤简单方便，节省时间且一次实验获得可稳定遗传的转基因不定芽数量多，概率高、转化效率大副提升。
附图说明
为了更清楚地说明本申请具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本申请的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本申请所使用的植株GL-3苹果组培苗适宜侵染的生长状态图；
图2为本申请质粒pCAMBIA1301-PGT2-Egfp的载体图谱；
图3为本申请质粒pCAMBIA1301-PGT2-Egfp经过烟草瞬时表达验证的荧光成像图；图中，G：含pCAMBIA1301-PGT2-Egfp质粒的菌株K599；ck：含pCAMBIA1301质粒的菌株K599；
图4为本申请侵染后的叶片转移至共培养培养基的状态图；
图5为本申请的侵染后的叶片在分化培养基中见光培养4周的状态图；
图6为本申请的侵染后的叶片分化出的部分不定芽，经过紫外光下初步荧光筛选后移入继代培养基继续培养4周的状态图；
图7为本申请中获得的部分不定芽通过PlantView100植物活体成像系统进行鉴定的荧光成像图，左图为合成图，右图为明场；
图8为本申请中获得的部分转基因植株PCR检测结果；图中，M：DL2000 DNA Marker；CK：未转化植株；1-11：转化植株；
图9为本申请中获得的部分转基因株系通过PlantView100植物活体成像系统进行鉴定的荧光成像图，左图为合成图，右图为明场；
图10为本申请实施例2获得的部分转基因株系通过PlantView100植物活体成像系统进行鉴定的荧光成像图，左图为明场，右图为合成图；
图11为本申请中实施例2获得的部分转基因植株PCR检测结果；图中，M：DL2000 DNA Marker；CK：未转化植株；1、2：获得的部分转化植株
图12为本申请中实施例3使用C58C1发根农杆菌菌株介导GL-3叶片获得的转基因不定芽通过PlantView100植物活体成像系统进行鉴定的荧光成像图，左图为合成图，右图为明场；
图13为本申请中实施例3使用C58C1发根农杆菌菌株介导GL-3叶片获得的转基因不定芽通过PlantView100植物活体成像系统进行鉴定的荧光成像图，左图为合成图，右图为明场；
图14为本申请中实施例3使用C58C1发根农杆菌菌株介导GL-3叶片获得的转基因不定芽通过PlantView100植物活体成像系统进行鉴定的荧光成像图，左图为合成图，右图为明场；
图15为本申请中实施例4使用的PK7WIWG2D-PGT1::GFP载体图谱；
图16为本申请中通过实施例4方式获得的部分转基因株系同野生型的表型对比图。
图17为本申请实施例4获得的PGT1-RNAi::GFP苹果转基因株系PCR检测结果；图中，M：DL2000 DNA Marker；CK：未转化植株；F1-1、F1-2：转化植株；
图18为本申请实施例5使用其他创伤方式介导GL-3叶片进行侵染获得的转基因不定芽通过PlantView100植物活体成像系统进行鉴定的荧光成像图，左图为合成图，右图为明场；
图19为本申请中实施例6使用传统根癌农杆菌介导的苹果转基因技术-“叶盘法”的操作流程图；
图20为本申请中使用发根农杆菌介导的苹果转基因技术的操作流程图；
图21为本申请中实施例6使用根癌农杆菌和发根农杆菌介导获得的转基因不定芽的能力通过PlantView100植物活体成像系统鉴定的荧光成像对比图，A为使用根癌农杆菌介导获得的不定芽通过PlantView100植物活体成像系统获得的合成图,图B为相应的明场，图C为使用发根农杆菌介导获得不定芽通过PlantView100植物活体成像系统获得的合成图，图D为相应的明场。
具体实施方式
下面将结合附图对本申请的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
以下术语或定义仅仅是为了帮助理解本申请而提供。这些定义不应被理解为具有小于本领域技术人员所理解的范围。
除非在下文中另有定义，本申请具体实施方式中所用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本申请。
如本申请中所使用，术语“包括”、“包含”、“具有”、“含有”或“涉及”为包含性的(inclusive)或开放式的，且不排除其它未列举的元素或方法步骤。术语“由…组成”被认为是术语“包含”的优选实施方案。如果在下文中某一组被定义为包含至少一定数目的实施方案，这也应被理解为揭示了一个优选地仅由这些实施方案组成的组。
在提及单数形式名词时使用的不定冠词或定冠词例如“一个”或“一种”，“所述”，包括该名词的复数形式。
本申请中的术语“大约”、“大体”表示本领域技术人员能够理解的仍可保证论及特征的技术效果的准确度区间。该术语通常表示偏离指示数值的±10％，优选±5％。
此外，说明书和权利要求书中的术语第一、第二、第三、(a)、(b)、(c)以及诸如此类，是用于区分相似的元素，不是描述顺序或时间次序必须的。应理解，如此应用的术语在适当的环境下可互换，并且本申请描述的实施方案能以不同于本申请描述或举例说明的其它顺序实施。
本申请中的方法，包括苹果及苹果属的不定芽诱导培养方法，以及苹果及苹果属稳定遗传转基因体系的构建方法，这些方法都是基于克服对于发根农杆菌的传统使用偏见，开创性使用苹果叶片作为受体，利用发根农杆菌介导，通过不定芽诱导培养获得稳定遗传的转基因不定芽，即首次利用“发根农杆菌+叶片侵染+诱导培养不定芽”的方式高效快速获得可稳定遗传的苹果转基因株系。因此，理论上但凡包括了上述“发根农杆菌+叶片侵染+诱导培养不定芽”的基础步骤的都在本申请的保护范围之内。
下面为具体的实施例。实施例以苹果属植物三叶甘有无性状相关的PGT2基因标记及其辅助育种技术的建立为例，对本申请的技术内容进行详细说明，该实施例仅用于解释本申请的方法思路，并不对保护内容进行限制。本领域技术人员可以预期，包括苹果品种、发根农杆菌种类、载体种类、目的基因和标记基因的选择等，通常都不会对本申请核心基础方法能够诱导稳定遗传转基因体系产生影响；同样的，一些基础培养基的选择或条件参数设置也不受限，比如在需要进行分化培养时，本领域能够选择相应的特定分化培养基及条件参数用于分化培养。
实施例1、以GL-3苹果材料为出发材料，利用发根农杆菌K599建立苹果稳转体系
1)本实施例以GL-3苹果材料为出发材料，GL-3苹果组培苗叶片的选择：选用当年3、4月份的嫩枝，建立GL-3苹果植株的外植体，将建立的外植体置于培养基中培养，每隔4周进行继代培养，继代3-4次，植株继续生长的继代培养基为：基本培养基MS(Murashige and Skoog)，添加外源激素6-BA即6-苄基腺嘌呤和IAA即3-吲哚乙酸，6-BA使用浓度为1mg/L，IAA使用浓度为0.5mg/L，再添加30g/L的蔗糖和7.5g/L的琼脂粉，调节pH为5.8，在25℃温度下生长4周，转化时挑选组培瓶中长势较好的植株，见图1，取下顶端两片嫩叶以下底端老叶以上的叶片置于组培瓶中作为转基因的受体。
2)载体的构建：使用质粒pCAMBIA1301为原始载体，质粒pCAMBIA1301含有CaMV 35S启动子的潮霉素磷酸转移酶基因HPT及将葡糖苷酸酶基因GUS，对该载体进行改造，使用酶切位点为XhoI进行单酶切，将潮霉素磷酸转移酶基因替换为使用的目的基因PGT2。其次，为方便后续转基因植株筛选，使用NcoI和BstEII进行双酶切，将葡糖苷酸酶基因GUS替换为Egfp增强绿色荧光蛋白，获得载体pCAMBIA1301-PGT2-Egfp，载体改造后的图谱，见图2。
3)发根农杆菌的转化：选用发根农杆菌菌株K599，将载体pCAMBIA1301-PGT2-Egfp转化到K599菌株中，将转化后含有目的基因和Egfp绿色荧光蛋白质粒的K599菌株，通过烟草瞬时表达进行验证，见图3。挑选含有目的质粒的农杆菌，涂布接种到的LB固体平板中(含有50mg/L卡那霉素(Kan)和50mg/L链霉素(Stre))，在28℃下暗培养48h，挑取单菌落于100ml液体LB中(含有50mg/L Kan和50mg/L Stre)，28℃，200rpm摇至OD600值＝1.5，将菌液置于50ml离心管内，室温，5500rpm离心5min去上清，将附在离心管壁上的菌体等体积重悬至pH＝5.6的含有乙酰丁香酮即Ace 的MES-KOH重悬液中，Ace的使用浓度为0.15mM，重悬后的液体转移至适当的容器中用来侵染，将农杆菌侵染液室温静置半小时以上即可用于侵染。MES-KOH重悬液:添加MES即MES一水合物和MgCl2即氯化镁，MES使用浓度为10mM，MgCl2使用浓度为10mM，pH＝5.6。
4)侵染及共培养：将准备好的苹果叶片放入农杆菌侵染液中，通过真空渗透处理的方式，将苹果叶片用重悬液浸泡，抽真空处理，在0.09Mpa压强下浸泡15min。在超净工作台中用无菌滤纸将叶片表面的菌液吸干，将农杆菌侵染后的叶片集中转移至共培养培养基中，23±2℃黑暗条件下培养3d。转移至共培养培养基中的叶片状态，结果见图4，叶片呈水渍状则表明侵染成功。共培养培养基：基本培养基MS(Murashige and Skoog)，添加外源激素TDZ即噻苯隆和NAA即α-萘乙酸，TDZ使用浓度为2mg/L，NAA使用浓度为0.5mg/L，再添加30g/L的蔗糖和7.5g/L的琼脂粉，调节pH为5.8，灭菌后在培养基中添加Ace即乙酰丁香酮和BT即甜菜碱，Ace的使用浓度为0.1mM，BT的使用浓度为1mM。
5)分化培养：三天后将共培养基取出，用含有250mg/L头孢霉素(Cef)的无菌水中清洗叶片2-3次，用无菌滤纸吸干叶片表面水分，将叶片转移至分化培养基上，温度25℃，光培养，光强2400lux，光周期16/8h，培养5-7周。培养基上的叶片会在见光培养4-6周后叶片表面分化出可见不定芽，将叶片转移至新的培养基上，温度25℃，光培养，光强2400lux，光周期16/8h继续培养2-3周，叶片状态见图5。分化培养基：基本培养基MS(Murashige and Skoog)，添加外源激素TDZ即噻苯隆和NAA即α-萘乙酸，TDZ使用浓度为2mg/L，NAA使用浓度为0.5mg/L，再添加30g/L的蔗糖和7.5g/L的琼脂粉，调节pH为5.8。灭菌后在培养基中添加抗生素Cef即头孢霉素，Cef的使用浓度为250mg/L。
6)荧光筛选及鉴定：首先使用UV365nm紫外灯对分化培养基上分化的不定芽进行初步的荧光筛选，将初步筛选出的不定芽置于含头孢霉素250mg/L继代培养基上培养，经过初步筛选移入继代培养基4周后不定芽的状态见图7。4周后将初步筛选获得的不定芽通过多光谱动态荧光成像系统对其进行再次荧光鉴定，将鉴定后具有荧光标记的不定芽再置于含头孢菌素继代培养基中，25℃，光强2400lux，光周期16/8h继续培养。继代培养基，见图6：基本培养基MS(Murashige and Skoog)，添加外源激素6-BA即6-苄基腺嘌呤和IAA即3-吲哚乙酸，6-BA使用浓度为1mg/L，IAA使用浓度为0.5mg/L，再添加30g/L的蔗糖和7.5g/L的琼脂粉，调节pH为5.8，灭菌后添加Cef即头孢霉素，Cef使用浓度为250mg/L。
7)生根培养：当分化培养出的再生植株长至4-6cm时移入生根培养基，先25℃，暗培养2周，2周后在光强2400lux，光周期16/8h，温度25℃的条件下继续生根培养2周左右，再生植株分化出根，将带根的再生植株种进土壤中，获得稳定遗传的转基因植株。生根培养基为：基本培养基MS(Murashige and Skoog)，添加外源激素IAA即3-吲哚乙酸和IBA即3-吲哚丁酸，IAA使用浓度为0.5mg/L，IBA使用浓度为1mg/L，再添加30g/L的蔗糖和7.5g/L的琼脂粉，调节pH为5.8。
8)转基因植株的鉴定：提取带荧光标记再生植株的DNA，对其进行PCR检测，根据载体序列和PGT2基因序列设计抗性苗的PCR检测的两个引物，分别为引物1：5’-CTCGAGATGGAGGCGACAGCTATAGTTTTATATCC-3’，引物2：5’-GATCTGGATTTTAGTACTGGATTTTGGTTTTAGGA–3’，PCR程序为：94℃预变性5min；然后94℃变性30s，56℃退火15s，72℃延伸20s，循环35次；72℃延伸10min。
9)实验结果如图8所示，所有再生植株(包括根、茎、叶)的电泳结果均表现为阳性，取其电泳产物测序表明目的基因已导入GL-3苹果的基因组中。同时，被提取DNA的部分转基因植株通过PlantView100植物活体成像系统进行再次鉴定，PlantView100植物活体成像系统激发滤光片参数设置为：480nm、发射滤光片参数设置为：520nm，部分转基因株系的荧光成像图见图9。
实施例2、使用皇家嘎拉(Royal Gala)苹果材料为出发材料，利用发根农杆菌K599建立苹果稳转体系
1)实验前期准备：建立皇家嘎拉(Royal Gala)外植体，该材料一次继代培养周期为4周，经过3-4次继代培养后的苹果植株可用于实验，挑选适合用于侵染的叶片于组培瓶中作为转基因受体。皇家嘎拉(Royal Gala)植株继续生长的继代培养基为：基本培养基MS(Murashige and Skoog)，添加外源激素6-BA，6-BA使用浓度为1mg/L，再添加30g/L的蔗糖和7.5g/L的琼脂粉，调节pH为5.8，灭菌后再加入0.5mg/L的维生素B5，在25℃温度下生长4周，转化时挑选组培瓶中长势较好的植株，取下顶端两片嫩叶以下底端老叶以上的叶片置于组培瓶中作为转基因的受体。
2)实验中所使用的发根农杆菌菌株、载体及侵染实验的流程、荧光筛选与鉴定、生根培养、转基因植株的鉴定方法步骤同实施例1一致。
3)侵染后叶片共培养：共培养时间、外部条件与以GL-3材料侵染后共培养一致。以下方法参考Yao(1995)，皇家嘎拉(Royal Gala)侵染后共培养培养基为基本培养基MS(Murashige and Skoog)，添加外源激素6-BA即6-苄基腺嘌呤和NAA即α-萘乙酸，6-BA使用浓度为5mg/L，NAA使用浓度为0.2mg/L，再添加30g/L的蔗糖和7.5g/L的琼脂粉，调节pH为5.8，灭菌后在培养基中添加0.5mg/L的维生素B5、Ace即乙酰丁香酮和BT即甜菜碱，Ace的使用浓度为0.1mM，BT的使用浓度为1mM。
4)不定芽诱导培养：培养时间、外部条件与以GL-3材料侵染后诱导培养一致，皇家嘎拉(Royal Gala)侵染后共培养培养基为基本培养基MS(Murashige and Skoog)，添加外源激素6-BA即6-苄基腺嘌呤和NAA即α-萘乙酸，6-BA使用浓度为5mg/L，NAA使用浓度为0.2mg/L，再添加30g/L的蔗糖和7.5g/L的琼脂粉，调节pH为5.8，灭菌后在培养基中添加0.5mg/L的维生素B5和250mg/L抗生素Cef即头孢霉素。使用皇家嘎拉(Royal Gala)作为受体获得的部分转基因植株见图10。
5)转基因植株的鉴定：鉴定方法以及所使用的引物与实施例1一致，结果如图11所示，部分再生植株(包括根、茎、叶)的电泳结果均表现为阳性，取其电泳产物测序表明目的基因已导入皇家嘎拉苹果的基因组中。
实施例3：基于不同发根农杆菌介导的苹果稳转体系建立
本实施例利用发根农杆菌8196、R1601和C58C1分别转化苹果转基因植株体系建立，包括以下步骤：
1)实验前期准备：以GL-3苹果材料为出发材料建立GL-3苹果植株的外植体，将建立的外植体置于培养基中培养，取下顶端两片嫩叶以下底端老叶以上的叶片置于组培瓶中作为转基因的受体。
2)转化并培养获得用于转化的菌株：载体使用pCAMBIA1301-PGT2-Egfp表达载体，将目的载体质粒使用热激法转入发根农杆菌感受态细胞8196、R1601、C58C1，涂布接种至LB固体培养基上培养，28℃暗培养48h，挑取单菌落于50ml液体LB中，28℃，200rpm摇至OD600值＝1.5，将菌液离心重悬至1.5倍体积的MES-KOH重悬液中，重悬后的液体转移至适当的容器中用来侵染，将农杆菌侵染液室温静置半小时以上即可用于侵染。
3)三种不同发根农杆菌菌株介导苹果叶片实验操作流程同实施例1，侵染后苹果叶片培养、不定芽诱导培养等步骤同实施例1一致。
4)实验结果:使用8196、R1601、C58C1发根农杆菌菌株均可诱导苹果叶片分化出带有荧光标记的转基因不定芽，详细结果参见图12、13、14。
实施例4基于不同载体的苹果稳转体系建立
1)实验前期准备：以GL-3苹果材料为出发材料，建立GL-3苹果植株的外植体，将建立的外植体置于培养基中培养，取下顶端两片嫩叶以下底端老叶以上的叶片置于组培瓶中作为转基因的受体。
2)载体的构建：创建RNAi沉默PK7WIWG2D-PGT1::GFP载体，使用pDONR222为中间载体，载体图谱见图15。
3)发根农杆菌转化、侵染及共培养、分化培养、荧光筛选与鉴定、生根培养方法同实施例1一致，最终获得部分转基因再生株系，见图16。
4)转基因植株的鉴定：提取带荧光标记再生植株的DNA，对PGT1进行DNA水平检测，根据载体序列和PGT1基因序列设计的PCR检测引物，分别为：引物3：5’-TGTTTGCAGGTCAGCTTGACACT-3’，引物4：5’-GTGACTCCCTTAATTCTCATGTATAATTCGC-3’，PCR程序为：94℃预变性5min；然后94℃变性30s，56℃退火15s，72℃延伸20s，循环35次；72℃延伸10min。
5)结果如图17所示，F1-1、F1-2均含有与目的片段大小一致的片段，即表明F1-1、F1-2株系均为PGT1-RNAi::GFP转基因株系。
实施例5、不同侵染处理方式下的苹果稳转体系建立
1)实验前期准备：实验所用材料、载体、发根农杆菌菌株同实施例1保持一致。
2)实验操作步骤：与实施例1的侵染处理方式-真空渗透处理不同，本实施例采用不同创伤处理方式，将苹果叶片使用灭菌刀片在叶片背面划3-4条1cm的伤口(对受体植物进行其他方式创伤处理也可以)，将带有伤口的叶片浸泡于侵染液中5-30分钟，5-30分钟后捞出用无菌滤纸吸干叶片表面液体，将叶片置于共培养基上培养，后续分化培养、荧光筛选与鉴定、生根培养、转基因植株鉴定同实施例1。
3)实验结果：对受体材料采用其他创伤方式进行发根农杆菌介导，虽然转化效率低于基于负压的转让方式，但也可以获得带有荧光标记的转基因不定芽，见图18。
实施例6、本申请方法与传统方法比较
1、实验前期准备：相同状态同一批苹果GL-3苗子16瓶，8瓶使用根癌农杆菌介导法侵染，其余用本申请申请保护的发根农杆菌介导法进行侵染。
2、实验所用菌株及载体：叶盘法侵染使用菌株为：GV3101根癌农杆菌菌株；发根发使用菌株为：K599发根农杆菌菌株。质粒均为载体pCAMBIA1301-PGT2-Egfp。
3、实验操作步骤：
1)叶盘法：参考Hongyan Dai(2013)，取适量叶片置于菌液中，使用灭菌刀片在叶片背面划3-4条1cm的伤口，创伤后的叶片浸于侵染液中8分钟，8分钟后捞出用无菌滤纸吸干叶片表面液体，将叶片置于共培养基上，每批实验流程重复7-8次，整个过程耗时3h，3天暗培养处理，将共培养上的叶片转移至延长培养基中暗培养4周后取出换板见光培养4-6周，创伤操作流程步骤，见图19。整个培养过程温度为22-25℃，光强2400lux，见光2-6周后统计侵染得到的转基因不定芽数量，一批15板，重复实验10次。
2)发根法：步骤如实施例1，耗时1.5h，3天暗培养处理，暗培养处理后转移至分化培养基见光2周可以分化愈伤和不定根，4周可以获得转基因不定芽，操作具体步骤，见图20。见光培养2-8周后统计转基因不定芽的数量；实验外部条件、每批数量与重复试验次数与叶盘法保持一致。
4、实验结果：
1)使用两种不同农杆菌侵染叶片，经过一定时间诱导获得带荧光标记的不定芽，通过PlantView100植物活体成像系统鉴定的荧光成像对比图，见图21。图中A为使用根癌农杆菌介导获得的不定芽通过PlantView100植物活体成像系统获得的合成图,图B为相应的明场，图C为使用发根农杆菌介导获得不定芽通过PlantView100植物活体成像系统获得的合成图，图D为相应的明场,图中除了未标记但仍有荧光的是具有分化能力但是还未分化出可见芽的愈伤组织，其中使用根癌农杆菌介导获得的不定芽为单生的不定芽，发根农杆菌介导的获得的部分不定芽为簇生的不定芽。
2)使用根癌农杆菌介导法进行十批仅有两批获得了转基因不定芽，共计3个。使用本申请的发根农杆菌介导法进行的十批侵染每一批次都有转基因不定芽，共获得转基因不定芽超过300个(该数字为肉眼可清晰分辨进行统计的相对较大的不定芽，其他簇生的较小的不定芽未统计)。最终获得可稳定遗传的转基因不定芽的效率提高了100倍以上。

参考文献：
[1]白永廷,唐惕,许智宏.根癌农杆菌(Agrobacterium tumefaciens)的Ti质粒[J].细胞生物学杂志,1984.
[2]曹庆丰.发根农杆菌K599侵染黄瓜形成转基因毛状根的初步研究及orf14基因的克隆[D].杭州师范大学,2012.
[3]丁雪.发根农杆菌介导的银中杨茎段遗传转化体系的建立及生理生化指标的测定[D].吉林师范大学,2017.
[4]方信武,冯凤娟,张良智.转基因技术在苹果遗传育种改良中的应用[J].生物技术世界,2014.
[5]胡菊.发根农杆菌介导朱砂根遗传转化体系的建立及发根中三萜皂苷的检测[D].四川农业大学,2015.
[6]李想韵.发根农杆菌介导桑遗传转化体系的建立及槲皮素在发根中的含量测定[D].西南大学,2010.
[7]吴萼,张瑞,张超,朱瑶,李晨曦,张娜娜,谌容,葛晓萍,谢恬,殷晓浦.发根农杆菌Ri质粒介导的植物基因工程及应用[J].杭州师范大学学报(自然科学版),2018
[8]王裕鹏.根癌农杆菌Ti质粒简介[J].生物学教学,2018,43(06):65-66.
[9]王艳.发根农杆菌介导八宝景天遗传转化体系的建立[D].河北农业大学,2009.
[10]肖璇.发根农杆菌介导的柑橘遗传转化体系建立及转基因柑橘溃疡病抗性分析[D].华中农业大学,2014.
[11]张改娜,贾敬芬.骆驼刺发根农杆菌转化系的原生质体培养和植株再生[J].植物生理学通讯,2009.
[12]张程程.发根农杆菌介导的棉花遗传转化的研究[D].中国农业科学院,2011.
[13]Dai H,Li W,Han G,et al.Development of a seedling clone with high regeneration capacity and susceptibility to Agrobacterium in apple[J].Scientia Horticulturae,2013,164(Complete):202–208。
[14]Yao JL,Cohen D,Atkinson R,Richardson K,Morris B.Regeneration of transgenic plants from the commercial apple cultivar Royal Gala.Plant Cell Rep.1995Apr；14(7):407-12.doi:10.1007/BF00234044.PMID:24185446。
前述对本申请的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本申请限定为所公开的精确形式，并且很显然，根据上述教导，可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本申请的特定原理及其实际应用，从而使得本领域的技术人员能够实现并利用本申请的各种不同的示例性实施方案以及各种不同的选择和改变。本申请的范围意在由权利要求书及其等同形式所限定。

Claims (10)

  1. 一种苹果或苹果属的不定芽诱导培养方法，其特征在于，包括如下步骤：
    1)转基因受体制备：所述受体为苹果叶片；
    2)表达载体构建；
    3)农杆菌的转化；
    4)叶片侵染及共培养；
    5)不定芽诱导培养。
  2. 根据权利要求1所述的方法，其特征在于，所述步骤3)中，所述农杆菌为发根农杆菌；优选的，所述发根农杆菌包括但不限于发根农杆菌K599、发根农杆菌8196、发根农杆菌R1601或发根农杆菌C58C1。
  3. 根据权利要求1所述的方法，其特征在于，所述步骤5)中，所述不定芽诱导培养获得不定芽，不获得毛状根。
  4. 根据权利要求1-3任一所述的方法，其特征在于，
    所述1)中的叶片为嫩叶片；
    优选的，所述步骤1)制备步骤为：选苹果嫩枝建立苹果植株外植体，外植体继代3-4次，取顶端嫩叶以下底端老叶以上的组培苗叶片作为转基因的受体。
  5. 根据权利要求1-4任一所述的方法，其特征在于，
    所述步骤2)中的载体构建为构建包含目的基因和/或标记基因的表达载体；
    优选的，所述表达载体包括但不仅限于pCAMBIA1301载体或RNAi沉默PK7WIWG2D-PGT1::GFP载体。
  6. 根据权利要求1-5任一所述的方法，其特征在于，
    所述步骤3)中的转化为将重组表达载体转化到发根农杆菌中，暗培养后至含有乙酰丁香酮的MES-KOH重悬液中进行侵染；
    优选的，所述MES-KOH重悬液为：8-12mM MES一水合物,5-15mM氯化镁,0.05-0.3mM乙酰丁香酮，pH＝5.3-5.6；
    更优选的，所述步骤3)转化步骤为：将重组表达载体转化到发根农杆菌中，在含有卡那霉素和链霉素的LB固体平板上，19-30℃暗培养13d，挑取单菌落于含有卡那霉素和链霉素的液体LB中，25-28℃，150-220rpm摇至OD600值达0.8-1.5范围，室温5000-7000rpm离心去上清，将菌体重悬至含有乙酰丁香酮的MES-KOH重悬液中侵染。
  7. 根据权利要求1-6任一所述的方法,其特征在于，
    所述步骤4)中侵染采用包括但不仅限于传统划伤或真空渗透；
    优选的，所述真空渗透方式进行侵染的步骤为：将组培苗叶片置于侵染液中，0.04-0.1Mpa压力下真空处理，之后将叶片转移至含有乙酰丁香酮和甜菜碱的共培养培养基上培养；
    更优选的，所述真空渗透方式进行侵染的步骤为：将组培苗叶片置于侵染液中，真空渗透时间1-60min，真空度0.04-0.1Mpa下进行渗透处理，真空渗透处理后，用无菌滤纸将叶片表面菌液吸干，将叶片转移至含有乙酰丁香酮和甜菜碱的共培养培养基上，叶片背面朝上正面紧贴培养基进行培养，培养基置于温度25±5℃的条件下，黑暗培养1-3d。
  8. 根据权利要求1-7任一所述的方法,其特征在于，
    所述步骤5)不定芽诱导培养为，将经共培养的苹果叶片转移至不定芽分化培养基，见光培养获得不定芽；
    优选的，所述不定芽分化培养基为：MS基础培养基中添加一定浓度的噻苯隆TDZ和/或6-苄基腺嘌呤6-BA，和α-萘乙酸NAA；
    更优选的，所述不定芽分化培养基为：MS基础培养基+0-5mg/L的噻苯隆TDZ和/或0-5mg/L的6-苄基腺嘌呤+0.05-3mg/L的α-萘乙酸NAA+25-35g/L蔗糖或35-45g/L山梨醇+6-8g/L的琼脂粉或2-3g/L植物凝胶，pH＝5.6-6.0；
    进一步优选的，所述步骤5)不定芽诱导培养的具体步骤为：将经共培养的苹果叶片转移至分化培养基，在光强500-5000lux，光周期12-18h光照/12-6h黑暗，温度25±5℃的条件下进行不定芽诱导培养，培养2-8周获得不定芽。
  9. 一种苹果及苹果属稳定遗传转基因体系的构建方法，其特征在于，所述方法包括权利要求1-7任一所述方法，并进一步包括如下步骤：
    6)转基因不定芽的筛选和鉴定；
    7)转基因植株生根与移栽。
  10. 根据权利要求8所述的方法，其特征在于，
    所述步骤6)的转基因不定芽的筛选与鉴定可基于荧光标记的筛选和鉴定；
    所述步骤7)转基因植株生根与移栽步骤为：把分化培养出的不定芽移入生根培养基，在光强500-5000lux，光周期12-18/12-6h，温度25±5℃的条件下进行生根培养，获得再生植株，将带根的再生植株移栽至土壤中，获得稳定遗传的完整转基因植株。
PCT/CN2023/127030 2022-10-28 2023-10-27 一种利用发根农杆菌高效快速获得苹果稳定转基因植株的方法 WO2024088372A1 (zh)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101457235A (zh) * 2008-08-05 2009-06-17 山东省林业科学研究院 一种真空渗透辅助农杆菌介导转化苜蓿的方法
CN102031270A (zh) * 2009-09-30 2011-04-27 西北农林科技大学 利用农杆菌介导高效转化体系培养苹果幼苗的方法
US20120137390A1 (en) * 2009-05-07 2012-05-31 Arborgen Inc. Materials and methods for regeneration and transformation of trees
CN105463014A (zh) * 2015-12-30 2016-04-06 中国农业科学院郑州果树研究所 RNAi抗苹果茎痘病毒表达载体及其在苹果抗病育种中的应用
CN114736920A (zh) * 2022-03-01 2022-07-12 中国计量大学 农杆菌侵染菜豆叶片实现目的基因瞬时过表达或瞬时沉默的方法
CN115896160A (zh) * 2022-10-28 2023-04-04 西北农林科技大学 一种利用发根农杆菌高效快速获得苹果稳定转基因植株的方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9600698D0 (en) * 1996-01-13 1996-03-13 British American Tobacco Co Genetic transformation of trees
CN110100735A (zh) * 2019-06-17 2019-08-09 山东农业大学 一种组培嘎啦苹果苗不定根离体再生不定芽的方法
CN111690678B (zh) * 2020-05-20 2023-04-07 山东农业大学 一种利用发根农杆菌转化获得整株转基因木本植株的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101457235A (zh) * 2008-08-05 2009-06-17 山东省林业科学研究院 一种真空渗透辅助农杆菌介导转化苜蓿的方法
US20120137390A1 (en) * 2009-05-07 2012-05-31 Arborgen Inc. Materials and methods for regeneration and transformation of trees
CN102031270A (zh) * 2009-09-30 2011-04-27 西北农林科技大学 利用农杆菌介导高效转化体系培养苹果幼苗的方法
CN105463014A (zh) * 2015-12-30 2016-04-06 中国农业科学院郑州果树研究所 RNAi抗苹果茎痘病毒表达载体及其在苹果抗病育种中的应用
CN114736920A (zh) * 2022-03-01 2022-07-12 中国计量大学 农杆菌侵染菜豆叶片实现目的基因瞬时过表达或瞬时沉默的方法
CN115896160A (zh) * 2022-10-28 2023-04-04 西北农林科技大学 一种利用发根农杆菌高效快速获得苹果稳定转基因植株的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
D. J. JAMES, A. J. PASSEY, S. PREDIERI, E. RUGINI: "Regeneration and Transformation of Apple Plants Using Wild-Type and Engineered Plasmids in Agrobacterium SPP", SOMATIC CELL GENETICS OF WOODY PLANTS : PROCEEDINGS OF THE IUFRO WORKING PARTY S2. 04-07 SOMATIC CELL GENETICS, HELD IN GROSSHANSDORF, FEDERAL REPUBLIC OF GERMANY, AUGUST 10 - 13, 1987, KLUWER, NL, 31 July 1988 (1988-07-31) - 13 August 1987 (1987-08-13), NL, pages 65 - 71, XP009556117, ISBN: 90-247-3728-1, DOI: 10.1007/978-94-009-2811-4_9 *

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