CN115896160A - Method for efficiently and quickly obtaining stable transgenic plants of apples by utilizing agrobacterium rhizogenes - Google Patents
Method for efficiently and quickly obtaining stable transgenic plants of apples by utilizing agrobacterium rhizogenes Download PDFInfo
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- CN115896160A CN115896160A CN202211342021.2A CN202211342021A CN115896160A CN 115896160 A CN115896160 A CN 115896160A CN 202211342021 A CN202211342021 A CN 202211342021A CN 115896160 A CN115896160 A CN 115896160A
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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
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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
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
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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
- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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)
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Abstract
The application belongs to the technical field of plant genetic engineering, and particularly discloses a method for efficiently and quickly obtaining an apple stable transgenic plant by utilizing agrobacterium rhizogenes. The method has the advantages of stable system, high conversion rate, simplicity in operation, time saving and the like.
Description
Technical Field
The application belongs to the technical field of plant genetic engineering, and particularly relates to a method for efficiently and quickly obtaining stable transgenic plants of apples by using leaves of apple plants as receptors and utilizing agrobacterium rhizogenes.
Background
There are two main types of agrobacterium: agrobacterium tumefaciens and Agrobacterium rhizogenes. The Agrobacterium plasmid is a natural system that enables DNA transfer and integration, and the most commonly used plasmids in plasmid vector systems are the Ti and Ri plasmids. The Ti plasmid is present in Agrobacterium tumefaciens (Agrobacterium tumefaciens), and the Ri plasmid is present in Agrobacterium rhizogenes (Agrobacterium rhizogenes). The Ti and Ri plasmids share many similarities in structure and function, with essentially identical properties. The Ti plasmid is between 160 and 240kB, the Ti plasmid is a region which is transferred and integrated into a plant receptor genome through a T-DNA region and can be expressed in a plant cell to cause crown gall and can be transmitted to a progeny cell through meiosis, the length of the T-DNA is between 12 and 24kB, two ends of the T-DNA are respectively provided with a boundary sequence containing a 25bp repetitive sequence, the T-DNA between the left and right boundary sequences can be transferred and integrated into the genome of the host cell during the integration process, and the research finds that only the boundary sequence is necessary for transferring the DNA, and the T-DNA between the boundary sequences does not participate in the transformation process, so that the boundary sequence can be replaced by an exogenous gene, and the exogenous gene can be introduced into the genome of the host (Bai Yongpeng et al 1984; wang Kung 2018); the agrobacterium rhizogenes infection can induce generation of hairy roots (Hair Root), which is caused by Ri (Root Inducing) Plasmid contained in the agrobacterium rhizogenes, wherein the Ri Plasmid is Root Inducing Plasmid; the Ri plasmid has a size of 200-800 kb, contains genes responsible for autonomous growth of hairy roots and opine synthesis, and has a structural virulence region (Vir region), a T-DNA region for transfer into plant cell nuclei, and an internal opine synthesis functional region. Ri plasmid is transferred into infected plant through T-DNA, so that T-DNA on the agropine type Ri plasmid generating hairy roots is discontinuously distributed, genes on TL-DNA, TR-DNA and TL-DNA are related to the formation of hairy roots, and the aboveground morphology and some physiological characters of regenerated plants are influenced (Wu calyx et al 2018).
At present, most of plant agrobacterium tumefaciens-mediated plant genetic transformation is mediated mainly by agrobacterium tumefaciens containing a target gene to finally obtain a transgenic regeneration plant. Taking apple transgenosis as an example, the leaf disc method is mainly adopted at present, agrobacterium tumefaciens is used for infecting leaves of an apple explant, the leaves are co-cultured in a short period, and an apple transgenic strain (Fujian et al 2014) is obtained by induction under different conditions in the later period. In a plant transgenic system, a mode also exists for obtaining transgenic hairy roots by using agrobacterium rhizogenes mediated plants, mainly using plant stalks as receptors and obtaining the transgenic hairy roots by using the agrobacterium rhizogenes mediated. However, this method has a limitation that only the obtained hairy root is transgenic material, and other tissues such as leaves and stems are still non-transgenic material, so that transgenic plants with stable inheritance cannot be obtained (Caoqing 2012; dixue 2017; xiaozu 2014). If a transgenic line capable of being stably inherited is further obtained by utilizing the obtained transgenic hairy roots, the hairy roots need to be subjected to isolated tissue culture, the hairy roots are firstly subjected to dedifferentiation induction to form callus, and then are subjected to redifferentiation induction to obtain transgenic regenerated plants (Lixiangyun 2010; wangyang 2009; zhangcheng 2011). However, the conditions for the isolated tissue culture of the hairy roots, which are used for obtaining transgenic regeneration plants capable of being stably inherited by the method, are further explored, the steps are complicated, and the time period is long. Therefore, it is very important to develop a method for efficiently and rapidly obtaining transgenic plants capable of being stably inherited by utilizing agrobacterium rhizogenes mediation.
In view of this, the present application is presented.
Disclosure of Invention
The application aims at solving the problems in the prior art, the application overcomes the traditional prejudice of agrobacterium rhizogenes, pioneering apple leaves are used as an acceptor, agrobacterium rhizogenes is used for mediating, and stable genetic transgenic adventitious buds are obtained through adventitious bud induction culture, namely, the stable genetic apple transgenic strain can be efficiently and quickly obtained by the mode of 'agrobacterium rhizogenes + leaf infection + induction culture of adventitious buds' for the first time.
Specifically, agrobacterium rhizogenes is used for mediating, the apple leaves which are in a better state and easy to infect are used as receptors for infecting, the infected leaves are co-cultured and differentiated to obtain a large number of adventitious buds with differentiated leaves, the adventitious buds are continuously cultured, the leaves are taken, and the regenerated plants of the transgenic apples are determined through PCR molecular detection. Compared with the reported agrobacterium rhizogenes-mediated apple transgenic method, the method disclosed by the application obtains stable genetic adventitious buds instead of hairy roots, and the stable genetic transgenic plant can be obtained after the adventitious buds are induced to root in the later stage. Has the advantages of simplicity, high efficiency, short culture period and the like.
The application specifically provides the following technical scheme:
the application firstly provides a quantitative adventitious bud induction culture method for apples or malus, which comprises the following steps:
1) Preparation of transgenic receptor: the receptor is apple leaves;
2) Constructing an expression vector;
3) Transforming agrobacterium;
4) Leaf infection and co-culture;
5) And (4) performing adventitious bud induction culture.
Further, the leaves in the step 1) are tender leaves;
preferably, the preparation step in the step 1) is specifically as follows: selecting apple twigs to establish an apple plant explant, carrying out subculture on the explant for 3-4 times, and taking tissue culture seedling leaves below top tender leaves and above bottom old leaves as transgenic receptors.
Further, the vector in the step 2) is constructed to construct an expression vector containing the target gene and/or the marker gene;
preferably, the marker gene is a fluorescent marker gene;
preferably, the expression vector includes but is not limited to pCAMBIA1301 vector or RNAi silencing PK7WIWG2D-PGT1:: GFP vector;
in some specific ways, the constructing step is specifically: plasmid pCAMBIA1301 is adopted as an original vector, hygromycin phosphotransferase gene and glucuronidase gene GUS are replaced by a target gene and a fluorescence marker gene, and a recombinant expression vector is obtained.
Further, in the step 3), the agrobacterium is agrobacterium rhizogenes;
preferably, the agrobacterium rhizogenes includes but is not limited to agrobacterium rhizogenes K599, agrobacterium rhizogenes 8196, agrobacterium rhizogenes R1601 or agrobacterium rhizogenes C58C1.
In some specific modes, the conversion in the step 3) is to convert the recombinant expression vector into agrobacterium rhizogenes, and the recombinant expression vector is infected into MES-KOH resuspension containing acetosyringone after dark culture;
preferably, the MES-KOH resuspension is: 8-12mM MES monohydrate, 5-15mM magnesium chloride, 0.05-0.3mM acetosyringone, pH =5.3-5.6;
in some more specific ways, the step 3) conversion step is specifically: transforming the recombinant expression vector into agrobacterium rhizogenes, performing dark culture at 19-30 ℃ for 13d on an LB solid plate containing kanamycin and streptomycin, selecting a single colony in liquid LB containing kanamycin and streptomycin, shaking at 25-28 ℃ and 150-220rpm until the OD600 value reaches 0.8-1.5 range, centrifuging at the room temperature of 5000-7000rpm to remove a supernatant, and suspending the thallus into MES-KOH heavy suspension containing acetosyringone for infection.
Further, the dip dyeing in the step 4) is carried out by adopting a traditional scratching or vacuum infiltration mode;
preferably, the infection is carried out in a vacuum infiltration mode, agrobacterium rhizogenes is introduced into apple leaf cells, and the T-DNA of a target gene carried by the agrobacterium rhizogenes is inserted into a plant genome;
in some specific modes, the vacuum infiltration mode carries out the infection step as follows: placing the leaves of the tissue culture seedling in an infection solution, performing vacuum treatment under the pressure of 0.04-0.1Mpa, and transferring the leaves to a co-culture medium containing acetosyringone and betaine for culture;
in some more specific forms, the vacuum infiltration form performs the step of infecting by: placing the leaves of the tissue culture seedling in an infection solution, carrying out infiltration treatment under the conditions of vacuum infiltration time of 1-60min and vacuum degree of 0.04-0.1Mpa, after vacuum infiltration treatment, sucking bacterial liquid on the surfaces of the leaves by using sterile filter paper, transferring the leaves to a co-culture medium containing acetosyringone and betaine, culturing the leaves with the back faces upwards and the front faces tightly attached to the culture medium, and culturing the culture medium in the dark at the temperature of 25 +/-5 ℃ for 1-3d.
Preferably, the co-culture medium is MS basal medium added with thidiazuron TDZ and/or 6-benzyladenine with certain concentration and alpha-naphthylacetic acid NAA; more preferably, the components are specifically: MS +0-5mg/L thidiazuron TDZ +0.05-3mg/L alpha-naphthylacetic acid NAA +25-35g/L sucrose or 35-45g/L sorbitol +6-8g/L agar powder or 2-3g/L plant gel, 0.05-0.3mM acetosyringone, 0.8-1.2mM betaine, and pH =5.6-6.0.
Further, the adventitious bud is obtained by the adventitious bud induction culture in the step 5), and a hairy root is not obtained; the step 5) of adventitious bud induction culture comprises the steps of transferring the co-cultured apple leaves to an adventitious bud differentiation culture medium, and performing visible light culture to obtain adventitious buds;
preferably, the adventitious bud differentiation medium is an MS basal medium added with thidiazuron TDZ and/or 6-benzyladenine with certain concentration and alpha-naphthylacetic acid NAA; more preferably, the components are specifically: MS +0-5mg/L thidiazuron TDZ +0.05-3mg/L alpha-naphthylacetic acid NAA +25-35g/L sucrose or 35-45g/L sorbitol +6-8g/L agar powder or 2-3g/L plant gel, and the pH is =5.6-6.0;
in some specific modes, the step 5) adventitious bud induction culture comprises the following specific steps: transferring the co-cultured apple leaves to a differentiation culture medium, performing adventitious bud induction culture under the conditions of light intensity of 500-5000lux, photoperiod of 12-18/12-6h and temperature of 25 +/-5 ℃, and culturing for 2-8 weeks to obtain adventitious buds.
The application also provides a method for constructing the stable genetic transgenic system of the apples and the malus, the method comprises any one of the steps of the method, and further comprises the following steps:
6) Screening and identifying the transgenic adventitious bud;
7) And (5) rooting and transplanting the transgenic plant.
Further, the screening and identification of the transgenic adventitious bud of the step 6) is based on the screening and identification of a fluorescent label;
preferably, the screening and identifying steps of the transgenic adventitious bud in the step 6) are specifically as follows: and (3) carrying out primary fluorescence screening on adventitious buds differentiated from leaves on a differentiation culture medium by using an ultraviolet lamp, placing the primarily screened adventitious buds on a subculture medium containing the cefuromycin for culturing, carrying out secondary fluorescence identification on the primarily screened adventitious buds after 2-4 weeks, and placing the identified adventitious buds with fluorescence labels in the subculture medium containing the cefuromycin for continuous culture.
Further, the specific steps of rooting and transplanting the transgenic plant in the step 7) are as follows: transferring the adventitious bud obtained by differentiation culture into a rooting culture medium, carrying out rooting culture under the conditions of light intensity of 500-5000lux, photoperiod of 12-18h illumination/dark of 12-6h and temperature of 25 +/-5 ℃ to obtain a regenerated plant, and transplanting the regenerated plant with roots into soil to obtain a complete transgenic plant with stable heredity;
preferably, the rooting medium is: MS +0.1-2.0 mg/L3-indoleacetic acid +0.02-2 mg/L3-indolebutyric acid +25-35g/L sucrose or 35-45g/L sorbitol +6-8g/L agar powder or 2-3g/L plant gel, and the pH =5.6-6.0.
Further, the method may further include the steps of:
8) Molecular level identification of transgenic plants:
and (3) carrying out DNA level detection on the obtained regenerated plant with the fluorescent marker by using a PCR method, determining that the target gene is integrated into the apple genome DNA, and finally determining the transgenic plant.
Compared with the prior art, the method has at least the following obvious technical advantages:
1) The existing method only can directly obtain transgenic hairy roots by utilizing the infection of agrobacterium rhizogenes, other tissues such as stems, leaves and the like cannot be infected by the agrobacterium rhizogenes, the tissues are still not transgenic tissues, materials of the transgenic tissues cannot be stably inherited, further in vitro tissue culture needs to be carried out on the transgenic hairy roots to induce the root systems to dedifferentiate and generate calluses, the calluses are redifferentiated and generate adventitious buds (the process is extremely difficult), and then the adventitious buds are subjected to subculture and rooting to obtain complete transgenic plants with stable inheritance capability. The technical process is complicated, the time period is long, and the efficiency is low. The agrobacterium rhizogenes is used for infecting the leaves, adventitious buds are directly obtained through co-culture and differentiation culture instead of the hairy roots of the previous people, and the obtained transgenic adventitious buds can obtain a complete transgenic plant with stable genetic ability after subculture and rooting. By using the method, the adventitious bud can be directly obtained by crossing the transgenic hairy root and the subsequent process of the dedifferentiation and differentiation of the hairy root, so that a complete transgenic plant can be obtained.
2) The existing method for infecting apple leaves by utilizing agrobacterium tumefaciens has low probability of obtaining transgenic adventitious buds capable of being stably inherited and complicated operation steps. For example, in the culture stage after the infection of the leaves, after the infection of the leaves by the agrobacterium tumefaciens, dark culture is required for 4 weeks, and then visible culture is required for 2-6 weeks to obtain the transgenic adventitious buds. However, according to the method, the infected apple leaves are dark-cultured for 3d and then directly transferred to a differentiation medium for 2-6 weeks to obtain the transgenic adventitious buds. Therefore, the steps of the genetic transformation method are more simplified, the whole experiment period is shortened by about half, and simultaneously the investment of resources such as manpower, material resources, financial resources and the like is greatly reduced.
3) The application overcomes the traditional technical concept, creatively uses apple leaves as a receptor, and obtains the stably inherited transgenic adventitious bud by utilizing the mediation of agrobacterium rhizogenes. Taking GL-3 apple transgenosis mentioned in the application as an example, when a transgenic plant is required to be obtained by using a traditional leaf disc method (mediated by agrobacterium tumefaciens), apple leaves are subjected to wound infection in a disposable culture dish containing an infection solution, so that the method can be repeatedly carried out a small amount of times, the technical requirement is high, the experiment steps are complicated, the time is long, the number of obtained transgenic adventitious buds is small, and the efficiency is low. However, by using the method for obtaining the transgenic plants by utilizing the agrobacterium rhizogenes mediation, a large number of leaves can be immersed into a tissue culture bottle (or a larger container) containing bacterial liquid at one time, the leaves do not need to be wounded, and the agrobacterium infection is carried out in a vacuum infiltration treatment mode. By using the vacuum infiltration treatment mode, as the vacuum drying vessel has large volume and can hold a plurality of tissue culture bottles (or larger containers), synchronous infection of a plurality of different transgenes can be realized. The method has the advantages of simple and convenient steps, time saving, large number of transgenic adventitious buds capable of being stably inherited and obtained through one experiment, high probability and great improvement of transformation efficiency.
Drawings
In order to more clearly illustrate the detailed description of the present application or the technical solutions in the prior art, the drawings used in the detailed description or the prior art description will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a diagram of a suitable infestation growth state of plant GL-3 apple tissue culture seedlings as used herein;
FIG. 2 is a vector map of the plasmid pCAMBIA1301-PGT2-Egfp of the present application;
FIG. 3 is a fluorescence imaging diagram of the plasmid pCAMBIA1301-PGT2-Egfp of the present application after tobacco transient expression verification; in the figure, G: a bacterial strain K599 containing pCAMBIA1301-PGT2-Egfp plasmid; ck: strain K599 containing pCAMBIA1301 plasmid;
FIG. 4 is a state diagram of leaf transfer to co-cultivation medium after infection according to the present application;
FIG. 5 is a diagram showing the infected leaves of the present application cultured in differentiation medium for 4 weeks under light;
FIG. 6 is a state diagram of part of adventitious buds differentiated from infected leaves of the present application, which are transferred to a subculture medium for continuous culture for 4 weeks after primary fluorescence screening under ultraviolet light;
FIG. 7 is a fluorescence image of a part of the adventitious buds obtained in the present application identified by a PlantView100 plant in vivo imaging system, the left image is a composite image, and the right image is a brightfield;
FIG. 8 shows the PCR detection results of the partially transgenic plants obtained in the present application; in the figure, M: DL2000DNA Marker; CK: untransformed plants; 1-11: transforming plants;
FIG. 9 is a fluorescence image of a part of the transgenic lines obtained in the present application, identified by the plantaVIEW 100 plant in vivo imaging system, the left image being a synthetic image and the right image being a bright field;
FIG. 10 is a fluorescence image of a part of the transgenic lines obtained in example 2 of the present application identified by a plant Living body imaging System of PlantView100, with the left image being a bright field and the right image being a synthetic image;
FIG. 11 shows the PCR detection result of the partial transgenic plant obtained in example 2 of the present application; in the figure, M: DL2000DNA Marker; CK: (ii) untransformed plants; 1. 2: obtaining partially transformed plants
FIG. 12 is a fluorescence imaging diagram of transgenic adventitious buds obtained by mediating GL-3 leaves using C58C1 Agrobacterium rhizogenes strain in the present application identified by the plant View100 in vivo imaging system, the left panel is a synthetic diagram, and the right panel is a bright field;
FIG. 13 is a fluorescence imaging diagram of transgenic adventitious buds obtained by mediating GL-3 leaves using C58C1 Agrobacterium rhizogenes strain in the present application identified by the plant View100 in vivo imaging system, the left panel is a synthetic diagram, and the right panel is a bright field;
FIG. 14 is a fluorescence imaging diagram of transgenic adventitious buds obtained by mediating GL-3 leaves using C58C1 Agrobacterium rhizogenes strain in the present application identified by the plant View100 in vivo imaging system, the left panel is a synthetic diagram, and the right panel is a bright field;
FIG. 15 is a GFP vector map of PK7WIWG2D-PGT1 used in example 4 of the present application;
FIG. 16 is a comparison of the phenotype of the partially transgenic lines obtained by way of example 4 with that of the wild type in the present application.
FIG. 17 shows the results of PCR detection of the GFP apple transgenic line in PGT1-RNAi obtained in example 4 of the present application; in the figure, M: DL2000DNA Marker; CK: (ii) untransformed plants; f1-1, F1-2: transforming plants;
FIG. 18 is a fluorescence imaging diagram of transgenic adventitious shoots obtained by invasion of GL-3 leaves mediated by other wound modes and identified by a plant in vivo imaging system of PlantView100 in example 5 of the present application, wherein the left diagram is a synthetic diagram and the right diagram is a bright field;
FIG. 19 is a flowchart showing the operation of example 6 in the present application, which employs the conventional Agrobacterium tumefaciens-mediated apple transgenic technology, the "leaf disk method";
FIG. 20 is a flow chart of the operation of the present application using Agrobacterium rhizogenes-mediated apple transgene technology;
FIG. 21 is a comparative fluorescence imaging plot identified by the plantaeview 100 plant in vivo imaging system using the capabilities of the transgenic adventitious shoots obtained using Agrobacterium tumefaciens and Agrobacterium rhizogenes mediation in example 6 of the present application, A is a synthetic plot obtained by the plantaeview 100 plant in vivo imaging system using the adventitious shoots obtained using Agrobacterium tumefaciens mediation, B is the corresponding brightfield, C is a synthetic plot obtained by the plantaeview 100 plant in vivo imaging system using the adventitious shoots obtained using Agrobacterium rhizogenes mediation, and D is the corresponding brightfield.
Detailed Description
The technical solutions of the present application will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The following terms or definitions are provided solely to aid in the understanding of the present application. These definitions should not be construed to have a scope less than understood by those skilled in the art.
Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present application are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.
As used in this application, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of 8230A" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.
Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.
The terms "about" and "substantially" in this application denote the interval of accuracy that a person skilled in the art can understand while still guaranteeing the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.
Furthermore, the terms first, second, third, (a), (b), (c) and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.
The method comprises an adventitious bud induction culture method for apples and malus and a construction method for a stable genetic transgenic system for the apples and the malus, and is based on overcoming the traditional use prejudice for agrobacterium rhizogenes, creatively uses apple leaves as a receptor, utilizes agrobacterium rhizogenes to mediate, and obtains stable genetic transgenic adventitious buds through adventitious bud induction culture, namely, an apple transgenic plant line capable of being stably inherited is efficiently and quickly obtained by a mode of 'agrobacterium rhizogenes, leaf infection and induction culture of adventitious buds' for the first time. Therefore, theoretically, all the basic steps including the above-mentioned "Agrobacterium rhizogenes + leaf infection + adventitious bud induction" are included in the scope of protection of the present application.
Specific examples are as follows. The technical contents of the present application will be described in detail by taking the PGT2 gene marker related to asexual shape of phyllanthus trifoliatus of the genus malus and the establishment of an auxiliary breeding technique thereof as an example, which is only used for explaining the method idea of the present application and is not intended to limit the protection contents. Those skilled in the art can expect that the selection of the variety of apple, the variety of agrobacterium rhizogenes, the variety of vector, the target gene and the marker gene, etc., will not affect the ability of the core basic method of the present application to induce a stable genetic transgenic system; also, the selection or condition parameter setting of some basic media is not limited, for example, when the differentiation culture is required, the corresponding specific differentiation media and condition parameters can be selected by the field for the differentiation culture.
Example 1 establishment of Stable apple turning line Using GL-3 apple Material as starting Material and Agrobacterium rhizogenes K599
1) In the embodiment, GL-3 apple material is used as a starting material, and GL-3 apple tissue culture seedling leaves are selected as follows: selecting tender branches of 3 and 4 months in the current year, establishing an explant of a GL-3 apple plant, placing the established explant in a culture medium for culture, carrying out subculture every 4 weeks for 3-4 times, wherein the subculture medium for continuous growth of the plant is as follows: adding exogenous hormones 6-BA (6-benzyladenine) and IAA (3-indoleacetic acid) into a basic culture medium MS (Murashige and Skoog), wherein the using concentration of the 6-BA is 1mg/L, the using concentration of the IAA is 0.5mg/L, adding 30g/L of cane sugar and 7.5g/L of agar powder, adjusting the pH to 5.8, growing for 4 weeks at the temperature of 25 ℃, selecting a plant with better growth vigor in a tissue culture bottle during transformation, and taking two leaves below a top tender leaf and above a bottom old leaf to place in the tissue culture bottle as a transgenic receptor as shown in figure 1.
2) Construction of the vector: the plasmid pCAMBIA1301 is used as an original vector, the plasmid pCAMBIA1301 contains hygromycin phosphotransferase gene HPT of CaMV35S promoter and glucuronidase gene GUS, the vector is modified, single enzyme digestion is carried out by using XhoI as an enzyme digestion site, and the hygromycin phosphotransferase gene is replaced by the used target gene PGT2. Secondly, in order to facilitate the subsequent screening of transgenic plants, double enzyme digestion is carried out by using NcoI and BstEII, glucuronidase gene GUS is replaced by Egfp enhanced green fluorescent protein, and a vector pCAMBIA1301-PGT2-Egfp is obtained, wherein a map obtained after vector modification is shown in figure 2.
3) And (3) transforming agrobacterium rhizogenes: an agrobacterium rhizogenes strain K599 is selected, a vector pCAMBIA1301-PGT2-Egfp is transformed into the K599 strain, and the transformed K599 strain containing a target gene and an Egfp green fluorescent protein plasmid is verified through tobacco transient expression, which is shown in figure 3. Agrobacterium containing the desired plasmid was selected, spread on an LB solid plate (containing 50mg/L kanamycin (Kan) and 50mg/L streptomycin (Stre)), cultured in the dark at 28 ℃ for 48 hours, and a single colony was picked up in 100ml of liquid LB (containing 50mg/L Kan and 50mg/L Stre), shaken at 28 ℃ and 200rpm to OD 600 Value =1.5, willPlacing the bacterial liquid in a 50ml centrifugal tube, centrifuging at room temperature and 5500rpm for 5min to remove supernatant, resuspending thalli attached to the wall of the centrifugal tube in equal volume to MES-KOH resuspension liquid containing acetosyringone, namely Ace, with the use concentration of 0.15mM, transferring the resuspended liquid to a proper container for infection, and standing the agrobacterium infection liquid at room temperature for more than half an hour for infection. MES-KOH resuspension solution adding MES, namely MES monohydrate and MgCl 2 Namely, magnesium chloride, MES was used at a concentration of 10mM 2 The concentration used was 10mM, pH =5.6.
4) Infection and co-culture: putting the prepared apple leaves into an agrobacterium tumefaciens staining solution, soaking the apple leaves in a heavy suspension by a vacuum infiltration treatment mode, vacuumizing, and soaking for 15min under the pressure of 0.09 Mpa. And (3) sucking the bacterial liquid on the surface of the leaf by using sterile filter paper in a clean bench, intensively transferring the infected leaf of the agrobacterium to a co-culture medium, and culturing for 3d at the temperature of 23 +/-2 ℃ in the dark. The cells were transferred to the leaf state in the co-culture medium, and the results are shown in FIG. 4, and the success of infection was indicated when the leaves were water-stained. Co-culture medium: basic culture medium MS (Murashige and Skoog), adding exogenous hormones TDZ (thidiazuron) and NAA (alpha-naphthylacetic acid), wherein the using concentration of TDZ is 2mg/L, the using concentration of NAA is 0.5mg/L, adding 30g/L of cane sugar and 7.5g/L of agar powder, adjusting the pH to 5.8, adding Ace (acetosyringone) and BT (betaine) into the culture medium after sterilization, wherein the using concentration of Ace is 0.1mM, and the using concentration of BT is 1mM.
5) Differentiation culture: taking out the co-culture medium after three days, washing the leaves with sterile water containing 250mg/L of cefamycin (Cef) for 2-3 times, drying the surface water of the leaves with sterile filter paper, transferring the leaves onto a differentiation culture medium, culturing at 25 ℃ under light intensity of 2400lux for 16/8h in a photoperiod, and culturing for 5-7 weeks. Visible adventitious buds are differentiated from the leaf surfaces of the leaves after the leaves on the culture medium are cultured for 4-6 weeks in the visible light, the leaves are transferred to a new culture medium, the temperature is 25 ℃, the leaves are cultured in the light, the light intensity is 2400lux, the light cycle is 16/8h, the leaves are continuously cultured for 2-3 weeks, and the leaf states are shown in figure 5. Differentiation medium: adding exogenous hormones TDZ (thidiazuron) and NAA (alpha-naphthylacetic acid) into a basic culture medium MS (Murashige and Skoog), wherein the using concentration of the TDZ is 2mg/L, the using concentration of the NAA is 0.5mg/L, adding 30g/L of cane sugar and 7.5g/L of agar powder, and adjusting the pH to be 5.8. After sterilization, the antibiotic Cef, i.e.cefamycin, was added to the medium at a Cef concentration of 250mg/L.
6) Fluorescence screening and identification: firstly, the adventitious buds differentiated on a differentiation culture medium are subjected to primary fluorescence screening by using a UV365nm ultraviolet lamp, the primarily screened adventitious buds are placed on a subculture medium containing 250mg/L of cefuroxime for culture, and the status of the adventitious buds after being primarily screened and transferred into the subculture medium for 4 weeks is shown in figure 7. And after 4 weeks, carrying out secondary fluorescence identification on the adventitious buds obtained by the primary screening by using a multispectral dynamic fluorescence imaging system, and placing the identified adventitious buds with fluorescence markers in a cephalosporin-containing subculture medium again, wherein the temperature is 25 ℃, the light intensity is 2400lux, and the photoperiod is 16/8h for continuous culture. Subculture medium, see fig. 6: adding exogenous hormones 6-BA (6-benzyladenine) and IAA (3-indoleacetic acid) into a basic culture medium MS (Murashige and Skoog), wherein the using concentration of the 6-BA is 1mg/L, the using concentration of the IAA is 0.5mg/L, adding 30g/L of cane sugar and 7.5g/L of agar powder, adjusting the pH to 5.8, and adding Cef (cephamycin) after sterilization, wherein the using concentration of the Cef is 250mg/L.
7) Rooting culture: when the regeneration plant grows to 4-6cm, the regeneration plant is transferred into a rooting culture medium, the regeneration plant is cultured in the dark at 25 ℃ for 2 weeks, the rooting culture is continued for about 2 weeks under the conditions of the light intensity of 2400lux, the photoperiod of 16/8h and the temperature of 25 ℃, the regeneration plant is differentiated to obtain roots, and the regeneration plant with the roots is planted into soil to obtain a transgenic plant with stable inheritance. The rooting culture medium comprises: adding exogenous hormones IAA (3-indoleacetic acid) and IBA (3-indolebutyric acid) into a minimal medium MS (Murashige and Skoog), wherein the using concentration of the IAA is 0.5mg/L, the using concentration of the IBA is 1mg/L, adding 30g/L of cane sugar and 7.5g/L of agar powder, and adjusting the pH to be 5.8.
8) Identification of transgenic plants: extracting DNA of a regeneration plant with a fluorescent marker, carrying out PCR detection on the DNA, and designing two primers for PCR detection of the resistant seedling according to a carrier sequence and a PGT2 gene sequence, wherein the two primers are respectively a primer 1:5' CTCGAGATGGAGGCGACAGCTATAGTTTTTTATATCC-: 5 'GATCTGGATTTTTTAGTACTGGATTTGGTTTTAGGA-3', and the PCR program is as follows: pre-denaturation at 94 deg.C for 5min; then denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 20s, and circulation for 35 times; extension for 10min at 72 ℃.
9) The experimental results are shown in FIG. 8, the electrophoresis results of all regeneration plants (including roots, stems and leaves) are positive, and the electrophoresis products are taken for sequencing, which shows that the target gene is introduced into the genome of GL-3 apple. Meanwhile, the partial transgenic plants of the extracted DNA are identified again by a PlantView100 plant living body imaging system, and the parameters of an excitation filter of the PlantView100 plant living body imaging system are set as follows: 480nm, emission filter parameters set to: the 520nm fluorescence image of the partially transgenic line is shown in FIG. 9.
Example 2 establishment of apple stable transgenic line using Royal Gala apple as starting material and Agrobacterium rhizogenes K599
1) Preparation in the early stage of the experiment: a Royal Gala explant is established, the one-time subculture period of the material is 4 weeks, apple plants subjected to 3-4 times subculture can be used for experiments, and leaves suitable for infection are selected to serve as transgenic receptors in tissue culture bottles. The subculture medium for the continued growth of the Royal Gala (Royal Gala) plants was: adding exogenous hormone 6-BA (6-BA) with the use concentration of 1mg/L into a basic culture medium MS (Murashige and Skoog), adding 30g/L of cane sugar and 7.5g/L of agar powder, adjusting the pH to 5.8, sterilizing, adding 0.5mg/L of vitamin B5, growing for 4 weeks at the temperature of 25 ℃, selecting a plant with better growth vigor in a tissue culture bottle during transformation, taking two leaves below the tender leaf at the top and above the old leaf at the bottom, and placing the leaves in the tissue culture bottle as a transgenic receptor.
2) The procedures of the agrobacterium rhizogenes strain, the vector used in the experiment, the flow of infection experiment, fluorescence screening and identification, rooting culture and identification of transgenic plants are the same as those of the example 1.
3) Co-culturing infected leaves: the co-cultivation time and external conditions were identical to those after infection with GL-3 material. The following method is referred to Yao (1995), the co-culture medium after Royal Gala (Royal Gala) infection is a minimal medium MS (Murashige and Skoog), exogenous hormones 6-BA, i.e., 6-benzyladenine and NAA, i.e., alpha-naphthylacetic acid, are added, the concentration of 6-BA used is 5mg/L, the concentration of NAA used is 0.2mg/L, 30g/L of sucrose and 7.5g/L of agar powder are further added, the pH is adjusted to 5.8, 0.5mg/L of vitamin B5, ace, i.e., acetosyringone and BT, i.e., betaine, the concentration of Ace used is 0.1mM, and the concentration of BT used is 1mM after sterilization.
4) Adventitious bud induction culture: the culture time and external conditions are consistent with those of induction culture after infection by GL-3 materials, a co-culture medium after infection by Royal Gala (Royal Gala) is a basic medium MS (Murashige and Skoog), exogenous hormones 6-BA, namely 6-benzyladenine and NAA, namely alpha-naphthylacetic acid are added, the using concentration of 6-BA is 5mg/L, the using concentration of NAA is 0.2mg/L, 30g/L of cane sugar and 7.5g/L of agar powder are added, the pH is adjusted to 5.8, 0.5mg/L of vitamin B5 and 250mg/L of antibiotic Cef, namely cefuromycin are added into the culture medium after sterilization. Partial transgenic plants obtained using the Royal Gala as recipient are shown in FIG. 10.
5) Identification of transgenic plants: the identification method and the primers used are the same as in example 1, the results are shown in fig. 11, the electrophoresis results of the partial regeneration plants (including roots, stems and leaves) are positive, and the electrophoresis products are taken for sequencing, which shows that the target gene is introduced into the genome of the royal gala apples.
Example 3: establishment of apple stable turning line based on different agrobacterium rhizogenes mediation
In this embodiment, the establishment of a system for transforming apple transgenic plants with agrobacterium rhizogenes 8196, R1601 and C58C1 respectively comprises the following steps:
1) Preparation in the early stage of the experiment: establishing an explant of a GL-3 apple plant by taking GL-3 apple material as a starting material, culturing the established explant in a culture medium, taking down two leaves below a tender leaf at the top end and above an old leaf at the bottom end, and placing the leaves in a tissue culture bottle to serve as a transgenic receptor.
2) Transformation and culture to obtain the strain used for transformation: the vector uses pCAMBIA1301-PGT2-Egfp expression vector, the target vector plasmid is transferred into agrobacterium rhizogenes competent cells 8196, R1601 and C58C1 by a heat shock method, the cells are coated and inoculated on LB solid culture medium for culturing, dark culture is carried out at 28 ℃ for 48h, a single colony is selected in 50ml liquid LB, the cell is shaken at 28 ℃ and 200rpm until OD600 value =1.5, the bacterial liquid is centrifugally resuspended in MES-KOH resuspension with 1.5 times of volume, the resuspended liquid is transferred to a proper container for infection, and the agrobacterium infection liquid is stood at room temperature for more than half an hour for infection.
3) The experimental operation flow of three different agrobacterium rhizogenes strains mediated apple leaf experiments is the same as that in example 1, and the steps of apple leaf culture after infection, adventitious bud induction culture and the like are the same as those in example 1.
4) Experimental results show that 8196, R1601 and C58C1 agrobacterium rhizogenes strains can induce apple leaves to differentiate transgenic adventitious buds with fluorescent markers, and detailed results are shown in figures 12, 13 and 14.
Example 4 establishment of stable apple rotation system based on different vectors
1) Preparation in the early stage of the experiment: establishing an explant of a GL-3 apple plant by taking GL-3 apple material as a starting material, culturing the established explant in a culture medium, taking down two leaves below a tender leaf at the top end and above an old leaf at the bottom end, and placing the leaves in a tissue culture bottle to serve as a transgenic receptor.
2) Construction of the vector: RNAi silencing PK7WIWG2D-PGT1:: GFP vector was created using pDONR222 as intermediate vector, and the vector map is shown in FIG. 15.
3) The agrobacterium rhizogenes transformation, infection and co-culture, differentiation culture, fluorescence screening and identification, and rooting culture methods are the same as example 1, and finally partial transgenic regeneration strains are obtained, as shown in fig. 16.
4) Identification of transgenic plants: extracting DNA of a regeneration plant with a fluorescent marker, carrying out DNA level detection on PGT1, and designing PCR detection primers according to a carrier sequence and a PGT1 gene sequence, wherein the PCR detection primers respectively comprise: primer 3:5' TGTTTGCAGGTCAGCTTGACACT-: 5 'GTGACTCCCTTTAATTCTCATGTTATAATTCGC-3', and the PCR program is as follows: pre-denaturation at 94 ℃ for 5min; then denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 20s, and circulation for 35 times; extension for 10min at 72 ℃.
5) The results are shown in FIG. 17, where both F1-1 and F1-2 contain fragments of the same size as the target fragment, indicating that both F1-1 and F1-2 strains are PGT1-RNAi strains, GFP transgenic strains.
Example 5 establishment of stable apple turning line under different infection treatment modes
1) Preparation in the early stage of the experiment: the materials, vectors and Agrobacterium rhizogenes strains used in the experiment were identical to those in example 1.
2) The experimental operation steps are as follows: different from the infection treatment mode-vacuum infiltration treatment of example 1, in this example, different wound treatment modes are adopted, 3-4 1cm wounds are cut on the back of the apple leaves by using a sterilization blade (other wound treatment can also be performed on the recipient plants), the leaves with the wounds are soaked in the infection solution for 5-30 minutes, the leaves with the wounds are fished out after 5-30 minutes, the liquid on the surfaces of the leaves is dried by using sterile filter paper, the leaves are placed on a co-culture medium for culture, and then, the steps of differentiation culture, fluorescence screening and identification, rooting culture and transgenic plant identification are the same as in example 1.
3) The experimental results are as follows: other invasive means of Agrobacterium rhizogenes-mediated transformation of the recipient material are used, and transgenic adventitious shoots with fluorescent markers can be obtained, although the transformation efficiency is lower than that of the negative pressure-based transfer method, see FIG. 18.
Example 6 comparison of the method of the present application with conventional methods
1. Preparation in the early stage of the experiment: in the same state, 16 bottles and 8 bottles of GL-3 seedlings of the same batch of apples are infected by an agrobacterium tumefaciens mediated method, and the rest are infected by the agrobacterium rhizogenes mediated method protected by the application.
2. Strains and vectors used in the experiments: the strains used for the leaf disc method infection are as follows: GV3101 agrobacterium tumefaciens strain; the strains used for rooting and hair are as follows: k599 Agrobacterium rhizogenes strain. The plasmids are all vectors pCAMBIA1301-PGT2-Egfp.
3. The experimental operation steps are as follows:
1) Leaf disc method: referring to Hongyan Dai (2013), a proper amount of leaves are taken and placed in bacterial liquid, 3-4 1cm wounds are scratched on the back sides of the leaves by using a sterilization blade, the wounded leaves are immersed in an infection liquid for 8 minutes, the leaves are fished out after 8 minutes and are dried by using sterile filter paper, the surface liquid of the leaves is sucked, the leaves are placed on a co-culture medium, each batch of experimental process is repeated for 7-8 times, the whole process takes 3 hours, 3 days of dark culture treatment, the co-cultured leaves are transferred to an extension medium for dark culture for 4 weeks and then are taken out for plate changing and light culture for 4-6 weeks, and the steps of the wound operation process are shown in figure 19. The temperature of the whole culture process is 22-25 ℃, the light intensity is 2400lux, the number of the transgenic adventitious buds obtained by infection is counted after the light is exposed for 2-6 weeks, a batch of 15 plates are used, and the experiment is repeated for 10 times.
2) Rooting: the procedure is as in example 1, consuming 1.5h, dark culture treatment for 3 days, transferring to differentiation medium after dark culture treatment, and differentiating callus and adventitious root after 2 weeks of light exposure, obtaining transgenic adventitious bud after 4 weeks, the operation is detailed, see fig. 20. Counting the number of the transgenic adventitious buds after culturing for 2-8 weeks by light; the external conditions of the experiment, the number of each batch and the times of repeated experiments are consistent with those of the leaf disc method.
4. The experimental results are as follows:
1) Two different agrobacteria are used to infect the leaf, adventitious buds with fluorescent markers are obtained after a certain time of induction, and a fluorescence imaging contrast diagram identified by a plant living body imaging system of PlantView100 is shown in figure 21. FIG. A is a synthetic diagram obtained by a PlantView100 plant in vivo imaging system using Agrobacterium tumefaciens mediated adventitious shoots, FIG. B is a corresponding brightfield, FIG. C is a synthetic diagram obtained by a PlantView100 plant in vivo imaging system using Agrobacterium tumefaciens mediated adventitious shoots, and FIG. D is a corresponding brightfield, in which callus having differentiation ability but not differentiating visible shoots is present except for unmarked but still fluorescent, wherein the Agrobacterium tumefaciens mediated adventitious shoots obtained are single adventitious shoots and the Agrobacterium tumefaciens mediated partial adventitious shoots obtained are clustered adventitious shoots.
2) Ten batches were conducted using Agrobacterium tumefaciens mediated method and only two of the ten batches obtained transgenic adventitious buds, for a total of 3. Ten batches of infection conducted by the agrobacterium rhizogenes mediated method have transgenic adventitious buds in each batch, and more than 300 transgenic adventitious buds are obtained in total (the number is a relatively large adventitious bud which can be clearly distinguished by naked eyes for statistics, and other clustered small adventitious buds are not counted). The efficiency of obtaining the transgenic adventitious bud which can be stably inherited is improved by more than 100 times.
Reference documents:
[1] the Ti plasmid of Agrobacterium tumefaciens (Agrobacterium tumefaciens) is the plasmid of cell biology, 1984.
[2] Preliminary research on formation of transgenic hairy roots by infecting cucumbers with agrobacterium rhizogenes K599 and cloning of orf14 gene [ D ]. University of hangzhou teachers, 2012.
[3] Establishment of a genetic transformation system of stem segments of populus alba mediated by agrobacterium rhizogenes and determination of physiological and biochemical indexes [ D ]. University of gillin, 2017.
[4] The application of transgenic technology in apple genetic breeding improvement [ J ]. Biotechnology world, 2014.
[5] The method comprises the steps of establishing a genetic transformation system of the aster scaber root mediated by agrobacterium rhizogenes and detecting triterpenoid saponin in the hairy root [ D ]. Sichuan university of agriculture, 2015.
[6] Plum rhyme, establishment of agrobacterium rhizogenes mediated mulberry genetic transformation system and content determination of quercetin in hairy roots [ D ]. Southwest university, 2010.
[7] Wucalyx, zuirkui, zhangchao, zhuying, yanna, janena, 3 35852o, gexiao nu, xiaoweu, agrobacterium tumefaciens Ri plasmid-mediated plant genetic engineering and applications [ J ] proceedings of hangzhou university (nature science edition), 2018
[8] Wangypeng. Agrobacterium tumefaciens Ti plasmid introduction [ J ] biological teaching, 2018,43 (06): 65-66.
[9] Establishment of Wangbuyan. Agrobacterium rhizogenes mediated Sedum spectabile genetic transformation system [ D ]. Hebei agricultural university, 2009.
[10] Xiaozi agrobacterium rhizogenes mediated citrus genetic transformation system establishment and transgenic citrus canker resistance analysis [ D ] university of china agriculture, 2014.
[11] Protoplast culture and plant regeneration of the agrobacterium rhizogenes transformed line zhangjiafen, zhangqiang, 2009.
[12] Zhang Cheng, study of Agrobacterium rhizogenes-mediated genetic transformation of Cotton [ D ]. Chinese academy of agricultural sciences, 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。
The foregoing descriptions of specific exemplary embodiments of the present application have been presented for purposes of illustration and description. It is not intended to limit the application to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the present application and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the present application and various alternatives and modifications. It is intended that the scope of the application be defined by the claims and their equivalents.
Claims (10)
1. An adventitious bud induction culture method for apples or malus is characterized by comprising the following steps:
1) Preparation of transgenic receptor: the receptor is apple leaves;
2) Constructing an expression vector;
3) Transforming agrobacterium;
4) Leaf infection and co-culture;
5) And (4) performing adventitious bud induction culture.
2. The method according to claim 1, wherein in the step 3), the agrobacterium is agrobacterium rhizogenes; preferably, the agrobacterium rhizogenes includes but is not limited to agrobacterium rhizogenes K599, agrobacterium rhizogenes 8196, agrobacterium rhizogenes R1601 or agrobacterium rhizogenes C58C1.
3. The method according to claim 1, wherein in the step 5), the adventitious bud induction culture obtains an adventitious bud without obtaining a hairy root.
4. The method according to any one of claims 1 to 3,
the leaves in the step 1) are tender leaves;
preferably, the preparation step in the step 1) is as follows: selecting apple twigs to establish an apple plant explant, carrying out subculture on the explant for 3-4 times, and taking tissue culture seedling leaves below top tender leaves and above bottom old leaves as transgenic receptors.
5. The method according to any one of claims 1 to 4,
the vector in the step 2) is constructed into an expression vector containing a target gene and/or a marker gene;
preferably, the expression vector includes, but is not limited to, pCAMBIA1301 vector or RNAi silencing PK7WIWG2D-PGT1:: GFP vector.
6. The method according to any one of claims 1 to 5,
the recombinant expression vector is transformed into agrobacterium rhizogenes in the transformation in the step 3), and the agrobacterium rhizogenes is infected into MES-KOH resuspension containing acetosyringone after dark culture;
preferably, the MES-KOH resuspension is: 8-12mM MES monohydrate, 5-15mM magnesium chloride, 0.05-0.3mM acetosyringone, pH =5.3-5.6;
more preferably, the step 3) of converting is: transforming the recombinant expression vector into agrobacterium rhizogenes, performing dark culture on an LB solid plate containing kanamycin and streptomycin at 19-30 ℃ for 13d, picking a single colony in liquid LB containing kanamycin and streptomycin, shaking at 25-28 ℃ and 150-220rpm until the OD600 value reaches 0.8-1.5 range, centrifuging at room temperature of 5000-7000rpm to remove a supernatant, and suspending the thallus into MES-KOH heavy suspension containing acetosyringone for infection.
7. The method according to any one of claims 1 to 6,
the infection in the step 4) includes, but is not limited to, traditional scratching or vacuum infiltration;
preferably, the vacuum infiltration method for infection comprises the following steps: placing the leaves of the tissue culture seedling in a staining solution, performing vacuum treatment under the pressure of 0.04-0.1Mpa, and then transferring the leaves to a co-culture medium containing acetosyringone and betaine for culture;
more preferably, the vacuum infiltration method comprises the following steps: placing the leaves of the tissue culture seedling in an infection solution, carrying out infiltration treatment under the conditions of vacuum infiltration time of 1-60min and vacuum degree of 0.04-0.1Mpa, after vacuum infiltration treatment, sucking bacterial liquid on the surfaces of the leaves by using sterile filter paper, transferring the leaves to a co-culture medium containing acetosyringone and betaine, culturing the leaves with the back faces upwards and the front faces tightly attached to the culture medium, and culturing the culture medium in the dark at the temperature of 25 +/-5 ℃ for 1-3d.
8. The method according to any one of claims 1 to 7,
the step 5) of adventitious bud induction culture is that the co-cultured apple leaves are transferred to an adventitious bud differentiation culture medium, and adventitious buds are obtained by visible light culture;
preferably, the adventitious bud differentiation medium is: adding thidiazuron TDZ and/or 6-benzyladenine 6-BA with certain concentration and alpha-naphthylacetic acid NAA into MS basic culture medium;
more preferably, the adventitious bud differentiation medium is: MS basal medium +0-5mg/L thidiazuron TDZ and/or 0-5 mg/L6-benzyladenine +0.05-3mg/L alpha-naphthylacetic acid NAA +25-35g/L sucrose or 35-45g/L sorbitol +6-8g/L agar powder or 2-3g/L plant gel, pH =5.6-6.0;
further preferably, the step 5) of adventitious bud induction culture comprises the following specific steps: transferring the co-cultured apple leaves to a differentiation culture medium, performing adventitious bud induction culture under the conditions of light intensity of 500-5000lux, photoperiod of 12-18h illumination/dark of 12-6h and temperature of 25 +/-5 ℃, and culturing for 2-8 weeks to obtain adventitious buds.
9. A method of constructing an apple and malus stable genetic transgene system, comprising the method of any one of claims 1 to 7, and further comprising the steps of:
6) Screening and identifying the transgenic adventitious bud;
7) And (5) rooting and transplanting the transgenic plant.
10. The method of claim 8,
the screening and identification of the transgenic adventitious bud in the step 6) can be based on the screening and identification of a fluorescent marker;
the rooting and transplanting steps of the transgenic plants in the step 7) are as follows: transferring the adventitious bud obtained by differentiation culture into a rooting culture medium, carrying out rooting culture under the conditions of light intensity of 500-5000lux, light cycle of 12-18/12-6h and temperature of 25 +/-5 ℃ to obtain a regenerated plant, and transplanting the regenerated plant with roots into soil to obtain a complete transgenic plant with stable heredity.
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