CN114158482B - Medicament concentration screening method applied to apple transgenosis - Google Patents
Medicament concentration screening method applied to apple transgenosis Download PDFInfo
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- CN114158482B CN114158482B CN202111250967.1A CN202111250967A CN114158482B CN 114158482 B CN114158482 B CN 114158482B CN 202111250967 A CN202111250967 A CN 202111250967A CN 114158482 B CN114158482 B CN 114158482B
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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
- A01H5/12—Leaves
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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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
- A01H6/7418—Malus domestica, i.e. apples
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- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of agricultural biology, and discloses an application of an apple transgenic medicament and a concentration screening method thereof, wherein the medicament concentration screening method applied to apple transgenesis comprises the following steps: the chlorsulfuron crude drug is prepared to obtain mother liquor, and the mother liquor is filtered and sterilized, so that the chlorsulfuron crude drug is applied to an apple genetic transformation system with aseptic operation. According to the drug concentration screening method applied to apple transgenes, the experimental result is used for finally determining that the use concentration of chlorsulfuron in GL-3 transgenes is 2-4 mug/L, and 2 mug/L is preferably selected; culture mediums below the concentration range have no inhibition effect on GL-3 budding, and can cause the occurrence of false positive seedlings, so that the workload of experiments is increased; culture media above this concentration range may cause damage to GL-3 leaves, possibly reducing genetic transformation efficiency by causing leaf necrosis.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a reagent concentration screening method applied to apple transgenosis.
Background
At present, apple fruits are rich in rich nutritional ingredients such as saccharides, organic acids, vitamins, flavonoids, dietary fibers, minerals and the like, and have the effects of resisting oxidation, aging and cancer, preventing cardiovascular diseases, promoting toxin expelling, losing weight and the like for human beings, so that the apple fruits are popular among people. At present, china is the first country of fruit tree industry, wherein the planting area and yield of apples are in the first world. The core element of the development of the apple industry is the breeding of good varieties, a plurality of world varieties with larger cultivation areas are cultivated internationally at present, and the breeding technology is developed into a mode of combining conventional breeding and molecular auxiliary breeding from single real-life seed selection, hybridization breeding and bud mutation seed selection. Molecular breeding includes transgenic breeding, i.e., a breeding method in which a new variety with certain requirements is cultivated by gene transfer. Transgenic technology has long existed in apple breeding, for example, the RNAi method is used for inhibiting the MdPPO gene of the Malus asiatica, so that the browning degree of pulp is greatly reduced; the BpMADS4 gene of the white birch is expressed, so that the transgenic apples successfully break the childhood phase and the flowering time is advanced.
Transgene technologies can be largely divided into four classes according to their transformation methods: agrobacterium-mediated methods, DNA direct insertion methods, pollen tube channel methods, and plant virus-mediated methods. The most widely applied method in apple transgenic technology is an agrobacterium-mediated method, firstly, genes to be researched are constructed in a binary expression vector, then the constructed vector is transformed into agrobacterium, and transgenic apple plants are obtained through leaf infection method transformation. The most important part of the binary expression vector is that it contains a marker gene for resistance screening against antibiotic agents, and the most widely used marker gene in apple transformation is the neomycin phosphotransferase II (nptII) gene, which is resistant to aminoglycoside antibiotics (e.g., kanamycin), and may raise public safety concerns over genes crossing natural disorders due to its bacterial origin. Meanwhile, since single kanamycin resistance screening has great limitation on application research of apples, for example, when researching the multi-gene functions in a certain channel, double transgenic plant materials cannot be obtained, so that the development of new marker genes and medicament screening are very important.
The plant-derived genes developed at present are used as screening markers including acetolactate synthase (acetolactate synthase, ALS) and belong to the targets of sulfonylurea herbicides and imidazolinone herbicides. Mutated ALSCan confer resistance to herbicides to a variety of plant species, including Arabidopsis, rice, apple, and the like. Previous studies have demonstrated that apple 'Royal Gala' can act as a marker gene for herbicides through mutated ALS
Resistance is generated so as to achieve the purpose of transgene. />
Is a sulfonylurea herbicide developed by Dupont in the beginning of the 20 th century in the 80 th year, the main ingredient of which is Chlorsulfuron (Chlorsulfuron), which belongs to systemic conduction herbicides, and by inhibiting the ALS enzyme activity of plants, the synthesis of proteins and the cell division and growth are destroyed, eventually leading to death of the plants. The common apple transgene material is the actual offspring GL-3 of Royal Gala, which is favored by the majority of apple researchers due to the efficient genetic transformation efficiency; since GL-3 is substantially different from "Royal Gala" in gene level, it is difficult to directly apply the former research results. The former study will be->
Is used as a selective agent, and +.>
As a preparation (which contains a plurality of other components, such as auxiliary agents and the like), the preparation is difficult to achieve aseptic conditions and is difficult to apply to aseptic plant tissue culture and genetic transformation systems; at the same time due to
Some new sulfonylurea herbicides have been replaced in China and are not easily available through purchase. Therefore, the former research results cannot be directly applied to the genetic transformation of apple GL-3. In summary, a new method for screening drug concentrations that can be applied to apple transgenes is needed.
Through the above analysis, the problems and defects existing in the prior art are as follows:
(1) The existing neomycin phosphotransferase II (nptII) gene has resistance to aminoglycoside antibiotics, and the gene is derived from bacteria, so that the safety concern of the public on the gene crossing natural obstacle can be raised.
(2) The single kanamycin resistance screening has great limitation on the application research of apples, for example, when researching the multi-gene function in a certain path, double transgenic plant materials cannot be obtained.
(3) Since GL-3 is substantially different from 'Royal Gala' in gene level, previous research results cannot be directly applied to genetic transformation of apple GL-3.
(4)
As a preparation, it is difficult to achieve sterile conditions and to apply it to sterile plant tissue culture and genetic transformation systems, and +.>
Is not easy to be purchased in China.
The difficulty of solving the problems and the defects is as follows:
1 the commercial herbicide Glean used in previous studies as a formulation (which contains many other ingredients, such as some adjuvants, etc.) is difficult to achieve sterile conditions and is difficult to apply to sterile plant tissue culture and genetic transformation systems; secondly, glean is not readily available from commercial sources as it has been replaced in China by some new sulfonylurea herbicides. Therefore, it is difficult to directly apply the former theoretical research results.
2-chlorsulfuron as a main component of the herbicide Glean damages the synthetic growth and cell division and growth of proteins by inhibiting the activity of acetolactate synthase (acetolactate synthase, ALS), a first key enzyme in the branched-chain amino acid synthesis pathway in plants, ultimately leading to death of the plants. Therefore, in the experimental process, the chlorsulfuron with higher concentration can cause necrosis of apple tissue culture seedling leaves; however, the regeneration and budding of the chlorsulfuron wild apple tissue culture leaves with lower concentration can not play a role in inhibiting, so that false positive seedlings appear in the apple transgenic process, and the workload of experiments is increased. The selection of the critical concentration and concentration gradient during the critical concentration screening process requires extensive experimentation to determine.
The meaning of solving the problems and the defects is as follows:
the target gene acetolactate synthase (acetolactate synthase, ALS) of 1 chlorsulfuron is one of plant-derived genes in the developed screening markers, and meets the requirement of the current society on biosafety.
The application of the 2-chlorsulfuron in determining the concentration of the apple transgenic agent enriches and develops the screening mark in the current apple genetic transformation system, and provides a feasibility thinking for obtaining the apple double-transgenic material.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a medicament concentration screening method applied to apple transgenosis.
The invention is realized in such a way that the drug concentration screening method applied to the apple transgenes comprises the following steps:
by combining
The main component chlorsulfuron is prepared into mother liquor, and the mother liquor is filtered and sterilized, so that the chlorsulfuron is applied to an apple genetic transformation system with aseptic operation.
Further, the agent concentration screening method applied to apple transgenesis comprises the following steps:
step one, obtaining a chlorsulfuron original drug by purchasing in a reagent company, dissolving the original drug by using DMSO and filtering and sterilizing the original drug by using a 0.22 mu m microporous filter membrane to obtain a chlorsulfuron original drug mother solution which can be used for aseptic operation;
preparing a resuspension and a culture medium I, and preparing mother liquor into culture media II with different concentrations; according to the pre-experiment result, the concentration gradient is 0.5-8 mug/L as the experiment treatment concentration, the culture medium provided by the step is critical to the development of the scheme, a proper growth environment is provided for genetically transformed apple leaves, and the set different concentration gradients can be used for detecting the regeneration effect of chlorsulfuron on the leaves;
thirdly, marking 3-5 wounds on GL-3 blades which are subcultured for about one month in the heavy suspension by using a sharp blade, and rotating and shaking the GL-3 blades in the heavy suspension for 15 minutes, wherein the step simulates a normal apple genetic transformation method and can provide guarantee for the application of the method in the follow-up process;
fourthly, placing the leaves on sterile paper to absorb water, then placing the leaves in a culture medium I in an inverted mode, simulating a normal apple genetic transformation method, and ensuring that agrobacterium infection to the leaves plays a positive role in subsequent application of the scheme;
step five, after the leaves are subjected to dark culture on a culture medium I for 5 days, transferring the leaves to a culture medium II with different chlorsulfuron concentrations, and carrying out dark culture in a 21 ℃ incubator for 4 weeks, wherein the wound parts of the apple leaves are regenerated into plants, and the chlorsulfuron with different concentrations plays a role in inhibiting the regeneration of the leaves;
step six, the sprouting condition and the phenotype of the blade wound on the culture medium with different chlorsulfuron concentrations are counted, and the use concentration of the chlorsulfuron in GL-3 transgenes is determined to be 2-4 mug/L, preferably 2 mug/L.
Further, in the first step, the raw medicine is dissolved, including:
100mg of chlorsulfuron is prepared into a raw medicine mother solution with the concentration of 10mg/mL by using 10mL of DMSO, diluted to 8mg/L by water and stored in a refrigerator at the temperature of minus 80 ℃.
Further, in the first step, the concentration of the reagent mother solution includes: 2mg/mL TDZ,1mg/mL NAA,100mM AS,1M Bet,250mg/mL Cef.
Further, in step two, the 1L volume of heavy suspension consists of 4.43g MS, 5.88g sodium citrate dihydrate and 20.00g sucrose, and ph=5.3 is adjusted with glacial acetic acid;
the culture medium I with the volume of 1L consists of 4.43g of MS, 30.00g of sucrose, 1.00mL of TDZ, 0.50mL of NAA, 7.50g of agar powder, 1.00mL of AS and 1.00mL of Bet; adjusting pH=5.8-6.0 with NaOH, and adding AS and Bet after sterilization;
the culture medium II with the volume of 1L consists of 4.43g of MS, 30.00g of sucrose, 1.00mL of TDZ, 0.50mL of NAA, 7.50g of agar powder, 1.00mL of Cef and chlorsulfuron; naOH is used for adjusting pH=5.8-6.0, and chlorsulfuron and Cef with different concentrations are added after sterilization.
Further, in the second step, the preparation of the culture medium II with the chlorsulfuron concentration of different concentrations comprises the following steps:
diluting the 10mg/mL of crude drug mother solution to 8mg/L by water, and then carrying out equal-ratio dilution on the 8mg/L of chlorsulfuron crude drug mother solution to 4mg/L, 2mg/L, 1mg/L and 0.5mg/L respectively; five gradients of crude drugs and culture medium were mixed according to a ratio of 1:1000 to medium II.
In the third step, the GL-3 leaves are middle leaves of GL-3 tissue culture seedlings which are subcultured for 1 month; 3 to 5 wounds are drawn from the GL-3 leaves in the heavy suspension by using a blade.
Further, in step three, the resuspension is free of agrobacterium.
Further, in the fifth step, the culturing method comprises the following steps: culturing in dark at 21deg.C for 5 days.
The invention also aims to provide an application of the agent concentration screening method applied to apple transgenesis in genetic transformation of apple GL-3 genes.
By combining all the technical schemes, the invention has the advantages and positive effects that: the invention provides a method for screening concentration of a medicament applied to apple transgenosis, and particularly relates to a method for screening in the apple transgenosis process, in particular to a method for determining the screening critical concentration of the medicament. According to the invention, the experimental result is used for finally determining that the use concentration of chlorsulfuron in GL-3 transgenosis is 2-4 mug/L, and 2 mug/L is preferably selected; culture mediums below the concentration range have no inhibition effect on GL-3 budding, and can cause the occurrence of false positive seedlings, so that the workload of experiments is increased; culture media above this concentration range may cause damage to GL-3 leaves, possibly reducing genetic transformation efficiency by causing leaf necrosis. The target gene acetolactate synthase (acetolactate synthase, ALS) of the adopted medicament chlorsulfuron is one of plant source genes in the developed screening marks, and meets the requirement of the current society on biosafety; the chlorsulfuron is applied to determining the concentration of the apple transgenic medicament, enriches and develops the screening mark in the current apple genetic transformation system, and provides a feasibility thought for obtaining the apple double-transgenic material.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a flowchart of a method for screening concentration of an agent applied to apple transgenes according to an embodiment of the present invention.
FIG. 2 is a schematic diagram showing the growth of adventitious buds at the wound of a leaf blade on a chlorsulfuron concentration medium of 0.5. Mu.g/L provided by the example of the present invention.
FIG. 3 is a schematic representation of the phenotype of leaf wounds on 1. Mu.g/L chlorsulfuron concentration medium provided by the examples of the present invention.
FIG. 4 is a schematic representation of the phenotype of leaf wounds on a 2. Mu.g/L chlorsulfuron concentration medium provided by an example of the present invention.
FIG. 5 is a schematic representation of the phenotype of a leaf wound on a 4. Mu.g/L chlorsulfuron concentration medium provided by an example of the present invention.
FIG. 6 is a schematic representation of the phenotype of leaf wounds on 8. Mu.g/L chlorsulfuron concentration medium provided by the example of the present invention.
FIG. 7 is a statistical schematic diagram of growth of leaf wounds on different chlorsulfuron concentration media provided by the embodiment of the invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Aiming at the problems existing in the prior art, the invention provides a medicament concentration screening method applied to apple transgenes, and the invention is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the method for screening the concentration of the medicament applied to the apple transgenes provided by the embodiment of the invention comprises the following steps:
s101, obtaining chlorsulfuron original drug by purchasing in a reagent company, dissolving the original drug by using DMSO and filtering and sterilizing by using a 0.22 mu m microporous filter membrane;
s102, preparing a resuspension and a culture medium I, and preparing mother liquor into culture media II with different concentrations; according to the pre-experiment result, selecting the concentration gradient of 0.5-8 mug/L as the experiment treatment concentration;
s103, simulating a normal apple genetic transformation method, drawing 3-5 wounds on GL-3 blades in the heavy suspension by using a sharp blade, and rotating and shaking the GL-3 blades in the heavy suspension for 15min;
s104, placing the leaves on sterile paper to absorb water, and then placing the leaves in a culture medium I in an inverted mode;
s105, transferring the leaves to a culture medium II with different chlorsulfuron concentrations after dark culture for 5 days on the culture medium I, and dark culturing for 4 weeks in a 21 ℃ incubator;
s106, counting the sprouting condition and the phenotype of the blade wound on the culture mediums with different chlorsulfuron concentrations, and determining that the use concentration of the chlorsulfuron in GL-3 transgenes is 2-4 mug/L, preferably 2 mug/L.
The technical scheme of the invention is further described below with reference to specific embodiments.
In view of the problems existing at present, the invention provides a screening method for medicament concentration by
The main component chlorsulfuron is prepared into mother liquor, and the mother liquor is filtered and sterilized, so that the chlorsulfuron is applied to an apple genetic transformation system with aseptic operation.
The invention is realized by obtaining a chlorsulfuron original drug (Alatine, product number C109991) by purchasing in a reagent company, dissolving the original drug by using DMSO, filtering and sterilizing by using a 0.22 mu m microporous filter membrane, and preparing mother liquor into culture medium II with different concentrations; simulating a normal apple genetic transformation method, drawing a wound of GL-3 blades in a heavy suspension (without agrobacterium) by using a sharp blade, rotating and shaking the GL-3 blades in the heavy suspension for 15 minutes, then placing the blades on sterile paper to absorb water, and then placing the blades in a culture medium I in an inverted manner; after 5 days of cultivation of the leaves on medium I (21℃incubator, dark), the leaves were transferred to medium II of different chlorosulfuron concentrations and then cultivated in 21℃incubator in dark for 4 weeks. And finally, counting the sprouting conditions of the leaves on the culture mediums with different chlorsulfuron concentrations.
1. Materials and reagents:
1.1 materials: apple tissue culture seedling GL-3
1.2 reagents used in the invention: MS (PhytoTech, murashige and Skoog, M519), TDZ (SIGMA, thidiazuron, P6186), NAA (SIGMA, 1-naphthylacetic acid, N1641), AS (Shanghai, acetosyringone, A60111-0001), bet (Methano, betaine, MB 4225), cef (Shanghai, cefotaxime sodium, A60276-0100). The chlorsulfuron is purchased from Allatin, product number C109991-100mg, prepared into mother liquor with concentration of 10mg/mL by DMSO, diluted to 8mg/L by water, and stored in a refrigerator at-80deg.C. The rest are conventional reagents.
Concentration of the reagent mother solution: TDZ (2 mg/mL), NAA (1 mg/mL), AS (100 mM), bet (1M), cef (250 mg/mL).
2. The specific method comprises the following steps:
(1) The chlorsulfuron original drug solution is prepared, and 100mg of the purchased chlorsulfuron original drug is dissolved by using 10mL of DMSO to prepare an original drug mother solution of 10mg/mL, and the original drug mother solution is filtered and sterilized by using a microporous filter membrane of 0.22 mu m and stored at the temperature of minus 80 ℃.
(2) According to the pre-experiment result, the concentration gradient of the invention is 0.5-8 mug/L as the experiment treatment concentration. The resuspension and the culture medium I were prepared according to the formulations shown in tables 1 and 2. Preparing a culture medium II with different chlorsulfuron concentrations according to a table 3, diluting the 10mg/mL of raw medicine mother solution to 8mg/L with water, then carrying out equal-ratio dilution on the 8mg/L chlorsulfuron raw medicine mother solution to 4mg/L, 2mg/L, 1mg/L and 0.5mg/L respectively, and then mixing the raw medicines with the culture medium according to a gradient of 1:1000 to medium II;
(3) Selecting a middle blade of GL-3 tissue culture seedling which is subcultured for about 1 month, marking 3-5 wounds on the blade by using a sharp blade, and then placing the blade into heavy suspension to rotationally shake for 15 minutes;
(4) Taking out the resuspended leaf from the resuspension, and placing the leaf on sterile paper to absorb water;
(5) Placing the leaves after moisture absorption in a culture medium I, wherein the front surfaces of the leaves are contacted with the culture medium;
(6) Leaves were grown on medium I and in the dark at 21 ℃ in an incubator for 5 days;
(7) The leaves were transferred to medium II and cultured in the dark at 21 ℃ in an incubator for 30 days;
(8) Budding and phenotyping at the wound of the leaf blade are counted.
The invention classifies the phenotype of the wound of the leaf into 4 categories, namely budding, growing callus, non-budding, growing callus and leaf necrosis. The difference between the non-budding and growing callus and the blade necrosis is the color of the blade wound, the non-budding and growing callus is still green, and the blade wound and other positions of the blade necrosis are discolored and necrotized.
This test was repeated three times.
According to the invention, the experimental result is used for finally determining that the use concentration of chlorsulfuron in GL-3 transgenosis is 2-4 mug/L, and 2 mug/L is preferably selected. Culture mediums below the concentration range have no inhibition effect on GL-3 budding, and can cause the occurrence of false positive seedlings, so that the workload of experiments is increased; culture media above this concentration range may cause damage to GL-3 leaves, possibly reducing genetic transformation efficiency by causing leaf necrosis.
Table 1 resuspension (1L volume, glacial acetic acid adjusted ph=5.3)
MS 4.43g
Sodium citrate dihydrate 5.88g
Sucrose 20.00g
Table 2 Medium I (1L volume, naOH adjusted pH=5.8-6.0, AS and Bet were added after sterilization)
Table 3 Medium II (1L volume, naOH adjusted pH=5.8-6.0, sterilized and added with chlorsulfuron and Cef of different concentrations)
FIG. 2 shows the growth of adventitious buds at the wound of leaf blade on a chlorsulfuron concentration medium of 0.5. Mu.g/L, all leaf blades growing adventitious buds. FIG. 2 (b) is an enlarged view showing the phenotype of adventitious buds growing at the wound of the leaf blade.
FIG. 3 shows the phenotype of leaf wounds on 1. Mu.g/L chlorsulfuron medium, with few leaves growing adventitious buds and most leaves growing callus. FIG. 3 (b) is an enlarged view showing the phenotype of adventitious buds and calli grown at the wound of the leaf blade.
FIG. 4 is a leaf wound phenotype on 2. Mu.g/L chlorsulfuron concentration medium with most leaves growing callus. FIG. 4 (b) is an enlarged view showing the phenotype of the growing callus.
FIG. 5 is a phenotype of leaf wounds on a 4. Mu.g/L chlorsulfuron concentration medium, with most leaves growing without growing callus and adventitious buds, and with some leaf wounds showing necrotic blackening. FIG. 5 (b) is an enlarged view showing the phenotype of non-growing calli and adventitious buds, which differ from leaf necrosis in that the knife edge is still green.
FIG. 6 shows leaf wound phenotype on 8. Mu.g/L chlorsulfuron concentration medium with most leaf necrosis. Fig. 6 (b) is an enlarged view showing the phenotype of leaf necrosis.
FIG. 7 is a statistical schematic of growth of leaf wounds on different chlorsulfuron concentrations of the culture medium.
The method is used for the subsequent apple transgenic process, a vector with a mutant MdAAM gene is constructed (namely, the MdAAM gene replaces kanamycin resistance gene nptII), and then a target gene to be researched is constructed in the vector. Genetic transformation of apples was performed according to the protocol described above.
The constructed vector was first transformed into Agrobacterium EHA105 and plated onto LB plates of the corresponding resistance. After 3 days of inversion incubation, the bacteria were resuspended using a resuspension (1L of resuspension was added with 1ml AS and 1ml Bet). The bacterial liquid concentration was adjusted to od=0.5.
Selecting middle blades of GL-3 tissue culture seedlings which are subcultured for about 1 month according to the method, marking 3-5 wounds on the blades by using a sharp blade, and then placing the blades into heavy suspension to rotationally shake for 15 minutes;
taking out the resuspended leaf from the resuspension, and placing the leaf on sterile paper to absorb water;
placing the leaves after moisture absorption in a culture medium I, wherein the front surfaces of the leaves are contacted with the culture medium;
leaves were grown on medium I and in the dark at 21 ℃ in an incubator for 5 days;
the leaves were transferred to medium II containing chlorsulfuron at a concentration of 2. Mu.g/L and were cultivated in the dark in an incubator at 21℃for 30 days;
and carrying out transgenic identification on the buds growing.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (7)
1. The method for screening the drug concentration applied to the apple transgenes is characterized in that the drug concentration screening method applied to the apple transgenes is characterized in that mother liquor is obtained by preparing chlorsulfuron-methyl raw drug, filtration sterilization is carried out, and the drug concentration screening method is applied to an apple genetic transformation system with aseptic operation;
the agent concentration screening method applied to apple transgenesis comprises the following steps:
step one, obtaining a chlorsulfuron original drug by purchasing in a reagent company, dissolving the original drug by using DMSO, and filtering and sterilizing by using a 0.22 mu m microporous filter membrane to prepare a chlorsulfuron original drug mother solution;
preparing a resuspension and a culture medium I, and preparing a chlorsulfuron original drug mother solution into a culture medium II with different concentrations; according to the pre-experiment result, selecting chlorsulfuron original medicine with concentration gradient of 0.5-8 mug/L as experiment treatment concentration; the 1L volume of the resuspension consisted of 4.43g MS, 5.88g sodium citrate dihydrate, and 20.00g sucrose, and ph=5.3 was adjusted with glacial acetic acid;
the culture medium I with the volume of 1L consists of 4.43g of MS, 30.00g of sucrose, 1.00mL of TDZ, 0.50mL of NAA, 7.50g of agar powder, 1.00mL of AS and 1.00mL of Bet; adjusting pH=5.8-6.0 with NaOH, and adding AS and Bet after sterilization;
the culture medium II with the volume of 1L consists of 4.43g of MS, 30.00g of sucrose, 1.00mL of TDZ, 0.50mL of NAA, 7.50g of agar powder, 1.00mL of Cef and chlorsulfuron; adjusting the pH value to be between 5.8 and 6.0 by NaOH, and adding chlorsulfuron and Cef with different concentrations after sterilization;
simulating a normal apple genetic transformation method, drawing 3-5 wounds on GL-3 blades in the heavy suspension by using a sharp blade, and rotating and shaking the GL-3 blades in the heavy suspension for 15min;
fourthly, placing the leaves on sterile paper to absorb water, and then placing the leaves in a culture medium I in an inverted mode;
step five, after the leaves are subjected to dark culture on the culture medium I for 5 days, transferring the leaves to the culture medium II with different chlorsulfuron concentrations, and carrying out dark culture in a 21 ℃ incubator for 4 weeks;
step six, counting the sprouting condition and phenotype of the blade wound on the culture mediums with different chlorsulfuron concentrations, and determining that the use concentration of chlorsulfuron in GL-3 transgenes is 2-4 mug/L.
2. The method for screening the concentration of the drug applied to the transgenes of apples according to claim 1, wherein in the first step, the raw drug is dissolved, comprising:
100mg of chlorsulfuron is prepared into a raw medicine mother solution with the concentration of 10mg/mL by using 10mL of DMSO, diluted to 8mg/L by water and stored in a refrigerator at the temperature of minus 80 ℃.
3. The method for screening concentration of drug for apple transgenesis according to claim 1, wherein in the second step, the preparation of the culture medium II with different concentration of chlorsulfuron comprises the following steps:
diluting the 10mg/mL of crude drug mother solution to 8mg/L by water, and then carrying out equal-ratio dilution on the 8mg/L of chlorsulfuron crude drug mother solution to 4mg/L, 2mg/L, 1mg/L and 0.5mg/L respectively; five gradients of crude drugs and culture medium were mixed according to a ratio of 1:1000 to medium II.
4. The method for screening the concentration of the drug applied to the transgenesis of apples according to claim 1, wherein in the third step, the GL-3 leaves are middle leaves of GL-3 tissue culture seedlings which are subcultured for 1 month; 3 to 5 wounds are drawn from the GL-3 leaves in the heavy suspension by using a blade.
5. The method of claim 1, wherein in step three, the resuspension is devoid of agrobacterium.
6. The method for screening the concentration of the drug applied to the transgenesis of apples according to claim 1, wherein in the fifth step, the culturing method comprises the following steps: culturing in dark at 21deg.C for 5 days.
7. Use of the agent concentration screening method for apple transgenes according to any one of claims 1-6 in genetic transformation of apple GL-3 genes.
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