CN112626105B - Method and device for obtaining transgenic apple tissue culture seedlings by apple leaf infection - Google Patents
Method and device for obtaining transgenic apple tissue culture seedlings by apple leaf infection Download PDFInfo
- Publication number
- CN112626105B CN112626105B CN202011459073.9A CN202011459073A CN112626105B CN 112626105 B CN112626105 B CN 112626105B CN 202011459073 A CN202011459073 A CN 202011459073A CN 112626105 B CN112626105 B CN 112626105B
- Authority
- CN
- China
- Prior art keywords
- apple
- disc
- bottom plate
- tissue culture
- face
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- 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
-
- 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
- A01H4/001—Culture apparatus for tissue culture
-
- 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
- A01H4/008—Methods for regeneration to complete plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/48—Holding appliances; Racks; Supports
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/16—Vibrating; Shaking; Tilting
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/18—Flow directing inserts
- C12M27/20—Baffles; Ribs; Ribbons; Auger vanes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Sustainable Development (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Environmental Sciences (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Clinical Laboratory Science (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention relates to the technical field of plant cultivation, in particular to a method for obtaining transgenic apple tissue culture seedlings by apple leaf infection, and also provides a device for obtaining transgenic apple tissue culture seedlings by apple leaf infection, which comprises a bottom plate, wherein a supporting plate is vertically and fixedly arranged on the upper end surface of the bottom plate, a top plate extending towards the front is fixedly arranged at the upper end of the supporting plate, a rotating disc is rotatably arranged at the position, close to the front, of the upper end surface of the bottom plate through a supporting structure, a driving disc is arranged at the position, close to the rear of the rotating disc, of the upper end surface of the bottom plate through a driving mechanism, a first connecting shaft and a second connecting shaft which are positioned on a straight line in the front-back direction respectively extend from the position of the edge of the lower end surface of the rotating disc and the edge of the upper end surface of the driving disc, and a transmission rod is movably sleeved on the outer surfaces of the first connecting shaft and the second connecting shaft together, so that the construction of the transgenic apple plants can be carried out at high efficiency, improve the survival rate of the tissue culture seedlings of the transgenic apples and improve the transformation efficiency.
Description
Technical Field
The invention relates to the technical field of plant culture, in particular to a method and a device for obtaining transgenic apple tissue culture seedlings by apple leaf infection.
Background
Transgenic plants have been obtained on apples, in order to reach the apple plants that improve functions such as apple plant insect-resistance, anti-medicament, in order to reach the purpose that improves promotion plant output, but in the transgenic culture process of apple blade, do not have the method of cultivateing out transgenic apple plant among the prior art, when cultivateing, liquid fungus liquid needs the shake cultivation, ensure in the abundant fungus liquid that gets into of oxygen, promote the reproductive growth of bacterial strain, shake through the oscillator among the prior art, the air gets into the effect not good when the culture bottle body is too high, inboard fungus liquid is shaken when the bottle body is short and is easily spilled over, the result of use is poor, after apple blade and fungus liquid mix, the apple blade need press from both sides and get out of tweezers, it is inconvenient because the blade is got in the fungus liquid clamp.
Disclosure of Invention
The invention aims to solve the defects in the background art and provides a method and a device for obtaining transgenic apple tissue culture seedlings by apple leaf infection.
In order to achieve the purpose, the technical scheme adopted by the invention is a method for obtaining transgenic apple tissue culture seedlings by apple leaf infection, which comprises the following steps:
s1, selecting 3-6 leaves just unfolded from the top of apple tree branches, cutting the leaves, and placing the cut leaves on sterilized filter paper;
s2, cutting all the leaves, cutting the leaves perpendicular to the main vein by a clean and aseptic blade for 2-3 times, cutting all the leaves, and culturing in a selective culture medium under a completely dark condition for 2-3 days;
s3, 2-3 days later, putting the leaves into diluted bacterium liquid, mixing and shaking for 15 minutes, taking out all the leaves with forceps, putting the leaves on a sterile filter paper board for moisture absorption, spreading the leaves with the back facing downwards on a selective culture medium, and culturing in a complete dark environment for 16-25 days in a tissue culture room;
s4, after 1 month, the yellow callus of the dense hemp is seen growing out at the cut, the leaves are spread on a new selective culture medium and put under the light for culture;
and S5, after the resistant buds are subjected to light differentiation for 1 month, transferring the resistant buds to a subculture medium, culturing under light, subculturing after the resistant buds grow into a normal shape, detecting and propagating.
Preferably, the selection medium comprises: 4.43-4.46g/L MS culture medium, 2-3mg/L thidiazuron, 0.5-0.6mg/L naphthylacetic acid, 30-32g/L sucrose and 7.5-8.0g/L agar, adjusting pH to 5.6, adding 25-28mg/L kanamycin and 500-550mg/L cephamycin after sterilization before plate inversion.
Preferably, the secondary culture medium comprises: 4.43-4.46g/L MS culture medium, 0.5-1.0 mg/L6-benzylaminopurine, 0.2-0.3mg/L indoleacetic acid, 30-32g/L sucrose and 7.05-7.2g/L agar, and adjusting the pH value to 5.8-6.0.
Preferably, the preparation of the bacterial liquid comprises the following steps:
streaking and plating the glycerol bacteria preserved at minus 80 ℃, and activating on a Rif + gene vector resistant LB solid culture medium for 2-3 days; scratching agrobacterium tumefaciens strains by using sterile toothpicks, adding a certain amount of acetosyringone into 100ml of LB liquid culture medium with Rif + carrier resistance, and shaking and culturing at 28 ℃ for overnight till OD600 is 1.5-2.0; centrifuging at 5000rpm for 5 min; the pellet was resuspended in sterile double-distilled water and diluted to OD600 ═ 0.6-1.0.
Preferably, the preparation of the bacterial liquid comprises the following steps:
transplanting the newly transformed and screened agrobacterium tumefaciens into a new LB liquid culture medium (60-80ml) with corresponding resistance again by using a sterile toothpick, and performing shake culture overnight at 28 ℃ until OD600 is 1.5-2.0; centrifuging at 5000rpm for 5 min; the pellet was resuspended in sterile double distilled water and diluted to OD600 ═ 0.6-1.0.
A device for obtaining transgenic apple tissue culture seedlings by apple leaf infection is used for preparing bacterial liquid and mixing and shaking the leaves and the bacterial liquid in the step S3 in the method for obtaining the transgenic apple tissue culture seedlings by apple leaf infection, and comprises a bottom plate, wherein a supporting plate is vertically and fixedly installed on the upper end face of the bottom plate, a top plate extending towards the front is fixedly installed at the upper end of the supporting plate, a rotating disc is rotatably installed at the position, close to the front, of the upper end face of the bottom plate through a supporting structure, a driving disc is installed at the position, close to the rear of the rotating disc, of the upper end face of the bottom plate through a driving mechanism, a first connecting shaft and a second connecting shaft which are positioned on a straight line in the front-back direction respectively extend from the edge of the lower end face of the rotating disc and the edge of the upper end face of the driving disc, a transmission rod is movably sleeved on the outer surfaces of the first connecting shaft and the second connecting shaft together, and a placing cylinder which is coaxial with the first connecting shaft is fixedly installed on the upper end face of the rotating disc, follow place a section of thick bamboo upper end movable insertion and have vibrate a section of thick bamboo, the up end of roof runs through and sets up the circular mouth coaxial with the rolling disc, the inboard of circular mouth has passed the dwang through meshing mechanism activity, the dwang with place a coaxial and lower extreme fixedly connected with connector, a plurality of drainage flabellum, a plurality of have been laid to the surface annular of connector the common fixedly connected with leak protection net in lower extreme border of drainage flabellum, a plurality of the common fixed cover in border of drainage flabellum is equipped with the housing, the surface of housing and the inner wall activity laminating of vibrating a section of thick bamboo, the upper end of dwang is provided with elevating system.
Preferably, the supporting structure comprises a supporting table fixedly installed on the upper end face of the bottom plate close to the front end, a notch circular groove is formed in the upper end face of the supporting table, an open notch is formed in the rear end face of the notch circular groove, and the rotating disc is rotatably installed on the inner side of the notch circular groove.
Preferably, actuating mechanism includes that fixed mounting is close to the motor of rear end department, connects the drive shaft on motor output shaft at the bottom plate up end, the drive shaft upwards extends and with the coaxial fixed connection of the lower terminal surface of driving-disc, the rear fixed mounting that the up end of bottom plate is close to the motor has the carriage, the up end of carriage and the lower terminal surface activity laminating of driving-disc.
Preferably, the meshing mechanism includes fixed ring gear, slip suit on the circular mouthful inner wall the dwang outside and with fixed ring gear engaged with ratchet, fixed mounting terminal surface about the roof in order to prevent that the ratchet breaks away from the inboard shrouding of circular mouthful, the antislip strip has been laid to the surface annular array of dwang, the inner wall of ratchet extends the connecting cylinder of slip cap in dwang and antislip strip outside, the tooth root radius size of ratchet is the same with the distance size between first connecting axle axis to the rolling disc axis.
Preferably, elevating system includes along vertical direction fixed mounting at the roof up end near the telescoping cylinder of rear end department, fixed connection at the telescoping cylinder upper end and towards connecting plate, the connection pad of fixed connection at the connecting plate front end that the place ahead extends, the upper end fixedly connected with cylinder card of dwang goes into the head, the lower extreme border of connection pad is buckled towards the inboard and is extended the interior turn-up, the lower terminal surface of connection pad extends T type post along the axial, cylinder card activity card goes into between T type post and the interior turn-up, the fixed guide bar that passes the connecting plate that extends the activity perpendicularly of up end of roof.
Compared with the prior art, the invention has the following beneficial effects:
1. the method for obtaining the transgenic apple tissue culture seedlings by apple leaf infection can efficiently construct transgenic apple plants, improve the survival rate of the transgenic apple tissue culture seedlings and improve the transformation efficiency.
2. The device for obtaining the transgenic apple tissue culture seedlings by apple leaf infection has the main technical effects that: in the fungus liquid preparation process, elevating system drives the dwang and the drainage flabellum shifts up, later can pour the LB liquid medium who has transplanted the bacterial strain into and shake a section of thick bamboo, later transfer the housing and stretch into the inboard that shakes a section of thick bamboo, the actuating mechanism drive, make driving-disc and rolling disc do the rotation with angular velocity jointly, it shakes a section of thick bamboo and vibrates and drive the housing and use the rolling disc axis to rotate as the axle simultaneously, engagement mechanism makes the dwang rotate, really the drainage flabellum rotates, blow in the inboard of shaking a section of thick bamboo with the air, promote the entering effect of air, promote the growth of bacterial strain, pivoted drainage flabellum has also effectively prevented the spilling over of fungus liquid simultaneously.
3. After 2-3 sword is drawn with clean aseptic blade with blade perpendicular to owner arteries and veins, can put the blade between the drainage flabellum, and be located the surface of leak protection net simultaneously, later make the housing move down through controlling elevating system, later the blade of being equipped with fall into the inboard of fungus liquid can, later control actuating mechanism operation, when making to vibrate a section of thick bamboo rotation and vibrate, the dwang rotates under the meshing mechanism effect, make blade and fungus liquid shake even mixture, it rises the mechanism in the control after shutting down, mention the housing again, make the blade fished out and vibrate in the section of thick bamboo, it is convenient to take out, it gets with artifical tweezers clamp, high efficiency.
4. In the preferred scheme, when the housing used the rolling disc axis as the axle rotation, the meshing transmission of ratchet and fixed ring gear, the drive dwang rotated, and elevating system when reciprocating the dwang, the ratchet will be hindered and can not break away from the inboard of circular mouth by the shrouding, and the dwang can slide from top to bottom relatively the connecting cylinder simultaneously to ensure to open the upper end of vibrating cylinder, be convenient for take off vibrating cylinder and pour out the fungus liquid or pour the liquid medium into, use scientific facility.
Drawings
FIG. 1 is a schematic structural diagram of an apparatus for obtaining transgenic apple tissue culture seedlings by apple leaf infection according to the present invention;
FIG. 2 is a partial cross-sectional view of an apparatus for obtaining transgenic apple tissue culture seedlings by apple leaf infestation according to the present invention;
FIG. 3 is a cross-sectional view of a support table of an apparatus for obtaining transgenic apple tissue culture seedlings by apple leaf infection according to the present invention;
FIG. 4 is a schematic view of an engaging mechanism of a device for obtaining transgenic apple tissue culture seedlings by apple leaf infestation according to the present invention;
FIG. 5 is an enlarged view of the portion A in FIG. 4 of the device for obtaining transgenic apple tissue culture seedlings by apple leaf infection according to the present invention;
FIG. 6 is a schematic structural view of a rotating rod of the device for obtaining transgenic apple tissue culture seedlings by apple leaf infestation of the present invention;
FIG. 7 is a cross-sectional view at the connecting disc of a device for obtaining transgenic apple tissue culture seedlings by apple leaf infection according to the present invention;
FIG. 8 is a schematic view of a rotating disc and a driving disc of the device for obtaining transgenic apple tissue culture seedlings by apple leaf infestation of the present invention;
FIG. 9 is a schematic view of another view angle of a rotating disk and a driving disk of the device for obtaining transgenic apple tissue culture seedlings by apple leaf infestation of the present invention;
FIG. 10 is a schematic view of a view below a leakage-proof net of a device for obtaining transgenic apple tissue culture seedlings by apple leaf infestation according to the present invention;
FIG. 11 is a cross-sectional view of a circular port of an apparatus for obtaining transgenic apple tissue culture seedlings by apple leaf infestation according to the present invention;
FIG. 12 is a schematic structural diagram of a support platform of a device for obtaining transgenic apple tissue culture seedlings by apple leaf infection.
In the figure: 1. a base plate; 2. a support plate; 3. a top plate; 4. a support table; 5. a notched circular groove; 6. rotating the disc; 7. a drive disc; 8. a transmission rod; 9. a first connecting shaft; 10. a second connecting shaft; 11. a drive shaft; 12. a motor; 13. a support frame; 14. placing the cylinder; 15. a vibrating cylinder; 16. a circular port; 17. rotating the rod; 18. anti-slip strips; 19. a connector; 20. drainage fan blades; 21. a housing; 22. a leakage-proof net; 23. a connecting cylinder; 24. a ratchet wheel; 25. a fixed gear ring; 26. closing the plate; 27. a telescopic cylinder; 28. a connecting plate; 29. a guide rod; 30. connecting the disc; 31. inward curling; 32. a T-shaped column; 33. the cylinder is snapped into the head.
Detailed Description
The following description is presented to disclose the invention so as to enable any person skilled in the art to practice the invention. The preferred embodiments in the following description are given by way of example only, and other obvious variations will occur to those skilled in the art.
Example one
A method for obtaining a transgenic apple tissue culture seedling by apple leaf infection comprises the following steps:
s1, selecting 3-6 leaves which are just unfolded from the top end of the apple branch, cutting the leaves, and putting the leaves on sterilized filter paper;
s2, cutting all the leaves, cutting the leaves perpendicular to the main vein by a clean and sterile blade for 2-3 times, and culturing in a selective culture medium under a complete dark condition for 2 days after all the leaves are cut;
s3, after 2 days, putting the leaves into diluted bacterium solution, mixing and shaking for 15 minutes, taking out all the leaves with forceps, putting the leaves on a sterile filter paper board, sucking water, spreading the leaves on a selective culture medium with the back facing downwards, and culturing for 16 days in a complete dark room;
s4, after 1 month, the yellow callus of the dense hemp is seen growing out at the cut, the leaves are spread on a new selective culture medium and put under the light for culture;
and S5, after the resistant buds are subjected to light differentiation for 1 month, transferring the resistant buds to a subculture medium, culturing under light, subculturing after the resistant buds grow into a normal shape, detecting and propagating.
The selection medium comprises: 4.43g/L MS culture medium, 2mg/L thidiazuron, 0.5mg/L naphthylacetic acid, 30g/L sucrose and 7.5g/L agar, adjusting pH to 5.6, adding 25mg/L kanamycin and 500mg/L cephamycin after sterilization before plate inversion.
The subculture medium comprises: 4.43g/L MS culture medium, 0.5 mg/L6-benzylaminopurine, 0.2mg/L indoleacetic acid, 30g/L sucrose, 7.05g/L agar, adjusting pH to 5.8-6.0.
The preparation of the bacterial liquid comprises the following steps:
streaking and plating the glycerol bacteria preserved at minus 80 ℃, and activating the glycerol bacteria on a Rif + gene vector resistant LB solid culture medium for 2 days; scratching a certain amount of agrobacterium tumefaciens strains by using a sterile toothpick into 100ml of LB liquid culture medium with Rif + carrier resistance, adding a proper amount of acetosyringone, and performing shake culture at 28 ℃ overnight until OD600 is 1.5; centrifuging at 5000rpm for 5 min; the pellet was resuspended in sterile double distilled water and diluted to OD600 ═ 0.6.
Or the preparation of the bacterial liquid comprises the following steps:
the newly transformed and screened agrobacterium tumefaciens is re-scratched into 60ml of a new LB liquid culture medium with corresponding resistance by using a sterile toothpick, and shake culture is carried out overnight at 28 ℃ until OD600 is equal to 1.5; centrifuging at 5000rpm for 5 min; the pellet was resuspended in sterile double distilled water and diluted to OD600 ═ 0.6.
Example two
A method for obtaining a transgenic apple tissue culture seedling by apple leaf infection comprises the following steps:
s1, selecting 3-6 leaves which are just unfolded from the top end of the apple branch, cutting the leaves, and putting the leaves on sterilized filter paper;
s2, cutting all the leaves, cutting the leaves perpendicular to the main vein by a clean and sterile blade for 2-3 times, and culturing in a selective culture medium under a complete dark condition for 2 days after all the leaves are cut;
s3, after 2 days, putting the leaves into diluted bacterium solution, mixing and shaking for 15 minutes, taking out all the leaves with forceps, putting the leaves on a sterile filter paper board, sucking water, spreading the leaves on a selective culture medium with the back facing downwards, and culturing for 16 days in a complete dark room;
s4, after 1 month, the yellow callus of the dense jute grows out from the incision, the leaves are laid on a new selective culture medium, and the culture is carried out under the light;
and S5, transferring the resistant bud to a subculture medium after the resistant bud is subjected to visible light differentiation for 1 month, culturing under light, carrying out subculture after the resistant bud grows into a normal form, detecting and propagating.
The selection medium comprises: 4.44g/L MS culture medium, 2.5mg/L thidiazuron, 0.5mg/L naphthylacetic acid, 31g/L sucrose and 7.6g/L agar, adjusting pH to 5.6, adding 26mg/L kanamycin and 520mg/L cephamycin after sterilization before plate inversion.
The subculture medium comprises: 4.44g/L MS culture medium, 0.7 mg/L6-benzylaminopurine, 0.2mg/L indoleacetic acid, 32g/L sucrose, 7.1g/L agar, adjusting pH to 5.8-6.0.
The preparation of the bacterial liquid comprises the following steps:
streaking and plating the glycerol bacteria preserved at minus 80 ℃, and activating the glycerol bacteria on a Rif + gene vector resistant LB solid culture medium for 2 days; scratching a certain amount of agrobacterium tumefaciens strains by using a sterile toothpick into 100ml of LB liquid culture medium with Rif + carrier resistance, adding a proper amount of acetosyringone, and performing shake culture at 28 ℃ overnight until OD600 is 1.8; centrifuging at 5000rpm for 5 min; the pellet was resuspended in sterile double distilled water and diluted to OD600 ═ 0.7.
Or the preparation of the bacterial liquid comprises the following steps:
the newly transformed and screened agrobacterium tumefaciens is re-scratched into 70ml of a new LB liquid culture medium with corresponding resistance by using a sterile toothpick, and shake culture is carried out overnight at 28 ℃ until OD600 is 1.8; centrifuging at 5000rpm for 5 min; the pellet was resuspended in sterile double distilled water and diluted to OD600 ═ 0.7.
EXAMPLE III
A method for obtaining a transgenic apple tissue culture seedling by apple leaf infection comprises the following steps:
s1, selecting 3-6 leaves just unfolded from the top of apple tree branches, cutting the leaves, and placing the cut leaves on sterilized filter paper;
s2, cutting all the leaves, cutting the leaves perpendicular to the main vein by a clean and sterile blade for 2-3 times, and culturing in a selective culture medium in complete darkness for 3 days after cutting all the leaves;
s3, 3 days later, putting the leaves into diluted bacterium solution, mixing and shaking for 15 minutes, taking out all the leaves with forceps, putting the leaves on a sterile filter paper board, sucking water, spreading the leaves on a selective culture medium with the back facing downwards, and culturing the leaves in a complete dark environment for 16-25 days in a tissue culture room;
s4, after 1 month, the yellow callus of the dense hemp is seen growing out at the cut, the leaves are spread on a new selective culture medium and put under the light for culture;
and S5, transferring the resistant bud to a subculture medium after the resistant bud is subjected to visible light differentiation for 1 month, culturing under light, carrying out subculture after the resistant bud grows into a normal form, detecting and propagating.
The selection medium comprises: 4.45g/L MS culture medium, 3mg/L thidiazuron, 0.55mg/L naphthylacetic acid, 31.5g/L sucrose, 7.8g/L agar, adjusting pH to 5.6, adding 27mg/L kanamycin and 530mg/L cephamycin after sterilization before plate inversion.
The subculture medium comprises: 4.45g/L MS culture medium, 0.9 mg/L6-benzylaminopurine, 0.28mg/L indoleacetic acid, 31.5g/L sucrose, 7.8g/L agar, adjusting pH to 5.8-6.0.
The preparation of the bacterial liquid comprises the following steps:
streaking and plating the glycerol bacteria preserved at minus 80 ℃, and activating for 3 days on a Rif + gene vector resistant LB solid culture medium; scratching the agrobacterium strain with sterile toothpicks into 100ml of LB liquid medium with Rif + carrier resistance, adding a proper amount of acetosyringone, shaking and culturing at 28 ℃ overnight until OD600 is 1.9; centrifuging at 5000rpm for 5 min; the pellet was resuspended in sterile double distilled water and diluted to OD600 ═ 0.9.
Or the preparation of the bacterial liquid comprises the following steps:
the newly transformed and screened agrobacterium tumefaciens is re-scratched into 75ml of a new LB liquid culture medium with corresponding resistance by using a sterile toothpick, and shake culture is carried out overnight at 28 ℃ until OD600 is 1.9; centrifuging at 5000rpm for 5 min; the pellet was resuspended in sterile double distilled water and diluted to OD600 ═ 0.9.
Example four
A method for obtaining transgenic apple tissue culture seedlings by apple leaf infection comprises the following steps:
s1, selecting 3-6 leaves just unfolded from the top of apple tree branches, cutting the leaves, and placing the cut leaves on sterilized filter paper;
s2, cutting all the leaves, cutting the leaves perpendicular to the main vein by a clean and sterile blade for 2-3 times, and culturing in a selective culture medium in complete darkness for 3 days after cutting all the leaves;
s3, after 3 days, putting the leaves into diluted bacteria liquid, mixing and shaking for 15 minutes, taking out all the leaves with a pair of tweezers, putting the leaves on a sterile filter paper board, sucking water, spreading the leaves onto a selective culture medium with the back of the leaves facing downwards, and culturing the leaves in a complete dark environment for 25 days in a tissue culture room;
s4, after 1 month, the yellow callus of the dense jute grows out from the incision, the leaves are laid on a new selective culture medium, and the culture is carried out under the light;
and S5, transferring the resistant bud to a subculture medium after the resistant bud is subjected to visible light differentiation for 1 month, culturing under light, carrying out subculture after the resistant bud grows into a normal form, detecting and propagating.
The selection medium comprises: 4.46g/L MS culture medium, 3mg/L thidiazuron, 0.6mg/L naphthylacetic acid, 32g/L sucrose, 8.0g/L agar, adjusting pH to 5.6, adding 28mg/L kanamycin and 550mg/L cephamycin after sterilization before plate inversion.
The subculture medium comprises: 4.46g/L MS culture medium, 1.0 mg/L6-benzylaminopurine, 0.3mg/L indoleacetic acid, 32g/L sucrose, 7.2g/L agar, adjusting pH to 5.8-6.0.
The preparation of the bacterial liquid comprises the following steps:
streaking and plating the glycerol strain preserved at minus 80 ℃, and activating for 3 days on a Rif + gene vector resistant LB solid culture medium; scratching a certain amount of agrobacterium tumefaciens strains by using a sterile toothpick into 100ml of LB liquid culture medium with Rif + carrier resistance, adding a proper amount of acetosyringone, and performing shake culture at 28 ℃ overnight until OD600 is 2.0; centrifuging at 5000rpm for 5 min; the pellet was resuspended in sterile double distilled water and diluted to OD600 ═ 1.0.
Or the preparation of the bacterial liquid comprises the following steps:
the newly transformed and screened agrobacterium tumefaciens is re-scratched into 80ml of a new LB liquid culture medium with corresponding resistance by using a sterile toothpick, and shake culture is carried out overnight at 28 ℃ until OD600 is 2.0; centrifuging at 5000rpm for 5 min; the pellet was resuspended in sterile double distilled water and diluted to OD600 ═ 1.0.
The method can be used for efficiently constructing the transgenic apple plants, improving the survival rate of the transgenic apple tissue culture seedlings and improving the transformation efficiency.
As shown in fig. 1-12, an apparatus for obtaining transgenic apple tissue culture seedlings by apple leaf infection, which is used for the preparation of bacterial liquid in the method for obtaining transgenic apple tissue culture seedlings by apple leaf infection and the mixing and shaking of leaves and bacterial liquid in step S3, comprises a bottom plate 1, wherein a supporting plate 2 is vertically and fixedly installed on the upper end surface of the bottom plate 1, a top plate 3 extending towards the front is fixedly installed on the upper end of the supporting plate 2, a rotating disc 6 is rotatably installed on the upper end surface of the bottom plate 1 close to the front through a supporting structure, a driving disc 7 is installed on the upper end surface of the bottom plate 1 close to the rear of the rotating disc 6 through a driving mechanism, a first connecting shaft 9 and a second connecting shaft 10 which are positioned on a straight line in the front-back direction respectively extend from the edge of the lower end surface of the rotating disc 6 and the edge of the upper end surface of the driving disc 7, a transmission rod 8 is sleeved on the outer surfaces of the first connecting shaft 9 and the second connecting shaft 10, the up end fixed mounting of rolling disc 6 has and places a section of thick bamboo 14 coaxial with first connecting axle 9, from placing a section of thick bamboo 14 upper end activity and inserting and vibrate a section of thick bamboo 15, the up end of roof 3 runs through sets up and has seted up the circular mouth 16 coaxial with rolling disc 6, the inboard of circular mouth 16 has passed dwang 17 through the meshing mechanism activity, dwang 17 with place a section of thick bamboo 14 coaxial and lower extreme fixedly connected with connector 19, a plurality of drainage flabellum 20 has been laid to the surface annular of connector 19, the common fixedly connected with leak protection net 22 in lower extreme border of a plurality of drainage flabellum 20, the common fixed cover in border of a plurality of drainage flabellum 20 is equipped with housing 21, the surface of housing 21 and the movable laminating of the inner wall of a vibration section of thick bamboo 15, the upper end of dwang 17 is provided with elevating system.
The bearing structure comprises a supporting table 4 fixedly installed on the upper end face of the bottom plate 1 and close to the front end, a notched circular groove 5 is formed in the upper end face of the supporting table 4, an open notch is formed in the rear end face of the notched circular groove 5, the rotating disc 6 rotates and is installed on the inner side of the notched circular groove 5, and the notch is used for providing a space for the movement of the transmission rod 8.
Actuating mechanism includes motor 12 that fixed mounting is close to rear end department at 1 up end of bottom plate, connect the drive shaft 11 at motor 12 output epaxial, drive shaft 11 upwards extend and with the coaxial fixed connection of lower terminal surface of driving-disc 7, the rear fixed mounting that the up end of bottom plate 1 is close to motor 12 has carriage 13, carriage 13's up end and the lower terminal surface activity laminating of driving-disc 7, motor 12 drives drive shaft 11 and rotates, and then make driving-disc 7 rotate, carriage 13 plays the effect of supporting driving-disc 7, and the increase of strength.
The meshing mechanism comprises a fixed toothed ring 25 fixedly arranged on the inner wall of the circular opening 16, a ratchet wheel 24 which is slidably sleeved on the outer side of the rotating rod 17 and is meshed with the fixed toothed ring 25, sealing plates 26 fixedly arranged on the upper end surface and the lower end surface of the top plate 3 to prevent the ratchet wheel 24 from being separated from the inner side of the circular opening 16, anti-slip strips 18 are distributed on the outer surface of the rotating rod 17 in an annular array, connecting cylinders 23 which are slidably sleeved on the outer sides of the rotating rod 17 and the anti-slip strips 18 extend out of the inner wall of the ratchet wheel 24, the radius of the root circle of the ratchet wheel 24 is the same as the distance between the central axis of the first connecting shaft 9 and the central axis of the rotating disk 6, when the housing 21 rotates by taking the central axis of the rotating disk 6 as the axis, the ratchet wheel 24 is in meshing transmission with the fixed toothed ring 25 to drive the rotating rod 17 to rotate, when the lifting mechanism moves the rotating rod 17 up and down, the ratchet wheel 24 is blocked by the sealing plates 26 and cannot be separated from the inner side of the circular opening 16, and the rotating rod 17 can slide up and down relative to the connecting cylinders 23, in order to ensure to open the upper end of shaking a section of thick bamboo 15, be convenient for take off and shake a section of thick bamboo 15 and pour the fungus liquid or with liquid medium and pour, use scientific convenience.
The lifting mechanism comprises a telescopic cylinder 27 fixedly arranged on the upper end face of the top plate 3 close to the rear end, a connecting plate 28 fixedly connected to the upper end of the telescopic cylinder 27 and extending towards the front, and a connecting plate 30 fixedly connected to the front end of the connecting plate 28, wherein the upper end of the rotating rod 17 is fixedly connected with a cylindrical clamping head 33, the lower end edge of the connecting plate 30 is bent towards the inner side to extend out an inner curled edge 31, the lower end face of the connecting plate 30 extends out a T-shaped column 32 along the axial direction, the cylindrical clamping head 33 is movably clamped between the T-shaped column 32 and the inner curled edge 31, the upper end face of the top plate 3 is fixedly provided with a guide rod 29 which movably penetrates through the connecting plate 28 in a vertical extending mode, the telescopic cylinder 27 drives the connecting plate 28 to move up and down, the rotating rod 17 to move up and down under the action of the cylindrical clamping head 33, and the cylindrical clamping head 33 also rotates between the inner curled edge 31 and the T-shaped column 32 in the process that the rotating rod 17 rotates by taking the central axis of the rotating plate 6 as a shaft, the separation phenomenon can not occur.
In the preparation process of the bacterial liquid used in the step S3, the worker lifting mechanism drives the rotating rod 17 and the drainage fan blade 20 to move upwards, then the LB liquid culture medium with the transplanted bacterial strains can be poured into the oscillation cylinder 15, then the encloser 21 is placed to extend into the inner side of the oscillation cylinder 15, the driving mechanism drives the driving disk 7 and the rotating disk 6 to rotate at the same angular speed, the oscillation cylinder 15 oscillates and simultaneously drives the encloser 21 to rotate around the central axis of the rotating disk 6, the meshing mechanism enables the rotating rod 17 to rotate and really causes the drainage fan blade 20 to rotate, air is blown into the inner side of the oscillation cylinder 15, the air entering effect is improved, the growth of the bacterial strains is promoted, and meanwhile, the rotating drainage fan blade 20 also effectively prevents the bacterial liquid from overflowing.
In the step S3, after the worker scribes the blades perpendicular to the main vein by using clean and sterile blades for 2-3 times, the blades can be placed between the drainage blades 20 and are simultaneously positioned on the surface of the leakage-proof net 22, then the housing 21 is moved downwards by controlling the lifting mechanism, the blades arranged later fall into the inner side of the bacteria liquid, then the driving mechanism is controlled to operate, the oscillating cylinder 15 rotates and oscillates, the rotating rod 17 rotates under the action of the meshing mechanism, so that the blades and the bacteria liquid are uniformly shaken and mixed, the mechanism is controlled to ascend after the machine is stopped, the housing 21 is lifted again, the blades are fished out of the oscillating cylinder 15, the taking out is convenient and fast, the clamping by using manual tweezers is not needed, and the efficiency is high.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (4)
1. The utility model provides an apple blade infects device that obtains transgenic apple tissue culture seedling, includes bottom plate (1), its characterized in that: the upper end face of the bottom plate (1) is vertically and fixedly provided with a supporting plate (2), the upper end of the supporting plate (2) is fixedly provided with a top plate (3) extending towards the front, the upper end face of the bottom plate (1) is provided with a rotary disc (6) in a rotating mode through a supporting structure at a position close to the front, the upper end face of the bottom plate (1) is provided with a driving disc (7) at a position close to the rear of the rotary disc (6) through a driving mechanism, a first connecting shaft (9) and a second connecting shaft (10) which are positioned on a straight line in the front-rear direction respectively extend from the edge of the lower end face of the rotary disc (6) and the edge of the upper end face of the driving disc (7), a transmission rod (8) is movably sleeved on the outer surfaces of the first connecting shaft (9) and the second connecting shaft (10) together, and a placing barrel (14) coaxial with the first connecting shaft (9) is fixedly arranged on the upper end face of the rotary disc (6), the vibration cylinder (15) is movably inserted from the upper end of the placing cylinder (14), a circular opening (16) coaxial with the rotating disc (6) is formed in the upper end face of the top plate (3) in a penetrating mode, a rotating rod (17) penetrates through the inner side of the circular opening (16) through a meshing mechanism, the rotating rod (17) and the placing cylinder (14) are coaxial, the lower end of the rotating rod (17) is fixedly connected with a connecting head (19), a plurality of drainage fan blades (20) are annularly distributed on the outer surface of the connecting head (19), a leakage-proof net (22) is fixedly connected to the lower end edges of the drainage fan blades (20) together, a housing (21) is fixedly sleeved on the edges of the drainage fan blades (20) together, the outer surface of the housing (21) is movably attached to the inner wall of the vibration cylinder (15), and a lifting mechanism is arranged at the upper end of the rotating rod (17);
elevating system includes along vertical direction fixed mounting at roof (3) up end near telescoping cylinder (27), fixed connection in telescoping cylinder (27) upper end and towards connecting plate (28), the connection pad (30) of fixed connection at connecting plate (28) front end that the place ahead extends, the upper end fixedly connected with cylinder card income head (33) of dwang (17), the lower extreme border of connection pad (30) is towards the inboard bending extension interior turn-up (31), the lower terminal surface of connection pad (30) extends T type post (32) along the axial, cylinder card income head (33) activity card is gone into between T type post (32) and interior turn-up (31), the fixed guide bar (29) that the activity passed connecting plate (28) of extending perpendicularly of up end of roof (3).
2. The device for obtaining transgenic apple tissue culture seedlings through apple leaf infection according to claim 1, wherein the device comprises: the supporting structure comprises a supporting table (4) fixedly installed on the upper end face of the bottom plate (1) and close to the front end, a notched circular groove (5) is formed in the upper end face of the supporting table (4), an open notch is formed in the rear end face of the notched circular groove (5), and the rotating disc (6) is rotatably installed on the inner side of the notched circular groove (5).
3. The device for obtaining transgenic apple tissue culture seedlings through apple leaf infection according to claim 1, is characterized in that: actuating mechanism includes motor (12), the connection drive shaft (11) of motor (12) output shaft that fixed mounting is close to rear end department at bottom plate (1) up end, drive shaft (11) extend upwards and with the coaxial fixed connection of the lower terminal surface of driving-disc (7), the rear fixed mounting that the up end of bottom plate (1) is close to motor (12) has carriage (13), the up end of carriage (13) and the lower terminal surface activity laminating of driving-disc (7).
4. The device for obtaining transgenic apple tissue culture seedlings through apple leaf infection according to claim 1, wherein the device comprises: meshing mechanism includes fixed ring gear (25), the slip suit on circular mouthful (16) inner wall and with fixed ring gear (25) engaged with ratchet (24), fixed mounting terminal surface about roof (3) in order to prevent that ratchet (24) from breaking away from circular mouthful (16) inboard shrouding (26) in the dwang (17) outside, antislip strip (18) have been laid to the surface annular array of dwang (17), the inner wall of ratchet (24) extends connecting cylinder (23) of slip cover in dwang (17) and antislip strip (18) outside, the root of tooth radius size of ratchet (24) is the same with first connecting axle (9) axis to the distance size between rolling disc (6) axis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011459073.9A CN112626105B (en) | 2020-12-13 | 2020-12-13 | Method and device for obtaining transgenic apple tissue culture seedlings by apple leaf infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011459073.9A CN112626105B (en) | 2020-12-13 | 2020-12-13 | Method and device for obtaining transgenic apple tissue culture seedlings by apple leaf infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112626105A CN112626105A (en) | 2021-04-09 |
| CN112626105B true CN112626105B (en) | 2022-07-26 |
Family
ID=75312254
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011459073.9A Active CN112626105B (en) | 2020-12-13 | 2020-12-13 | Method and device for obtaining transgenic apple tissue culture seedlings by apple leaf infection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112626105B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997025434A1 (en) * | 1996-01-13 | 1997-07-17 | Advanced Technologies (Cambridge) Limited | Genetic transformation of trees |
| CN104686355A (en) * | 2015-03-04 | 2015-06-10 | 鲁东大学 | Method for increasing callus rate and sprouting ratio of leaves of aseptic apple seedlings |
| CN106636180A (en) * | 2016-09-18 | 2017-05-10 | 山东农业大学 | Plasmid vector used for obtaining plant with hypersensitivity to salt stress and method |
| CA3003867A1 (en) * | 2017-05-24 | 2018-11-24 | Guifang Jia | A transgenic plant and the method for producing the same |
| CN109169287A (en) * | 2018-10-19 | 2019-01-11 | 中国科学院新疆生态与地理研究所 | A method of the Malus sieversii Regeneration System based on callus differentiation |
| CN111690678A (en) * | 2020-05-20 | 2020-09-22 | 山东农业大学 | Method for obtaining whole transgenic woody plant by utilizing agrobacterium rhizogenes transformation |
-
2020
- 2020-12-13 CN CN202011459073.9A patent/CN112626105B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997025434A1 (en) * | 1996-01-13 | 1997-07-17 | Advanced Technologies (Cambridge) Limited | Genetic transformation of trees |
| CN104686355A (en) * | 2015-03-04 | 2015-06-10 | 鲁东大学 | Method for increasing callus rate and sprouting ratio of leaves of aseptic apple seedlings |
| CN106636180A (en) * | 2016-09-18 | 2017-05-10 | 山东农业大学 | Plasmid vector used for obtaining plant with hypersensitivity to salt stress and method |
| CA3003867A1 (en) * | 2017-05-24 | 2018-11-24 | Guifang Jia | A transgenic plant and the method for producing the same |
| CN109169287A (en) * | 2018-10-19 | 2019-01-11 | 中国科学院新疆生态与地理研究所 | A method of the Malus sieversii Regeneration System based on callus differentiation |
| CN111690678A (en) * | 2020-05-20 | 2020-09-22 | 山东农业大学 | Method for obtaining whole transgenic woody plant by utilizing agrobacterium rhizogenes transformation |
Non-Patent Citations (2)
| Title |
|---|
| Tina Schäfer等.Chitinase activities, scab resistance, mycorrhization rates and biomass of own-rooted and grafted transgenic apple.《Genet Mol Biol》.2012, * |
| 影响农杆菌基因转化效率因素的研究;郑进等;《黄石理工学院学报》;20081015;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112626105A (en) | 2021-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107557381A (en) | A kind of foundation and its application of Chinese cabbage CRISPR Cas9 gene editing systems | |
| JP5013143B2 (en) | Production method of cuttings of Pinaceae trees | |
| KOZAI et al. | Effects of the number of air changes per hour of the closed vessel and the photosynthetic photon flux on the carbon dioxide concentration inside the vessel and the growth of strawberry plantlets in vitro | |
| CN110604059B (en) | Method for in vitro culture of regeneration plants by using stem segments of camellia oleifera | |
| CN112626105B (en) | Method and device for obtaining transgenic apple tissue culture seedlings by apple leaf infection | |
| CN207383057U (en) | A kind of cultivation greenhouse with automatic closure ceiling | |
| CN111893133A (en) | Agrobacterium-mediated cabbage heart genetic transformation method | |
| CN107155896A (en) | A kind of method for promoting African Chrysanthemum tissue culture to transplant seedling rooting | |
| CN109122276A (en) | A kind of vegetable cultivation device for raising seedlings facilitating extraction | |
| CN109479726A (en) | The quick breeding method for tissue culture of the red early cherry in capital | |
| CN105532453A (en) | Two-step culturing method for seeding formation tissue of Tylophora kerrii | |
| CN107125005B (en) | Mycorrhizal seedling raising method for paphiopedilum harderi | |
| CN109984043B (en) | Method for culturing tissue of vaccinium camphorate by taking leaves as explants | |
| JP2001186814A (en) | Method for preparing new rooted cutting | |
| CN1109706A (en) | Method for producing potato microtubers | |
| CN116250482B (en) | Method for tissue culture and rapid propagation of coconuts | |
| CN108770690A (en) | A kind of method for building up carrying out efficient stable regeneration system using Dendrocalamus hamiltonii bud point | |
| CN110651715B (en) | Industrial seedling growing method for actinidia arguta | |
| CN214316591U (en) | Device for improving growth and transplanting survival rate of tissue culture seedlings of cercis chinensis | |
| CN105850740A (en) | Screening method for pear homologous transgene functional verification material | |
| CN112772415B (en) | Method for rapid propagation of banana seedlings | |
| CN112493126B (en) | Method for induction of lagerstroemia indica somatic embryo and plant regeneration | |
| CN1319436C (en) | Limonium suffruticosum test tube rapid breeding method for factory | |
| CN103229717A (en) | Method for improving zantedeschia hybrida tissue-culture clustered bud proliferation rate | |
| CN107549013A (en) | Improve the embryo processing method and special culture media of plum blossom matured cotyledons adventitious bud induction frequency |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |