CN116171862B - Genetic transformation system, genetic transformation method and application of solanaceae crops - Google Patents
Genetic transformation system, genetic transformation method and application of solanaceae crops Download PDFInfo
- Publication number
- CN116171862B CN116171862B CN202310251630.5A CN202310251630A CN116171862B CN 116171862 B CN116171862 B CN 116171862B CN 202310251630 A CN202310251630 A CN 202310251630A CN 116171862 B CN116171862 B CN 116171862B
- Authority
- CN
- China
- Prior art keywords
- culture medium
- genetic transformation
- culture
- hypocotyl
- screening
- 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
- 230000002068 genetic effect Effects 0.000 title claims abstract description 50
- 238000011426 transformation method Methods 0.000 title claims abstract description 24
- 230000009466 transformation Effects 0.000 title abstract description 40
- 241000208292 Solanaceae Species 0.000 title abstract description 22
- 239000001963 growth medium Substances 0.000 claims abstract description 81
- 244000061458 Solanum melongena Species 0.000 claims abstract description 47
- 235000002597 Solanum melongena Nutrition 0.000 claims abstract description 46
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 20
- 238000012216 screening Methods 0.000 claims description 53
- 208000015181 infectious disease Diseases 0.000 claims description 31
- 241000589158 Agrobacterium Species 0.000 claims description 30
- 239000007787 solid Substances 0.000 claims description 25
- 239000007788 liquid Substances 0.000 claims description 22
- 239000002609 medium Substances 0.000 claims description 21
- 230000006698 induction Effects 0.000 claims description 20
- 238000012258 culturing Methods 0.000 claims description 19
- 230000001580 bacterial effect Effects 0.000 claims description 17
- 238000005286 illumination Methods 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 14
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 13
- 239000012881 co-culture medium Substances 0.000 claims description 13
- 230000009261 transgenic effect Effects 0.000 claims description 13
- 238000003501 co-culture Methods 0.000 claims description 8
- 239000012883 rooting culture medium Substances 0.000 claims description 8
- 238000002791 soaking Methods 0.000 claims description 8
- 230000004069 differentiation Effects 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 230000009545 invasion Effects 0.000 claims description 5
- 238000009630 liquid culture Methods 0.000 claims description 3
- 238000010362 genome editing Methods 0.000 abstract description 4
- 230000006872 improvement Effects 0.000 abstract description 2
- 230000008929 regeneration Effects 0.000 description 23
- 238000011069 regeneration method Methods 0.000 description 23
- 241000196324 Embryophyta Species 0.000 description 20
- 230000004083 survival effect Effects 0.000 description 15
- 230000012010 growth Effects 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 238000011282 treatment Methods 0.000 description 11
- 230000002829 reductive effect Effects 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 239000012882 rooting medium Substances 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 229940027257 timentin Drugs 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000035784 germination Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 101100178213 Arabidopsis thaliana HMGB6 gene Proteins 0.000 description 2
- 101100317406 Arabidopsis thaliana WRKY53 gene Proteins 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 206010061217 Infestation Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 229960002523 mercuric chloride Drugs 0.000 description 2
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 238000011533 pre-incubation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- RLGNNNSZZAWLAY-UHFFFAOYSA-N 2-(2,3-dimethoxy-4-methylsulfanylphenyl)ethanamine Chemical compound COC1=C(CCN)C=CC(SC)=C1OC RLGNNNSZZAWLAY-UHFFFAOYSA-N 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 102000007156 Resistin Human genes 0.000 description 1
- 108010047909 Resistin Proteins 0.000 description 1
- 235000002634 Solanum Nutrition 0.000 description 1
- 241000207763 Solanum Species 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000011369 optimal treatment Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000004017 vitrification Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- 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
- A01H4/002—Culture media 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
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a genetic transformation system, a genetic transformation method and application of solanaceae crops, and relates to the technical field of genetic transformation. The invention provides a culture medium combination of solanaceous crops, and constructs a genetic transformation system and a genetic transformation method of the solanaceous crops based on the culture medium combination, and the transformation efficiency is greatly improved compared with the transformation efficiency by taking cotyledons as explants by selecting eggplant hypocotyls as the explants. The genetic transformation method can better study the gene function of the eggplant, improve the yield, quality and resistance of the eggplant, and plays a vital role in realizing innovation and utilization of germplasm resources of the eggplant, directional improvement of varieties and the like by using biotechnology such as gene editing and the like.
Description
Technical Field
The invention belongs to the technical field of genetic transformation, and particularly relates to a genetic transformation system, a genetic transformation method and application of solanaceae crops.
Background
Eggplant (Solanum melongena L.) is annual herb plant of Solanum genus of Solanaceae family, china is the largest eggplant producing country in the world, and eggplant has the characteristics of high yield, strong adaptability, long supply time and the like, and is one of main vegetables in summer and autumn. However, the eggplant is often affected by bad environment and plant diseases and insect pests in the cultivation process, and the yield and quality of the eggplant are seriously reduced. With the development of molecular biology and biotechnology, the cultivation of new eggplant varieties with adverse environment resistance, insect resistance and disease resistance by using biotechnology means is a direction of future development.
Germplasm resources are a source of germplasm technology innovation, and innovation and application of breeding technology, particularly biotechnology, are important means for improving quality and enhancing efficiency of varieties. At present, biotechnology represented by gene editing has become the leading edge and core of international breeding, and the application of technologies such as transgenesis, gene editing and the like requires an efficient and mature plant genetic transformation system as a premise. As a key step of transgenic technology, the establishment of a plant genetic transformation system is particularly important, and is an essential step for exploring the functions of genes in plants. Agrobacterium-mediated methods are the most mature and most mechanistically distinct transgenic methods at present. After infection of plants by Agrobacterium, the T-DNA fragment carried by the self-vector may be inserted into the plant genome. By utilizing the natural characteristic, ti plasmid can be artificially modified, the carrier nonessential sequence is replaced by exogenous gene, and stable inheritance can be realized after plant transformation.
At present, the regeneration system of eggplants mostly takes cotyledons as explants, and no complete genetic transformation system is reported.
Disclosure of Invention
The invention aims to provide a genetic transformation system, a genetic transformation method and application of solanaceae crops, and the hypocotyl is an explant and has higher efficiency than other parts.
The invention provides a culture medium combination of solanaceae crops, which comprises the following independently packaged reagents: induction medium, co-culture medium, screening medium, redifferentiation screening medium and rooting medium;
the induction culture medium takes an MS solid culture medium as a basic culture medium and also comprises 2.0mg/LZT, and the pH value is 5.8-6.0;
The co-culture medium takes an MS solid culture medium as a basic culture medium and also comprises 2.0mg/LZT, and the pH value is 5.8-6.0;
The screening culture medium takes an MS solid culture medium as a basic culture medium, and also comprises 2.0mg/LZT, 200mg/LTim, 75mg/Lkan and 20g/L active carbon, wherein the pH value is 5.8-6.0;
the redifferentiation screening culture medium takes an MS solid culture medium as a basic culture medium, and also comprises 3.0mg/LZT and 75mg/Lkan, and the pH value is 5.8-6.0;
The rooting culture medium takes 1/2MS solid culture medium as basic culture medium, and also comprises 75mg/Lkan and pH value of 5.8-6.0.
The invention also provides a genetic transformation system of the solanaceae crops, which comprises the culture medium combination and a carrier capable of connecting exogenous genes.
The invention also provides a genetic transformation method of the solanaceae crops based on the genetic transformation system, which comprises the following steps: (1) Taking the hypocotyl of the seedling of the solanaceae crop as an explant, and placing the explant on an induction culture medium for dark culture to obtain a pretreated hypocotyl;
(2) Infecting the pretreated hypocotyl by using agrobacterium containing a vector of an exogenous gene, and then co-culturing the infected hypocotyl on a co-culture medium to obtain a co-cultured hypocotyl;
(3) Transferring the co-cultured hypocotyl into a screening culture medium for screening culture, transferring the grown callus into a redifferentiation screening culture medium for differentiation culture, transferring the emitted buds onto a rooting culture medium for rooting culture, and obtaining the genetically transformed seedlings.
Preferably, the solanaceous crop seedlings in step (1) are sterile solanaceous crop seedlings.
Preferably, the method for obtaining the aseptic solanaceae crop seedlings comprises the following steps: soaking the seeds of the solanaceae crops in warm water at 55-60 ℃ for 15min, then soaking the seeds in water at 18-22 ℃ for 8-12 h, sterilizing, inoculating the seeds on a 1/2MS solid culture medium, culturing the seeds in a dark place at 25 ℃, and culturing the seeds under illumination conditions after the seeds are exposed to white, thus obtaining the aseptic solanaceae crop seedlings.
Preferably, the infection of step (2) is an infection of the pretreated hypocotyl with an infection solution prepared by using Agrobacterium having an OD 600 value of 0.8.
Preferably, the method for preparing the dyeing liquor comprises the following steps: inoculating agrobacterium containing exogenous gene vector into LB culture medium, transferring bacterial liquid into YEP liquid culture medium containing Rif and Kan for culturing, centrifuging to collect bacterial body when OD 600 is 0.8, and re-suspending with equal volume suspension to obtain the invasion liquid.
Preferably, the suspension is a 1/2MS liquid medium containing AS.
Preferably, the temperature of the co-culture in the step (2) and the screening culture and the differentiation culture in the step (3) are 25+/-1 ℃, the illumination intensity is 2500Lx, and the illumination time is 16h/d.
The invention also provides application of the culture medium combination or the genetic transformation system or the genetic transformation method in cultivating transgenic solanaceous crops.
The beneficial effects are that: the invention provides a genetic transformation system of solanaceous crops, and a genetic transformation method of the solanaceous crops is constructed based on the genetic transformation system, and by selecting the hypocotyl of eggplant as an explant, the transformation efficiency is greatly improved compared with that of cotyledons as the explant, and compared with the cotyledons, the regeneration rate of resistant buds is about 58.7% higher, and the seedling rate of resistant seedlings is about 38.3% higher; by adding active carbon into the culture medium, the proliferation of buds, the growth of stems and seedlings are promoted, and the browning phenomenon of tissue culture is effectively prevented; effectively inhibit the growth of agrobacterium by using a suitable timentin Tim; the transformation efficiency is made higher by using a suitable pre-incubation time, time of Agrobacterium infection and time of co-incubation. The whole callus induction process mainly uses MS solid culture medium and 2.0mg/LZT culture medium, and the components of the culture medium are simpler and more efficient; and by utilizing the resistin with proper concentration, the bud of the unsuccessful transformation gene is whitened and dead, and the transgenic eggplant seedling containing the exogenous gene vector fragment can be differentiated and grown, so that the genetic transformation system of the eggplant taking the hypocotyl as an explant is perfected. The genetic transformation method can better study the gene function of the eggplant, improve the yield, quality and resistance of the eggplant, and plays a vital role in realizing innovation and utilization of germplasm resources of the eggplant, directional improvement of varieties and the like by using biotechnology such as gene editing and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a photograph of a transgenic eggplant seedling;
FIG. 2 is a graph showing the effect of different preculture times on explant growth;
FIG. 3 shows the growth of different explant types in culture medium;
FIG. 4 is a graph showing the DNA detection results of resistant plants.
Detailed Description
The invention provides a culture medium combination of solanaceae crops, which comprises the following independently packaged reagents: induction medium, co-culture medium, screening medium, redifferentiation screening medium and rooting medium;
the induction culture medium takes an MS solid culture medium as a basic culture medium and also comprises 2.0mg/LZT, and the pH value is 5.8-6.0;
The co-culture medium takes an MS solid culture medium as a basic culture medium and also comprises 2.0mg/LZT, and the pH value is 5.8-6.0;
The screening culture medium takes an MS solid culture medium as a basic culture medium, and also comprises 2.0mg/LZT, 200mg/LTim, 75mg/Lkan and 20g/L active carbon, wherein the pH value is 5.8-6.0;
the redifferentiation screening culture medium takes an MS solid culture medium as a basic culture medium, and also comprises 3.0mg/LZT and 75mg/Lkan, and the pH value is 5.8-6.0;
The rooting culture medium takes 1/2MS solid culture medium as basic culture medium, and also comprises 75mg/Lkan and pH value of 5.8-6.0.
The invention also provides a genetic transformation system of the solanaceae crops, which comprises the culture medium combination and a carrier capable of being connected with exogenous genes. The vector of the present invention preferably comprises pCambia2301. In performing genetic transformation, an exogenous gene is inserted into the vector, and agrobacterium is transformed with the vector containing the exogenous gene. The active carbon in the screening culture medium has the functions of promoting the proliferation of buds, the growth of stems and seedlings and resisting browning. In the examples, the transcription factor WRKY53 (sme2.5_00013.1_g00025.1wrky 53) was used for genetic transformation, but it cannot be regarded as the full scope of the present invention.
The invention also provides a genetic transformation method of the solanaceae crops based on the genetic transformation system, which comprises the following steps: (1) Taking the hypocotyl of the seedling of the solanaceae crop as an explant, and placing the explant on an induction culture medium for dark culture to obtain a pretreated hypocotyl;
(2) Infecting the pretreated hypocotyl by using agrobacterium containing a vector of an exogenous gene, and then co-culturing the infected hypocotyl on a co-culture medium to obtain a co-cultured hypocotyl;
(3) Transferring the co-cultured hypocotyl into a screening culture medium for screening culture, transferring the grown callus into a redifferentiation screening culture medium for differentiation culture, transferring the emitted buds onto a rooting culture medium for rooting culture, and obtaining the genetically transformed seedlings.
The invention takes hypocotyl of Solanaceae crop seedling as explant, and places the explant on induction culture medium for dark culture to obtain pretreated hypocotyl. The solanaceous crop of the invention preferably comprises eggplant and the explant is preferably derived from a sterile solanaceous crop seedling. The method for acquiring the aseptic solanaceae crop seedlings preferably comprises the following steps: soaking the seeds of the solanaceae crops in warm water at 55-60 ℃ for 15min, then soaking the seeds in water at 18-22 ℃ for 8-12 h, sterilizing, inoculating the seeds on a 1/2MS solid culture medium, culturing the seeds in a dark place at 25 ℃, and culturing the seeds under illumination conditions after the seeds are exposed to white, thus obtaining the aseptic solanaceae crop seedlings.
According to the invention, the seeds are soaked in water at different temperatures, so that the germination rate of the seeds can be improved. The sterilization according to the present invention preferably comprises sterilization with 0.1% mercuric chloride for 3min, followed by rinsing with sterile water 3 times, and pouring out the sterile water to complete the sterilization. The invention pours the sterilized seeds on sterilized filter paper, sucks water to dry, transfers the seeds on a 1/2MS solid culture medium, and cultures the seeds in a dark place at 25 ℃. After the seeds are exposed to white, the seeds are moved to an environment with the illumination time of 16h/d and the light intensity of 2500Lx for cultivation. After 10-15 d, the cotyledons of the aseptic seedlings of the eggplants can be fully stretched.
Preferably, after the sterile solanaceae crop seedlings are obtained, the hypocotyl is cut in a sterile environment, the hypocotyl is cut into small sections of about 1cm, inoculated onto an induction medium and subjected to dark culture for 3 days to obtain the pretreated hypocotyl. The temperature of the dark culture according to the invention is preferably 25.+ -. 1 ℃.
After obtaining the pretreated hypocotyl, the invention utilizes agrobacterium containing a vector of an exogenous gene to infect the pretreated hypocotyl, and then the infected hypocotyl is subjected to co-culture on a co-culture medium to obtain the co-cultured hypocotyl. In the present invention, it is preferable that the pretreatment hypocotyl is infected when the value of the agrobacterial liquid OD 600 is 0.8. The preparation method of the invasion solution for invasion, which is disclosed by the invention, preferably comprises the following steps: inoculating agrobacterium containing exogenous gene vector into LB culture medium, transferring bacterial liquid into YEP liquid culture medium containing Rif and Kan for culturing, centrifuging to collect bacterial body when OD 600 is 0.8, and re-suspending with equal volume suspension to obtain the invasion liquid. The YEP liquid medium of the invention preferably comprises 50mg/LRif and 75mg/LKan. The invention preferably cultures agrobacterium under the condition of 200rpm of a 28 ℃ incubator, when the bacterial liquid OD 600 reaches 0.8, the bacterial liquid is centrifuged for 10min at 4000rpm, the supernatant is discarded, and the collected bacterial body is resuspended by an equal volume of suspension (1/2MS+AS50mg/L), thus obtaining the invaded solution. The time of infestation according to the invention is preferably 15 minutes. After the infection, the hypocotyl after the infection is preferably transplanted to a co-culture medium for co-culture, wherein the temperature of the co-culture is preferably 25+/-1 ℃, the illumination intensity is preferably 2500Lx, and the illumination time is preferably 16h/d. The time of the co-cultivation according to the present invention is preferably 4d.
After obtaining the co-cultured hypocotyl, the co-cultured hypocotyl is transferred into a screening culture medium for screening culture, the grown callus is transferred into a redifferentiation screening culture medium for differentiation culture, and the emitted buds are transferred onto a rooting culture medium for rooting culture, so that the genetically transformed seedlings are obtained. The co-cultured hypocotyl is preferably transferred into a screening culture medium for screening, and the cultured hypocotyl is subjected to secondary culture every 14 days, and after 4 weeks of screening, the grown callus is transferred into a redifferentiation screening culture medium for secondary culture, and sprouting is performed for about 4-6 weeks. The culture temperature is preferably 25+/-1 ℃, the illumination intensity is 2500Lx, and the illumination time is 16h/d.
The invention preferably cuts off buds growing to 2-4 cm, moves the buds to a rooting culture medium for rooting culture, and when the regenerated seedlings grow a plurality of main roots and lateral roots, acclimates the seedlings by opening the tissue culture bottle cap, gradually and completely opens the seedlings over time, and after the seedlings are adapted, takes out the seedlings, washes off the root culture medium and moves the seedlings to soil. The temperature, illumination intensity, illumination time and the like of the culture are preferably the same as those described above, and are not described herein.
The invention also provides application of the culture medium combination or the genetic transformation system or the genetic transformation method in cultivating transgenic solanaceous crops.
Through statistics, the genetic transformation method provided by the invention has the transformation efficiency reaching about 18.8%, and provides an effective method for genetic transformation of eggplants, so that the genetic function research and breeding process of eggplants are accelerated.
For further explanation of the present invention, the genetic transformation system, genetic transformation method and application of the present invention to solanaceous crops will be described in detail with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
Obtaining aseptic seedlings: soaking eggplant seeds in warm water of 55-60 ℃ for 15min, and then soaking in water of about 20 ℃ for overnight to improve the germination rate of the seeds. Sterilizing with 0.1% mercuric chloride for 3min, washing with sterile water for 3 times, pouring out the sterile water, pouring the sterilized seeds on sterilized filter paper, sucking water to dry, transferring the seeds onto 1/2MS medium, and culturing at 25deg.C in dark place. After the seeds are exposed to white, the seeds are moved to an environment with the illumination time of 16h/d and the light intensity of 2500Lx for cultivation. After 10-15 d, the cotyledons of the aseptic seedlings of the eggplants can be fully stretched.
Example 2
Screening of optimal Kan concentration: to reduce the work of testing and identifying transgenic later plants, genes with selectable markers are typically joined to an artificially engineered T-DNA region along with the gene of interest to co-transform plant cells. In the invention, a pCambia2301 carrier is adopted to carry a Kan resistance marker, in order to determine the critical concentration of the explant on Kan tolerance, the explant is inoculated on an induction medium containing different Kan concentrations (0, 25, 50, 75 and 100 mg/L) respectively, and the culture is carried out for 15 days at 26 ℃, 80 explants are cultivated in each treatment, and each treatment is repeated for 3 times. The explants were counted for recovery and survival after screening to determine the appropriate Kan concentration.
As shown in Table 1, kan has a certain inhibition effect on the growth of the normal explants, and the cure rate and the bud ratio of the explants are obviously reduced with the increase of Kan concentration. When Kan is not added in the culture medium, the explant grows normally, callus is generated for 2-3 weeks, bud points grow after 4 weeks, and bud elongation and rooting can be further carried out to obtain a complete regenerated plant. When Kan concentration is 25mg/L, the explant yield is more than 75%, and the growth state of the explant is good; when Kan concentration reaches 50mg/L, the recovery rate is greatly reduced. When the concentration is increased to 75mg/L, the growth inhibition effect of Kan on the explant is more obvious, the callus yield is only 10.4%, a small part of callus is subdivided into bud points, the bud differentiation is slower, the culture is continued for a period of time, and the leaves are found to be whitish, so that the complete plant is difficult to form. When the Kan concentration is continuously increased to 100mg/L, the plant growth is severely inhibited, and the formed few calli grow weakly and cannot induce regeneration buds. Thus 75mg/LKan was chosen as screening concentration for transgenic eggplants.
TABLE 1 growth of explants at different Kan concentrations
Example 3
Screening conditions for transforming eggplants by agrobacterium-mediated method:
a. screening of Pre-incubation time
Pre-culture refers to the time of culture before the infection of the explant by Agrobacterium, which can increase the tolerance of the explant to Agrobacterium. 5 different treatments were set by controlled variable method, 1,2, 3, 4, 5d pre-cultures, all other infections and culture conditions were identical. After 7d of delay screening, photographing is carried out, and the growth condition of the explant is observed.
Pre-culturing: after obtaining aseptic seedlings, the hypocotyl was cut on an ultra-clean bench, the hypocotyl was cut into small pieces of about 1cm, and inoculated onto an induction medium for dark culture. Induction medium: MS solid medium +2.0mg/LZT.
The effect of different preculture times on the explants is shown in fig. 2 and table 2, the explants of preculture 1d grow weakly, almost no callus is formed, the explants are soft after being infected, and the later stage is gradually brown, so that adventitious buds cannot be induced; pre-culturing for 2d, wherein a few explants can form callus at the incision, the explants are harder than the pre-culturing for 1d, but the whole explants are yellow brown, the callus induction rate is 23.9%, and adventitious buds can not be induced in the later period; when the strain is pre-cultured for 3 days, the wound healing degree of the explant can resist the injury after the infection of agrobacterium, the healing rate reaches 69.0%, and 19.0% of the explant forms resistant buds after screening and bud induction; pre-culturing for 4d, wherein the cure rate of the explant after delayed screening culture is 61.2%, which is slightly lower than that of the explant after pre-culturing for 3d, but the regeneration rate of the resistant buds under the treatment is far lower than that of the explant after pre-culturing for 3d, and only 5.5%; pre-culture for 5d, the wound edge of the explant is hard, infection transformation is difficult to carry out, and resistant buds are not induced. Therefore, it is considered that the preculture 3d can better resist the attack of Agrobacterium and the transformation efficiency is optimal.
TABLE 2 Effect of different pretreatment times on explant growth
B. screening of infection conditions
The infection condition of the agrobacterium mainly comprises infection concentration and infection time. The infection concentration can be generally represented by the absorbance value of the bacterial liquid at the wavelength of 600nm, namely OD 600, which is proportional to the concentration of the agrobacterium and can be used for judging the growth condition of the agrobacterium. Infecting eggplant explants with activated 5 concentration gradients (OD 600 is 0.4, 0.6, 0.8, 1.0, 1.2) for 15min respectively to form 5 treatments; the infection time is also one of the influencing factors influencing the infection effect. Eggplant explants 5, 10, 15 and 20min were infected with the same concentration of bacterial liquid, forming 4 treatments. The infected explants were inoculated onto a co-culture medium and dark cultured at 25 ℃.
As shown in Table 3, when OD 600 was 0.4, the survival rate of the explant was the highest, up to 89.3%, but the induced regeneration buds could not grow normally on the Kan-containing medium, and the regeneration buds appeared yellow and white, and finally the regeneration rate of the resistant buds against Kan was low, which was only 15.4%. When the bacterial liquid OD 600 is 0.6, the bacterial liquid activity is increased, and the regeneration rate of the resistant buds reaches 82.4%. When the bacterial liquid OD 600 reaches 0.8, the pollution rate of the explant is further increased, the survival rate of the explant is reduced to 67.1%, but the regeneration rate of the resistant buds is increased to 35.4%. At this time, the agrobacterium growth is in logarithmic phase and the activity is higher. When the bacterial liquid OD 600 reached 1.0, the survival rate of the explant was only 30.4%, and the regeneration rate of the resistant buds was reduced by 25.0% compared with OD 600 at 0.8. The concentration of the bacterial liquid is increased again on the basis of 1.0, and the survival rate of the explant and the regeneration rate of the resistant buds are found to be in a decreasing trend. From table 4 it can be seen that the longer the infestation time, the lower the survival of the explants, while the transformation of resistant shoots showed a tendency to rise before fall. When the infection time is 5min, the survival rate of the explant is highest and is 93.4%, but the regeneration rate of the resistant buds is only 12.9%; when the time is prolonged to 10min, compared with 5min of infection, the survival rate of the explant is reduced by 9%, and the regeneration rate of the resistant buds is increased; when the infection is carried out for 15min, the pollution rate and the death rate of the explant are further increased, and the regeneration rate of the resistant buds is also increased to 41.3%; when the infection is carried out for 20min, the survival rate of the explant is the lowest and is 52.5%, and compared with the infection for 15min, the regeneration rate of the resistant buds is reduced, but no obvious difference exists between the two treatments. In summary, when the OD 600 of the infectious microbe liquid is 0.8 and the infection lasts for 15 minutes, the regeneration rate of the resistant buds is highest, and the infection effect is best.
TABLE 3 influence of different bacterial liquid concentrations on conversion efficiency
TABLE 4 influence of different infection times on conversion efficiency
C. Co-cultivation time screening
The kan75mg/L screening and bacteriostasis treatment are not carried out immediately after the agrobacterium infects the explant, and the agrobacterium and the explant need to be co-cultured for a period of time. The time of co-cultivation greatly influences the transformation efficiency, since after infection with Agrobacterium, the exogenous gene needs at least 16h to integrate with the plant genome. Co-culture was set up with 5 time gradients: 1.2, 3, 4, 5d. Each treatment described above was inoculated with 80 explants and repeated 3 times. The survival rate of the explants after the screening culture is statistically delayed and the probability of obtaining resistant buds finally is determined, so that the optimal treatment is determined.
Co-cultivation: transferring the infected hypocotyl into a co-culture medium, wherein the co-culture medium is: MS solid medium +2.0mg/LZT.
As can be seen from Table 5, the longer the co-culture time, the lower the survival rate of the explants, while the regeneration rate of the resistant shoots tended to increase and then decrease. When co-cultured for 1d, the survival rate of the explant is 75% at the highest, and the regeneration rate of the resistant bud is 15.3% at the lowest; when the co-cultivation time was prolonged to 4d, the survival rate of the explant was reduced to 53%, and at this time, the regeneration rate of the resistant bud was highest, reaching 34.5%; when co-cultured for 5d, the survival rate of the explant is the lowest, 30%, and the regeneration rate of the resistant buds is also reduced by 24.6% compared with that of co-cultured for 4 d. In summary, the co-cultivation time is optimal at 4 d.
TABLE 5 influence of different Co-cultivation times on transformation efficiency
Example 4
Screening of the concentration of timentin: after preculture, agrobacterium infection and co-culture, the agrobacterium attached to the surface of the explant needs to be removed in time. The timentin (Tim) is a novel bacteriostatic agent, and has small influence on plant growth while effectively inhibiting the propagation of agrobacterium. Setting 4 Tim concentration treatments in the delay screening culture medium, namely 0mg/L, 100 mg/L, 200 mg/L and 300mg/L, transferring the explants into a culture medium added with Kan after 7d, starting screening culture, and counting the pollution rate of the explants after 7d and 14d of transferring the explants into the screening culture medium.
Induction screening culture: the hypocotyls obtained in example 3 were transferred to a screening medium for screening, and subcultured every 14 days. Screening the culture medium: MS solid culture medium +2.0mg/LZT+Tim200mg/L+75mg/Lkan +20g/L active carbon.
Each treatment was inoculated with 80 explants and repeated 3 times. As can be seen from Table 6, there was no contamination both after 7 days and after 14 days at a Tim concentration of 200mg/L, and the survival rate after 14 days was as high as 85.5%, so that 200mg/L was taken as the optimum concentration.
TABLE 6 Effect of different timentin concentrations on explants
Example 5
Screening of different explant types of eggplants: according to the optimal parameters obtained in examples 1-4, eggplant cotyledons and hypocotyls are used as explants, and transformation effects of different parts of eggplant seedlings are explored through the above explored eggplant optimal genetic transformation method. The two explants are respectively inoculated with 80 explants on a culture medium, repeated for 3 times, photographed after 4 weeks of bud induction, counted to obtain the regeneration rate of the resistant buds and the seedling rate of the resistant buds at the end of the experiment, and the optimal explant type is determined.
After the induction screening culture of example 4, the following operations were continued:
And (3) redifferentiation screening culture: after four weeks of induction culture, the grown calli are transferred to a redifferentiation medium for two weeks for subculture and germination for about 4-6 weeks. Redifferentiation screening media: MS solid culture medium +2.0mg/LZT +75mg/Lkan;
Rooting cultivation: cutting off the buds growing to 2-4cm in the step (5) on a rooting medium, and carrying out rooting culture. Rooting medium: 1/2MS solid medium +75mg/Lkan;
Transplanting regenerated seedlings: and (3) after the regenerated seedlings grow into a plurality of main roots and lateral roots, hardening seedlings at the opening part of the tissue culture bottle cap, gradually and completely opening the tissue culture bottle cap along with time, taking out the seedlings after the seedlings are adapted, washing off root culture medium, and transferring the seedlings into soil.
As can be seen from FIG. 3, cotyledons are more prone to induce resistant calli, but leaves of tissue culture seedlings induced from cotyledon calli are prone to vitrification and later rooting is difficult. And the callus induced from hypocotyl has healthy growth state of the resistant bud induced in later stage, and is easy to generate root to form complete resistant seedling. Analysis of the rate of regeneration of resistant shoots and the rate of emergence of resistant eggplants revealed (Table 7) that the rate of regeneration of resistant shoots and the rate of emergence of resistant shoots of the hypocotyl were significantly higher than those of cotyledons, and therefore the hypocotyl was selected as the explant for optimal genetic transformation.
TABLE 7 Effect of different explant types on eggplant transformation
Example 6
Identification of transgenic positive eggplants: complete transgenic eggplant seedlings are selected through screening with the critical kan concentration of 75mg/L, and the numbers of the complete transgenic eggplant seedlings are shown in figure 1. The DNA of leaf is extracted by CTAB method, and the DNA of non-transgenic eggplant plant of the same variety is used as negative control. Primer amplification products were designed based on the WRKY53 (SEQ ID NO: sme2.5_00013.1_g00025.1WRKY53) vector sequence and were subjected to 10g/L agarose gel electrophoresis and photographed under observation under a gel imaging system.
Positive plant identification primer:
OE-WR53YZ-F(SEQ ID NO.1)：5'CCCTTCCTCTATATAAGGAAGTTCA3'
OE-WR53YZ-R(SEQ ID NO.2)：5'CTCCAGCTATATCCATCATCAGTAG3'
the amplified product was 500bp and proved to be a positive plant.
The DNA detection result of the resistant plants is shown in fig. 4, 198 complete plants are obtained when the hypocotyl is taken as the explant, 100 plants are randomly extracted for DNA detection, 85 plants are identified as positive, and the transformation efficiency of the explant by taking the hypocotyl is calculated to reach 18.8%.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (8)
1. A genetic transformation method of eggplants, which is characterized by comprising the following steps:
(1) Taking the hypocotyl of eggplant seedlings as an explant, and placing the explant on an induction medium for dark culture to obtain a pretreated hypocotyl; the induction culture medium consists of an MS solid culture medium and 2.0mg/L ZT, and the pH value is 5.8-6.0; the time of the dark culture is 3 days;
(2) Infecting the pretreated hypocotyl by using agrobacterium containing a vector of an exogenous gene, and then co-culturing the infected hypocotyl on a co-culture medium for 4 days to obtain a co-cultured hypocotyl; the co-culture medium consists of an MS solid culture medium and 2.0mg/L ZT, and the pH value is 5.8-6.0; the infection is to infect the pretreated hypocotyl for 15min by using an infection liquid prepared by agrobacterium with an OD 600 value of 0.8;
(3) Transferring the co-cultured hypocotyl into a screening culture medium for screening culture, transferring the grown callus into a redifferentiation screening culture medium for differentiation culture, transferring the emitted buds with the length of 2-4 cm onto a rooting culture medium for rooting culture, and obtaining genetically transformed seedlings; the screening culture medium consists of an MS solid culture medium, 2.0mg/L ZT, 200mg/L Tim, 75mg/L kan and 20g/L active carbon, and the pH value is 5.8-6.0; the redifferentiation screening culture medium consists of an MS solid culture medium, 2.0mg/L ZT and 75mg/L kan, and the pH value is 5.8-6.0;
The rooting culture medium consists of a 1/2MS solid culture medium and 75mg/L kan, and the pH value is 5.8-6.0.
2. The genetic transformation method according to claim 1, wherein the eggplant seedlings of step (1) are sterile eggplant seedlings.
3. The genetic transformation method according to claim 2, wherein the method for obtaining the sterile eggplant seedlings comprises: and (3) soaking eggplant seeds in warm water at 55-60 ℃ for 15min, then soaking in water at 18-22 ℃ for 8-12 h, sterilizing, inoculating to a 1/2MS solid culture medium, culturing in a dark place at 25 ℃, and culturing under illumination after the seeds are exposed to white, thus obtaining the aseptic eggplant seedlings.
4. The genetic transformation method according to claim 1, wherein the temperature of the dark culture in step (1) is 25.+ -. 1 ℃.
5. The genetic transformation method according to claim 1, wherein the method for preparing the invader solution of step (2) comprises: inoculating agrobacterium containing exogenous gene vector into LB culture medium, transferring the obtained bacterial liquid into YEP liquid culture medium containing Rif and Kan, culturing until OD 600 is 0.8, centrifuging to collect bacterial body, and resuspending with equal volume suspension to obtain the invasion liquid.
6. The genetic transformation method according to claim 5, wherein the suspension is a 1/2MS liquid medium containing AS.
7. The genetic transformation method according to claim 1, wherein the temperature of the co-culture in the step (2) and the selection culture, the differentiation culture and the rooting culture in the step (3) are all 25.+ -.1 ℃, the illumination intensity is 2500Lx, and the illumination time is 16h/d.
8. Use of the genetic transformation method according to any one of claims 1 to 7 for the cultivation of transgenic eggplants.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310251630.5A CN116171862B (en) | 2023-03-15 | 2023-03-15 | Genetic transformation system, genetic transformation method and application of solanaceae crops |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310251630.5A CN116171862B (en) | 2023-03-15 | 2023-03-15 | Genetic transformation system, genetic transformation method and application of solanaceae crops |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116171862A CN116171862A (en) | 2023-05-30 |
| CN116171862B true CN116171862B (en) | 2024-05-10 |
Family
ID=86434563
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310251630.5A Active CN116171862B (en) | 2023-03-15 | 2023-03-15 | Genetic transformation system, genetic transformation method and application of solanaceae crops |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116171862B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101191127A (en) * | 2006-11-28 | 2008-06-04 | 上海市农业科学院园艺研究所 | Tomato explant highly effective conversion system induced by agrobacterium |
| CN101946711A (en) * | 2010-08-28 | 2011-01-19 | 中国农业科学院草原研究所 | High-efficiency tissue culture and regeneration method for Medicago sativa |
| CN108004268A (en) * | 2018-01-24 | 2018-05-08 | 金华职业技术学院 | Tomato conversion method and application |
-
2023
- 2023-03-15 CN CN202310251630.5A patent/CN116171862B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101191127A (en) * | 2006-11-28 | 2008-06-04 | 上海市农业科学院园艺研究所 | Tomato explant highly effective conversion system induced by agrobacterium |
| CN101946711A (en) * | 2010-08-28 | 2011-01-19 | 中国农业科学院草原研究所 | High-efficiency tissue culture and regeneration method for Medicago sativa |
| CN108004268A (en) * | 2018-01-24 | 2018-05-08 | 金华职业技术学院 | Tomato conversion method and application |
Non-Patent Citations (7)
| Title |
|---|
| 农杆菌介导抗根结线虫Bt cry6A基因转化茄子的研究;张明华;陈钰辉;刘富中;张映;陈国华;谢丙炎;连勇;;核农学报(第04期);第685-691页 * |
| 农杆菌介导的三月茄遗传转化体系的优化研究;胡鑫;蓝宇涵;陈思焓;谢有林;朱黎黎;陈松林;刘慧欣;苏承刚;;西南大学学报(自然科学版)(第09期);全文 * |
| 农杆菌介导的番茄遗传转化研究进展;高佩;尹锐;林彦萍;张美萍;王康宇;王义;;北方园艺(第14期);全文 * |
| 发根农杆菌介导番茄遗传转化研究;黄彩红, 孙洪祥!黑龙江省鹤岗市东山区蔬园乡, 154101, 周淑香, 于广建;北方园艺(第03期);第32-33页 * |
| 提高农杆菌介导番茄遗传转化效率的研究;曹慧颖;夏润玺;吕淑霞;马镝;姜义仁;秦萍;;北方园艺(第01期);全文 * |
| 番茄遗传转化体系的建立;葛艳辉;赵俊英;崔继哲;;吉林工程技术师范学院学报(自然科学版)(第06期);全文 * |
| 番茄遗传转化体系的建立及LC和GFP标记基因的表达;苏彩霞;李君明;霍秀文;范维强;刘磊;;中国农学通报(第06期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116171862A (en) | 2023-05-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Koroch et al. | In vitro regeneration of Echinacea purpurea from leaf explants | |
| CN101457235B (en) | Method for converting lucerne by vacuum penetrating aided agrobacterium-mediation | |
| CN111657143B (en) | Passion fruit detoxification and rapid propagation method | |
| CN111454983B (en) | Preparation and transformation method of papaya callus agrobacterium transformation receptor | |
| CN100362908C (en) | Strawberry virus elimination method | |
| CN113584072A (en) | Construction method of genetic transformation system of strawberries | |
| CN112753582B (en) | Method for sterilizing and rapidly proliferating stem segments of aleurites montana | |
| CN102487829A (en) | Method of comprehensive detoxification and rapid propagation for starch-type water chestnut | |
| CN112119912B (en) | Method for quickly and efficiently establishing genetic transformation system of sesame hairy roots | |
| CN116411021B (en) | Conversion method of tomato glyphosate screening system | |
| CN111500620B (en) | Preparation and transformation method of papaya hypocotyl agrobacterium transformation receptor | |
| CN113604497A (en) | Genetic transformation method of gramineous plants | |
| CN103340180B (en) | Method for identifying resistance function of aphid-resisting genes through transforming hairy roots of soybean | |
| CN116171862B (en) | Genetic transformation system, genetic transformation method and application of solanaceae crops | |
| CN110558234A (en) | Method for culturing embryonic calluses of catalpa bungei based on stem segments with leaves | |
| CN114606257B (en) | Genetic transformation method for pomegranate agrobacterium | |
| CN113755521B (en) | Construction method of agrobacterium-mediated strawberry 'sweet Charles' genetic transformation system | |
| CN115136893A (en) | Callus regeneration system establishment method for picking of macaque macrosperma | |
| CN102002512A (en) | Genetic transformation method for soybean | |
| CN109479712A (en) | It is a kind of using the non-lignifying side shoot young sprout of China fir as the root induction method of explant | |
| CN113215191B (en) | Agrobacterium-mediated genetic transformation method for toona sinensis | |
| JP2008259497A (en) | Method for creating transformant of domestic variety of soybean through agrobacterium and method for acquiring seed of current generation and progeny of transformant in short period of time | |
| CN100334203C (en) | Establishent of acacia crassicarpa high efficiency transforming system induced by agricultural bacillus | |
| CN106636196B (en) | A kind of peanut method of optimization, efficient mediated by agriculture bacillus | |
| CN106386492B (en) | Induction method of loose embryonic callus of potato |
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 |