CN102286526B - Method for quickly obtaining capsicum transgenic plant - Google Patents
Method for quickly obtaining capsicum transgenic plant Download PDFInfo
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- CN102286526B CN102286526B CN 201110231054 CN201110231054A CN102286526B CN 102286526 B CN102286526 B CN 102286526B CN 201110231054 CN201110231054 CN 201110231054 CN 201110231054 A CN201110231054 A CN 201110231054A CN 102286526 B CN102286526 B CN 102286526B
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Abstract
The invention relates to a method for quickly obtaining a capsicum transgenic plant, which comprises the following steps: sampling; activating and infecting agrobacterium; preparing a regeneration culture medium and a screening culture medium; inoculating the immature embryo, and culturing; and forming adventitious bud and regenerating the plant, thereby efficiently obtaining the capsicum transgenic regenerated plant. In the invention, it usually takes 7 weeks from the immature embryo infected by agrobacterium to the acquisition of the regenerated plant, the formation rate of the regenerated plant in the culture process is up to 50% to the maximum, and the positive rate of the transgenic plant is up to 80%. In the invention, the immature embryo is used as a receptor material to infect the agrobacterium and carry out inoculation and culture so as to directly form the regenerated plant, thereby shortening the culture period and enhancing the culture effect and genetic transformation efficiency. If applied to genetic functional verification and breeding practice, the invention can greatly accelerate the research into capsicum functional genomics and the development of breeding practice.
Description
Technical field
The present invention relates to the acquisition methods of transfer-gen plant in biological technical field, relate in particular to and a kind ofly by Agrobacterium, infect the method that the capsicum immature embryo obtains transfer-gen plant fast.
Background technology
Transgenic technology is to utilize the technology such as recombinant DNA, cell tissue cultivation or the conversion of germplasm system, by exogenous gene transfered plant cell or tissue, makes it directed genetic recombination material, and the improvement plant trait, cultivate the good quality and high output New Crop Varieties.From the first transfer-gen plant birth of nineteen eighty-three, the natural plant gene engineering starts the inherited character of orderly improvement plant under Artificial Control.With the conventional breeding method, compare, it has following Some features: 1) be not subject to the restriction of sibship, can realize exchanging of genetic material between animal, plant and microorganism, thereby take full advantage of the various genetic resourcess that nature exists; 2) effectively break the chain of beneficial gene and unfavorable gene, take full advantage of useful gene; 3) accelerate breeding process, shortening the breeding cycle.
Since the first case transgene tobacco comes out, plant transgene research and application development are rapid.Till 1997, whole world transgenic plant relate to more than 200 kinds of at least 35 sections.At present, the plant transgenosis has become the strong laboratory facilities of molecular biology of plants research; Gene clone especially, the requisite experimental tool of functional genome research.In recent years, genetic transforming method is constantly brought forth new ideas, and the plant transgene operation system of having delivered has nearly 10 kinds, is broadly divided into three classes with the principle of conversion system: 1) agriculture bacillus mediated transgenosis; 2) take the direct transfer that protoplastis, cell or tissue be acceptor, as electricity swashs, microinjection, PEG mediated method; 3) transgenosis of germplasm system, as utilize ovary injection, embryo, somatic embryo and pollen.What application was maximum at present is Agrobacterium tumefaciens mediated transgenic method, in more than 200 kind of transgenic plant obtaining so far, more than 80%, utilizes the method to obtain.By 1999, at least 30 countries carried out amounting to the field test of the genetically modified crops more than 30,000 times, and the economic characters of improvement have more than 10; The transformed variety of 9 kinds of crops such as existing soybean, corn, cotton, rape, tomato, potato, tobacco, summer squash and papaya has dropped into to be commercially produced, and has obtained good Social benefit and economic benefit.
Capsicum is Solanaceae Capsicum vegetable crop, is one of vegetable crop of China's cultivated area maximum.Since carrying out first tissue culture of pepper work, different capsicum explants has both at home and abroad appearred adopting in succession, as cotyledon, stem apex, stem section, blade, hypocotyl, cotyledon petiole, flower pesticide and protoplastis thereof etc. carry out the report of tissue culture.But tissue culture of pepper has very strong Serotype-dependent, regeneration efficiency is undesirable, in majority test, the regeneration period of plant is long or differentiation frequency is low, the energy for growth of its indefinite bud is poor, have even rest on the bud induction period and can not further growth, this has limited the progress of Gentic Engineering on Capsium annuum to a great extent.At present, about capsicum transgenosis work sutdy, mainly concentrate in the foundation of genetic conversion system, but the application of capsicum transgenic technology in actual breeding that the genetic transformation process has still existed some subject matters to limit, as: culture cycle is long, plant regeneration is difficult and the genetic transformation rate is low etc.Therefore, how setting up ripe genetic conversion system, improving the genetic transformation rate is to be badly in need of the difficult problem solved in the work of capsicum transgenosis.
Summary of the invention
technical problemthe purpose of this invention is to provide and a kind ofly by Agrobacterium, infect the method that the capsicum immature embryo obtains transfer-gen plant fast.The method can be used for the checking of new gene function and the acquisition of good transgenic breeding material, and also the transgenic research for other crops provides reference.
technical schemethe invention provides and a kind ofly by Agrobacterium, infect the method that the capsicum immature embryo obtains transfer-gen plant fast.Its process is the capsicum immature embryo first to be carried out to Agrobacterium infect, then is cultivated, and then the regeneration plant obtained is carried out to the PCR evaluation, obtains in a short time transfer-gen plant.Concrete steps are as follows:
1) sampling: at picking fruit phase fine day, from healthy and strong plant, take from the haw (pollination after 40 ~ 50 days) of right maturation.
2) Agrobacterium activates and infects: will contain goal gene (Rhizome of Pedate Pinellia agglutinin gene PPA, full length gene 777bp) Agrobacterium LBA4404 is inoculated on the solid medium of the YEB that contains 45mg/L kantlex and 50mg/L Rifampin, after 27 ℃ of dark cultivation 2d, choose single colony inoculation to the YEB liquid nutrient medium that contains 45mg/L kantlex and 45mg/L Rifampin, 27 ℃, the 250rpm shaking culture is to OD
600value is 0.4 ~ 06, bacterium liquid is proceeded in the 50mL centrifuge tube, after the centrifugal bacterium liquid of 4000rpm, with standby after isopyknic sterilized water suspension bacteria liquid.Get 1) in the seed of fruit, with scalper, break it into two, take out subsequently immature embryo and be transferred in off-the-shelf Agrobacterium bacterium liquid, submergence 5 minutes.
3) regeneration culture medium (R substratum) and screening culture medium (S substratum) preparation: R and S substratum minimum medium used are the MS substratum, and carbon source used is sucrose, and peptizer is agar.The moiety of R substratum is MS+2mg/L 6-BA+3% sucrose+0.8% agar, and the moiety of S substratum is MS+2mg/L 6-BA+3% sucrose+0.8% agar+60mg/L kantlex.
4) after the immature embryo infected immature embryo inoculation and cultivating: by 2) drains bacterium liquid on sterilizing filter paper, be placed in 3) cultivate on the R substratum and first cultivate under 27 ℃ of dark conditions after 3 days with aseptic water washing explant for several times, transfer on screening culture medium, dark culturing 1 week, after under 27 ℃ of illuminance 2000lx, light application time 12h, room temperatures, continue to cultivate, every triangular flask is put 30 immature embryos.
5) adventitious bud formation and plant regeneration: latter one week of inoculation can be observed immature embryo and expands.Continue to cultivate after three weeks, can observe successively the appearance of indefinite bud, form subsequently root, bud, the various whole plant of leaf.
6) PCR of transfer-gen plant identifies: the blade of regeneration plant of take is the material extraction genomic dna, utilizes the sequences Design special primer of institute's transforming gene to be increased to DNA, take plasmid and water as contrast.Having or not of expansion band means that transgenosis infects success or not.
beneficial effect
1) in the capsicum transgenic research, utilize first immature embryo to obtain fast transgenic regenerated plant for the examination material.Transgenosis inoculation regeneration incubation time has been shortened in this invention, has improved transformation efficiency, and the transfer-gen plant of acquisition can be used for the functional verification of new gene.Simultaneously, the transfer-gen plant of acquisition can be used as excellent breeding material and applies to breeding practice.
2) capsicum (
capsicum annuuml.) belonging to the Solanaceae Capsicum, is a kind of important vegetable be widely cultivated.Yet, because the capsicum hereditary basis is narrow, germ plasm resource is limited, only adopts that conventional breeding means seed selection new variety not only take time and effort, efficiency is low, and be difficult to make new variety to be significantly increased at aspects such as resistance and qualities.And utilize the inventive method transfer-gen plant fast, not only can shorten the incubation time of genetic transformation in the transgenosis process, improve transformation efficiency, just can be completed in a short time the functional verification of goal gene, and new germ plasm resource can be provided, new strain is significantly improved at aspects such as specific trait (resistance and quality).The transfer-gen plant that application the present invention obtains can be used as the cultivation that breeding material is directly used in new variety.
3) the present invention is based upon and take the capsicum immature embryo and cultivate and to comprise on genotype, sampling time of explant, inoculation bacterial concentration and the systematic study working foundations such as time and medium component as the examination material carries out transgenosis.Infecting immature embryo acquisition transgenic regenerated plant by Agrobacterium can shorten incubation time, improve transformation efficiency.The present invention infects acquisition general need 7 time-of-weeks of immature embryo to regeneration plant from Agrobacterium, and in culturing process, the regeneration plant rate of formation reaches as high as 50%, and the transfer-gen plant positive rate is up to 80%.And immature embryo has the advantages such as the aspect of drawing materials, simple in structure, genotype is abundant, immature embryo is carried out to isolated culture can study exactly the plant regeneration condition, and the transgenic breeding material that can obtain fast Multi-genotype.Simultaneously, by Agrobacterium, infect the screening that Immature embryo culture can carry out good capsicum somaclonal variation material, formulate new germ plasm resource, enlarge the capsicum gene pool.In a word, present method has solid theoretical foundation, and science is strong, method is easy, easy handling and practical application.
The accompanying drawing explanation
Fig. 1 utilizes immature embryo to carry out capsicum transgenic research schema
Fig. 2 embodiment Agrobacterium is infected immature embryo and obtains transfer-gen plant.A. rigidly connect the capsicum immature embryo of planting; B
.the capsicum immature embryo expanded; C
.adventitious bud formation; D. the various transgenic regenerated plant of radical bud leaf;
Fig. 3 embodiment transfer-gen plant PCR the result, the 750bp vicinity has the purpose band to be expressed as transfer-gen plant.
Embodiment
Plant transgenic technology improving biological resistance, improve biological quality, create new germ plasm or the aspects such as kind, human health care and medicine have larger application prospect.Biotechnology can solve the insoluble problem of some routine techniquess as supplementary means, makes agricultural new Industrial Revolution occur once.Transgenic technology is not subject to limit between Crop Species, and the purpose of controlling certain biological character is had from biology and takes out, and by genetic manipulation, it is imported in host cell to be improved and expresses, thereby cultivate new variety.This technology can improve breeding effect greatly, accelerates breeding process, enriches nutrition and the function of farm crop, extends the shelf life, and removes allergic component, improves the resistance such as crop is high temperature resistant, saline and alkaline, disease.The foundation that regeneration system is cultivated by plant tissue is the basis of carrying out the gene genetic conversion.Since carrying out first tissue culture of pepper work, different capsicum explants has both at home and abroad appearred adopting in succession, as cotyledon, stem apex, stem section, blade, hypocotyl, cotyledon petiole, flower pesticide and protoplastis thereof etc. carry out the report of tissue culture.But tissue culture of pepper has very strong Serotype-dependent, regeneration efficiency is undesirable, in majority test, the regeneration period of plant is long or differentiation frequency is low, the energy for growth of its indefinite bud is poor, have even rest on the bud induction period and can not further growth, this has limited the progress of Gentic Engineering on Capsium annuum to a great extent.And utilize immature embryo as transgenic acceptor have regenerative power strong, draw materials conveniently, the advantage such as genotype is abundant, Population is large, therefore immature embryo is carried out to isolated culture can control genetic transformation and plant regeneration condition better, obtain fast the transgenic line of Multi-genotype, and easily obtain the somaclonal variation material.We apply method of the present invention, infect the capsicum immature embryo by Agrobacterium and can obtain at short notice transgenic regenerated plant.
This example just be take the capsicum prematurity as material, adopts the inventive method to infect immature embryo by Agrobacterium and obtained transgenic regenerated plant within 50 days.The Agrobacterium LBA4404 that contains goal gene Rhizome of Pedate Pinellia agglutinin gene PPA can buy from market or adopt classical molecular biology working method obtain (referring to Sambrook J, Fristsh E F, Maniatis T. Molecular Cloning; A Laboratory Manual 2nd ed.NY:Cold Spring Harbor Laboratory Press, 1989).Implementation process is as follows:
1) sampling: take from the haw of right maturation at picking fruit phase fine day from healthy and strong plant, after pollination 40 ~ 50 days;
2) Agrobacterium activates and infects: will contain goal gene (Rhizome of Pedate Pinellia agglutinin gene PPA, full length gene 777bp) Agrobacterium LBA4404 is inoculated on the solid medium of the YEB that contains 45mg/L kantlex and 45mg/L Rifampin, 27 ℃ of dark cultivations after 2 days, choose single colony inoculation to the YEB liquid nutrient medium that contains 45mg/L kantlex and 45mg/L Rifampin, 27 ℃, the 250rpm shaking culture is to OD
600value is 0.4 ~ 06, bacterium liquid is proceeded in the 50mL centrifuge tube, after the centrifugal bacterium liquid of 4000rpm, with standby after isopyknic sterilized water suspension bacteria liquid.Get 1) in the seed of fruit, with scalper, break it into two, take out subsequently immature embryo and be transferred in off-the-shelf Agrobacterium bacterium liquid, submergence 5 minutes;
3) regeneration culture medium (R substratum) and screening culture medium (S substratum) preparation: R and S substratum minimum medium used are the MS substratum, and carbon source used is sucrose, and peptizer is agar.The moiety of R substratum is MS+2mg/L 6-BA+3%wt sucrose+0.8%wt agar, and the moiety of S substratum is MS+2mg/L 6-BA+3%wt sucrose+0.8%wt agar+60mg/L kantlex;
4) after the immature embryo infected immature embryo inoculation and cultivating: by 2) drains bacterium liquid on sterilizing filter paper, be placed in 3) cultivate on the R substratum and first cultivate under 27 ℃ of dark conditions after 3 days with aseptic water washing explant for several times, transfer on screening culture medium, dark culturing 1 week, after under 27 ℃ of illuminance 2000lx, light application time 12h, room temperatures, continue to cultivate, every triangular flask is put 30 immature embryos;
5) adventitious bud formation and plant regeneration: latter one week of inoculation can be observed immature embryo and expands.Continue to cultivate after three weeks, can observe successively the appearance of indefinite bud, form subsequently root, bud, the various whole plant of leaf;
6) PCR of transfer-gen plant identifies: the blade of regeneration plant of take is the material extraction genomic dna, utilizes the sequences Design special primer of institute's transforming gene to be increased to DNA, take plasmid and water as contrast.
Claims (2)
1. one kind obtains the capsicum transgenic plant method fast, it is characterized in that comprising the following step:
1) sampling: at picking fruit phase fine day, from healthy and strong plant, take from the haw of right maturation;
2) Agrobacterium activates and infects: the Agrobacterium LBA4404 that will contain goal gene is inoculated on the solid medium of the YEB that contains 45mg/L kantlex and 45mg/L Rifampin, 27 ℃ of dark cultivations after 2 days, choose single colony inoculation to the YEB liquid nutrient medium that contains 45mg/L kantlex and 45mg/L Rifampin, 27 ℃, the 250rpm shaking culture is to OD
600value is 0.4, bacterium liquid is proceeded in the 50mL centrifuge tube, after the centrifugal bacterium liquid of 4000rpm, with standby after isopyknic sterilized water suspension bacteria liquid; Get 1) in the seed of fruit, take out immature embryo and be transferred in off-the-shelf Agrobacterium bacterium liquid, submergence 5 minutes;
3) regeneration culture medium and screening culture medium preparation: regeneration and screening culture medium minimum medium used are the MS substratum, and carbon source used is sucrose, and peptizer is agar; The moiety of regeneration culture medium is MS+2mg/L 6-BA+3%wt sucrose+0.8%wt agar, and the moiety of screening culture medium is MS+2mg/L 6-BA+3%wt sucrose+0.8%wt agar+60mg/L kantlex;
4) after the immature embryo infected immature embryo inoculation and cultivating: by 2) drains bacterium liquid on sterilizing filter paper, be placed in 3) on regeneration culture medium, use the aseptic water washing explant after cultivating under 27 ℃ of dark conditions, transfer to dark culturing on screening culture medium, then under 27 ℃ of illuminance 2000lx, light application time 12h, room temperatures, continue to cultivate, every triangular flask is put 30 immature embryos;
5) adventitious bud formation and plant regeneration: after inoculation, surrounding forms whole plant.
2. the method for quick acquisition capsicum transgenic plant according to claim 1, it is characterized in that in immature embryo inoculation and culturing process, by 2) in after the immature embryo that infects drains bacterium liquid on sterilizing filter paper, be placed in 3) on regeneration culture medium, use the aseptic water washing explant after first under 27 ℃ of dark conditions, cultivating 1 ~ 5 day, transfer on screening culture medium dark culturing 5 ~ 10 days.
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| CN102618556B (en) * | 2012-04-12 | 2013-09-25 | 重庆大学 | Pepper CaCOI1.2 gene, recombinant expression vector and application thereof |
| CN106102452B (en) | 2014-03-07 | 2018-09-28 | 先锋国际良种公司 | Method and system for extracting monocotyledon plumule |
| CN110982837B (en) * | 2019-12-16 | 2022-08-02 | 北京市农林科学院 | Preparation method of pepper genetic transformation system |
| CN110951776B (en) * | 2019-12-19 | 2023-02-10 | 江苏省农业科学院 | Agrobacterium-mediated pepper genetic transformation method |
| CN113667690B (en) * | 2021-08-19 | 2023-03-31 | 西北农林科技大学 | Rapid and efficient pepper transgenic method |
| CN117535343A (en) * | 2023-11-16 | 2024-02-09 | 武汉市农业科学院 | Method for rapidly, efficiently and stably verifying functions of capsicum gene and application |
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