CN102293153A - High efficiency genetic transformation method for rubber trees based on secondary embryogenesis - Google Patents
High efficiency genetic transformation method for rubber trees based on secondary embryogenesis Download PDFInfo
- Publication number
- CN102293153A CN102293153A CN201110198304XA CN201110198304A CN102293153A CN 102293153 A CN102293153 A CN 102293153A CN 201110198304X A CN201110198304X A CN 201110198304XA CN 201110198304 A CN201110198304 A CN 201110198304A CN 102293153 A CN102293153 A CN 102293153A
- Authority
- CN
- China
- Prior art keywords
- embryoid
- medium
- callus
- genetic transformation
- induce
- 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.)
- Granted
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a high efficiency genetic transformation method for rubber trees based on secondary embryogenesis. The method is characterized by comprising the processes of acquisition of a genetic transformation acceptor--somatic embryoid, genetic transformation of somatic embryoid, propagation of transformed embryoid, plant regeneration of transformed embryoid and transplant of transformed plants. The invention has the characteristics of simple operation and high efficiency, enables repeated, poorly effective and heavy manual labor for continuous inflorescence collection, disinfection and callus induction in conventional genetic transformation and proneness of acquisition of the genetic transformation acceptor to influence of environmental factors like weather and season to be avoided, and allows full utilization of the fact that high efficiency acquisition of embryoid and cyclic propagation of somatic embryo can be obtained through secondary embryogenesis, thereby overcoming the defect of low efficiency in acquiring transformed embryoid and transformed plants by using conventional methods, greatly improving genetic transformation efficiency of rubber trees and playing an important role of technical support in transgenic breeding of rubber trees and functional verification of genes.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of bamboo grows high-efficiency genetic transforming method that takes place based on secondary body embryo.
Background technology
Natural rubber has the incomparable elasticity of synthetic rubber, ductility and thermal conductivity, is the important raw material of industry, is mainly derived from Para rubber tree (Hevea brasiliensis Muell.Arg.).By the conventional hybridization breeding technique, cultivating bamboo grows new varieties needs 20～30 years.Transgenic technology is to shorten bamboo grows breeding cycle, the highly effective approach of quickening rearing new variety.
The acceptor that is used for the bamboo grows Agrobacterium-mediated Transformation at present has two kinds: flower pesticide embryo callus and long-term subculture friable embryogenic callus.
There is following defective in the flower pesticide embryo callus as transformation receptor: drawing materials is subject to seasonal restrictions, it is time-consuming to peel off stamen, the flower pesticide regeneration capacity is poor, therefore can't satisfy conversion requirement throughout the year, can not obtain abundant transfer-gen plant, be difficult to satisfy growing research and production demand.
There is following defective in long-term subculture friable embryogenic callus as transformation receptor: more than the acquisition of friable embryogenic callus needed half a year, the callus that propagation rapidly, embryo is strong was that ratio is very low, induced, screening time is longer; Friable embryogenic callus regeneration plant deformity, growing way, output lack application prospect not as stereotype bud grafting tree.
Therefore, introduce new receptor system, the bamboo grows genetic conversion system is most important efficiently for setting up.Hua Yuwei etc. (2010) have set up rubber tree secondary body embryo generation system, and this system is made up of three processes: 1) inducing of elementary body embryo: flower pesticide, inner integument, tree root or other explant induction callus, and induce elementary somatic embryo to take place then; 2) inducing of secondary body embryo: nascent embryo or secondary embryo are explant, and it is cut to the embryo piece of 0.2-0.5 * 0.2-0.5cm, and evoked callus induces secondary somatic embryo to take place again then, and this process can circulate; 3) plant regeneration: the cotyledon shape embryoid of selecting maturation changes the medium of emerging over to and induces plantlet regeneration.Use this system, an experienced operator can produce 8000 strains in 1 year from root clone plant.The foundation that is established as the efficient genetic conversion system of bamboo grows of this system provides a brand-new approach.Embryoid has clear superiority as transformation receptor: 1 all has material throughout the year, can satisfy at any time to transform needs; 2 save the time and the recruitment of peeling off stamen or inducing, screening stable propagation friable embryogenic callus; 3 secondary body embryos have very strong regeneration capacity, and each workman can produce 8000 strain tissue cultivating seedling every year, can satisfy the research and production needs fully; 4 regeneration plant well developed root systems, growing way is vigorous; 5 conversions need the quantity of explant few, the time of save conversion, recovering to cultivate the gripping callus, improve conversion rate greatly.
The bamboo grows transgenic line propagation mode of having reported has three kinds: 1 transforms friable embryogenic callus propagation back inductor embryo takes place and plant regeneration; 2 transfer-gen plant stem with bud micro cuttages are realized propagation; 3 is scion with the transfer-gen plant, expands numerous by bud grafting.Callus after mode 1 propagation takes place through suspension culture inductor embryo, and vitrification phenomenon is serious, and the regenerated transgenic plant deformity, and all not as contrast, application prospect is pessimistic for growth potential, output; Mode the 2, the 3rd, as transformation receptor, this receptor major defect is that resistance embryoid induction rate is low with anther callus, and normal resistance embryoid ratio is low, and therefore most transgenosis embryoids can't regeneration plant.Transfer-gen plant has just been expanded in little numerous, bud grafting numerous, and it is low really not solve resistance embryoid induction rate, the technical bottleneck that regeneration frequency is too low.Rubber tree secondary body embryo is to be explant with the embryoid, takes place by the loop body embryo, realizes the circulation propagation of embryo to embryo, finally realizes the method for body embryo plant propagation.This regenerating system embryoid induction rate height, normal chick embryo ratio height, and complete three kinds of propagation modes different from the past, be that the resistance embryo that obtains is bred, induce abundant normal embryoid to emerge again, guarantee that as far as possible each transgenosis embryoid all can emerge, it is low thoroughly to solve flower pesticide regenerating system embryoid induction rate, the difficult problem that emergence rate is low.
The present invention is a receptor system with secondary body embryo generation system, sets up a kind of genetic transforming method of having reported of bamboo grows efficiently that is different from.
Summary of the invention
The purpose of this invention is to provide a kind of bamboo grows high-efficiency genetic transforming method that takes place based on secondary body embryo, it occurs as the basis with secondary body embryo, the somatic cell embryoid is that Agrobacterium is infected acceptor, by inducing secondary body embryo to take place, efficient acquisition transforms embryoid and transformed plant, and verifying for the transgenosis of bamboo grows transgenic breeding and functional gene provides genetic transforming method efficiently.
The technical scheme that the present invention is adopted for its technical problem of solution is:
A kind of bamboo grows high-efficiency genetic transforming method that takes place based on secondary body embryo is characterized in that: comprise the acquisition of genetic transformation acceptor-somatic cell embryoid, the genetic transformation of somatic cell embryoid, the propagation that transforms embryoid, the plant regeneration that transforms embryoid and five steps of transplanting of transformed plant:
(a) acquisition of genetic transformation acceptor-somatic cell embryoid: select stalwartness, embryoid that vitality is vigorous, it is separated as transformation receptor from original medium;
(b) genetic transformation of somatic cell embryoid: its process comprises and infects, cultivates altogether, recovers cultivation, callus of induce and atomization;
Infect: OD
600=0.1-0.6 Agrobacterium is infected 3-10min, puts aseptic filter paper and blots bacterium liquid;
Cultivate altogether: the embryoid after infecting is changed on the callus of induce medium that adds the 1-50mg/L silver nitrate, and its pH value is 5.2, cultivates altogether 3-6 days for 20-28 ℃;
Recover to cultivate: the embryoid after cultivating is altogether changed over to add the 1-50mg/L silver nitrate and suppress Agrobacterium and grow on the antibiotic callus of induce medium 23-28 ℃ of dark the cultivation 3-10 days;
Callus of induce: the embryoid after will recovering to cultivate is cut into the embryo piece of 0.2-0.5 * 0.2-0.5cm, change over to then and added the 1-50mg/L silver nitrate, on the antibiotic of inhibition Agrobacterium growth and the callus of induce medium of corresponding selective agent, its pH value is 5.8,23-28 ℃ of dark the cultivation 15-40 days;
Differentiation: change the embryo piece of callusization over to the antibiotic of interpolation inhibition Agrobacterium growth and the differential medium of corresponding selective agent; Its pH value is 5.8,23-28 ℃ of dark the cultivation 30-70 days;
(c) transform the propagation of embryoid: the embryoid of GUS stained positive is cut into the embryo piece of 0.2-0.5 * 0.2-0.5cm, the described callus of induce of repeating step (b), atomization, but remove the silver nitrate of the used medium of callus of induce process; This process is capable of circulation, obtains enough ripe cotyledon shape embryoids until transformant; After circulation 1-3 time, antibacterial antibiotic can be removed;
(d) plant regeneration of conversion embryoid: forward the cotyledon shape embryoid of maturation to the medium of emerging, its pH value is 5.8, and 23-28 ℃ of illumination cultivation emerged in 20-60 days;
(e) transplanting of transformed plant: (1) bud grafting: as scion, seedling or tissue cultivating seedling be as stock with the transformed plant of regeneration, by bud grafting, separate and tie up, pack a series of processes and realize transplanting from the laboratory to the land for growing field crops; (2) husky bed is transplanted: move into husky bed after the transformed plant of regeneration is cleaned medium, control temperature, humidity, disease are lifted canopy gradually behind the two weeks, and transformed plant was extracted sprouting out, grown new root in one month, treated to pack after blade grows to stationary phase.
The employed genetic transformation acceptor of described step (a) is the embryoid that acquisition takes place by the secondary body embryo of generation of explant body embryos such as flower pesticide, inner integument and tree root and body embryo thereof.
The callus of induce medium of described step (b) is to be basal medium with the MS medium, and be potassium dihydrogen phosphate 350-500mg/L, anhydrous calcium chloride 150-400mg/L, four water manganese sulphate 10-40mg/L, epsom salt 400-600mg/L with the content furnishing of the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, epsom salt, and add 2,4-D 0.5-3mg/L, KT 0.5-3mg/L, NAA 0.5-3mg/L, Phytagel 2-3g/L, sucrose 50-90g/L and Sucus Cocois 40-100ml;
The active ingredient of the differential medium of described step (b) is for being minimal medium with the MS medium, and the content of the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, epsom salt is adjusted into potassium dihydrogen phosphate 350-500mg/L, anhydrous calcium chloride 150-400mg/L, four water manganese sulphate 10-40mg/L, epsom salt 400-600mg/L, and add 6-BA 0.5-3mg/L, KT 0.5-3mg/L, NAA0.1-2mg/L, GA31-5mg/L, Phytagel 2-3g/L, sucrose 50-90g/L, activated carbon 0.5-3g/L and Sucus Cocois 40-100ml.
Described step (d) is emerged the active ingredient of medium for being minimal medium with the MS medium, and the content of the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, epsom salt is adjusted into potassium dihydrogen phosphate 350-500mg/L, anhydrous calcium chloride 150-400mg/L, four water manganese sulphate 10-40mg/L, epsom salt 400-600mg/L, and add KT 0.1-1.0mg/L, 6A30.5-3mg/L, IAA 0.5-4mg/L, Phytagel 2-4g/L, sucrose 30-70g/L, activated carbon 0.5-3g/L and Sucus Cocois 40-100ml.
The invention has the beneficial effects as follows:
1, transformation receptor of the present invention was with report was all different in the past, and embryoid has clear superiority as transformation receptor: a, throughout the year material is arranged all, can satisfy the conversion needs at any time; B, save the time and the recruitment of peeling off stamen or inducing, screening stable propagation friable embryogenic callus; C, body embryo have very strong regeneration capacity, and each workman can produce 8000 strain tissue cultivating seedling every year, can satisfy the research and production needs fully; D, regeneration plant well developed root system, growing way is vigorous; E, conversion need the quantity of explant few, the time of save conversion, recovering to cultivate the gripping callus, improve conversion rate greatly.
2, the present invention adopts transformant propagation mode and reports all different in the past, be that the resistance embryo that obtains is bred, induce abundant normal embryoid to emerge again, guarantee that as far as possible each transgenosis embryoid all can emerge, it is low thoroughly to solve flower pesticide regenerating system embryoid induction rate, the difficult problem that emergence rate is low.
3, all very healthy and strong through plant root, stem, the leaf of secondary body embryo generation regeneration, thin and weak different fully with the plant of long-term subculture callus regeneration, and the vitrifying seedling of some can appear in the strongest RITA training method of long-term subculture callus regeneration power, and cotyledon shape embryoid is then avoided this phenomenon fully through secondary body embryo generation regeneration plant.The transfer-gen plant stalwartness has improved transplanting survival rate greatly, thereby further improves conversion ratio.
4, regenerated transgenic plant has the root system next by radicle growth after propagation takes place secondary body embryo, and this root system is identical with the seedling root system, has flourishing main root, helps to improve the wind loading rating of Para rubber tree behind field planting.And the root of the transfer-gen plant that obtains by micro-propagation method is a regenerated root, can develop into similar main root behind field planting, but the fragility that is connected of main root and stem is unfavorable for the Para rubber tree wind resistance.
Embodiment
Embodiment 1:
Below with Agrobacterium tumefaciems EHA105 infect be example the invention will be further described, need to prove that the Molecular Detection and the former method of preparation, transformed plant of engineering bacteria is as broad as long.
(a) acquisition of genetic transformation acceptor-somatic cell embryoid: select the vigorous embryoid of stalwartness, vitality, it is separated as transformation receptor from original medium by flower pesticide, inner integument, tree root cultivation, the generation of secondary body embryo or the differentiation of other body embryogenesis paths.Incubation time is of a specified duration excessively, and the embryoid of vitrifying, albefaction, brownization is not suitable for as transformation receptor.
(b) genetic transformation of somatic cell embryoid: infect: Agrobacterium OD value is transferred to 0.5, and the embryoid that picks out with (a) method infects 5min as acceptor, puts aseptic filter paper and blots bacterium liquid; Cultivate altogether: cultivation temperature is 25 ℃ altogether, and medium is that pH value transfers to 5.2 altogether, adds the callus of induce medium of 5mg/L silver nitrate; Recover to cultivate: with the embryoid after transforming change over to add 5mg/L silver nitrate and 500mg/L spy vanish fourth (Ticarcillin: the callus of induce medium (PH5.8) of potassium clavulanate=15:1), 25 ℃ of darkrooms recover to cultivate 10 days; Callus of induce: the embryoid after will recovering to cultivate is cut into the embryo piece of 0.2 * 0.2cm, changes over to then and has added the 5mg/L silver nitrate, the vanish callus of induce medium (PH5.8) of fourth and 50mg/L kanamycin of 500mg/L spy, 25 ℃ of dark cultivations 20 days.Differentiation: kanamycin-resistant callus tissue changed over to add the vanish differential medium (PH5.8) of fourth and 50mg/L kanamycin of 500mg/L spy, 25 ℃ of dark cultivations 50 days obtain ripe embryoid.Described callus of induce medium is to be basal medium with the MS medium, and be potassium dihydrogen phosphate 350mg/L, anhydrous calcium chloride 200mg/L, four water manganese sulphate 20mg/L, epsom salt 400mg/L with the content furnishing of the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, epsom salt, and add 2,4-D 2mg/L, KT 1mg/L, NAA 1.5mg/L, Phytagel 2g/L, sucrose 80g/L, Sucus Cocois 100ml; The active ingredient of described differential medium is for being minimal medium with the MS medium, and the content of the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, epsom salt is adjusted into potassium dihydrogen phosphate 350mg/L, anhydrous calcium chloride 200mg/L, four water manganese sulphate 20mg/L, epsom salt 400mg/L, and add 6-BA 0.5mg/L, KT 1.0mg/L, NAA 0.1mg/L, GA32mg/L, Phytagel 2.2g/L, sucrose 50g/L, activated carbon 1.0g/L, Sucus Cocois 40ml.
(c) transform the propagation of embryoid: GUS stained positive embryoid is cut into the embryo piece of 0.2 * 0.2cm, repeats (b) described callus of induce, atomization, but remove the silver nitrate of the used medium of callus of induce process.This process is capable of circulation, obtains enough ripe cotyledon shape embryoids until transformant.After circulation 1-3 time, antibacterial antibiotic can be removed.
(d) plant regeneration of conversion embryoid: forward the cotyledon shape embryoid of maturation to the medium of emerging (PH5.8), 25 ℃ of illumination cultivation were emerged in 20-60 days.Emerge the active ingredient of medium for being minimal medium with the MS medium, and the content of the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, epsom salt is adjusted into potassium dihydrogen phosphate 350mg/L, anhydrous calcium chloride 200mg/L, four water manganese sulphate 20mg/L, epsom salt 400mg/L, and add KT 0.5mg/L, GA32mg/L, IAA 0.5mg/L, Phytagel 2g/L, sucrose 50g/L, activated carbon 0.5g/L, Sucus Cocois 100ml.
(e) transplanting of transformed plant: (1) bud grafting: as scion, seedling or tissue cultivating seedling be as stock with the transformed plant of regeneration, by bud grafting, separate and tie up, pack a series of processes and realize transplanting from the laboratory to the land for growing field crops; (2) husky bed is transplanted: move into husky bed after the transformed plant of regeneration is cleaned medium, control temperature, humidity, disease are lifted canopy gradually behind the two weeks, and transformed plant was extracted sprouting out, grown new root in one month, treated to pack after blade grows to stationary phase.
Embodiment 2:
Below with another agrobacterium tumefaciens lba4404 infect be example the invention will be further described, the preparation of same engineering bacteria, the Molecular Detection of transformed plant and former method are as broad as long.
(a) acquisition of genetic transformation acceptor-somatic cell embryoid: select the vigorous embryoid of stalwartness, vitality, it is separated as transformation receptor from original medium by flower pesticide, inner integument, tree root cultivation, the generation of secondary body embryo or the differentiation of other body embryogenesis paths.Incubation time is of a specified duration excessively, and the embryoid of vitrifying, albefaction, brownization is not suitable for as transformation receptor.
(b) genetic transformation of somatic cell embryoid: infect: Agrobacterium OD value is transferred to 0.3, and the embryoid that picks out with (a) method infects 5min as acceptor, puts aseptic filter paper and blots bacterium liquid; Cultivate altogether: cultivation temperature is 20 ℃ altogether, and medium is that pH value transfers to 5.2 altogether, adds the callus of induce medium of 10mg/L silver nitrate; Recover to cultivate: with the embryoid after transforming change over to add 10mg/L silver nitrate and 300mg/L spy vanish fourth (Ticarcillin: callus of induce medium (PH5.8) potassium clavulanate=15: 1), 25 ℃ of darkrooms recover to cultivate 5 days; Callus of induce: the embryoid after will recovering to cultivate is cut into the embryo piece of 0.2 * 0.2cm, changes over to then and has added the 5mg/L silver nitrate, the vanish callus of induce medium (PH5.8) of fourth and 5mg/L hygromycin of 300mg/L spy, 25 ℃ of dark cultivations 20 days.Differentiation: kanamycin-resistant callus tissue changed over to add the vanish differential medium (PH5.8) of fourth and 5mg/L hygromycin of 300mg/L spy, 25 ℃ of dark cultivations 60 days obtain ripe embryoid.Described callus of induce medium is to be basal medium with the MS medium, and be potassium dihydrogen phosphate 500mg/L, anhydrous calcium chloride 400mg/L, four water manganese sulphate 20mg/L, epsom salt 400mg/L with the content furnishing of the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, epsom salt, and add 2,4-D 1mg/L, KT1mg/L, NAA 1mg/L, Phytagel 2g/L, sucrose 60g/L, Sucus Cocois 40ml; The active ingredient of described differential medium is for being minimal medium with the MS medium, and be potassium dihydrogen phosphate 500mg/L, anhydrous calcium chloride 400mg/L, four water manganese sulphate 20mg/L, epsom salt 400mg/L, and add 6-BA1.0mg/L, KT 1.0mg/L, NAA 0.2mg/L, GA3 3mg/L, Phytagel 2.2g/L, sucrose 70g/L, activated carbon 1.0g/L, Sucus Cocois 40ml with the content furnishing of the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, epsom salt.
(c) transform the propagation of embryoid: GUS stained positive embryoid is cut into the embryo piece of 0.2 * 0.2cm, repeats (b) described callus of induce, atomization, but remove the silver nitrate of the used medium of callus of induce process.This process is capable of circulation, obtains enough ripe cotyledon shape embryoids until transformant.After circulation 1-3 time, antibacterial antibiotic can be removed.
(d) plant regeneration of conversion embryoid: forward the cotyledon shape embryoid of maturation to the medium of emerging (PH5.8), 25 ℃ of illumination cultivation were emerged in 20-60 days.Emerge the active ingredient of medium for being minimal medium with the MS medium, and be potassium dihydrogen phosphate 500mg/L, anhydrous calcium chloride 400mg/L, four water manganese sulphate 20mg/L, epsom salt 400mg/L with the content furnishing of the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, epsom salt,, and add KT 0.7mg/L, GA3 2.5mg/L, IAA 0.3mg/L, Phytagel 2.2g/L, sucrose 50g/L, activated carbon 0.5g/L, Sucus Cocois 50ml.
(e) transplanting of transformed plant: (1) bud grafting: as scion, seedling or tissue cultivating seedling be as stock with the transformed plant of regeneration, by bud grafting, separate and tie up, pack a series of processes and realize transplanting from the laboratory to the land for growing field crops; (2) husky bed is transplanted: move into husky bed after the transformed plant of regeneration is cleaned medium, control temperature, humidity, disease are lifted canopy gradually behind the two weeks, and transformed plant was extracted sprouting out, grown new root in one month, treated to pack after blade grows to stationary phase.
Claims (4)
1. bamboo grows high-efficiency genetic transforming method that takes place based on secondary body embryo is characterized in that: comprise the acquisition of genetic transformation acceptor-somatic cell embryoid, the genetic transformation of somatic cell embryoid, the propagation that transforms embryoid, the plant regeneration that transforms embryoid and five steps of transplanting of transformed plant:
(a) acquisition of genetic transformation acceptor-somatic cell embryoid: select stalwartness, embryoid that vitality is vigorous, it is separated as transformation receptor from original medium;
(b) genetic transformation of somatic cell embryoid: its process comprises and infects, cultivates altogether, recovers cultivation, callus of induce and atomization;
Infect: OD
600=0.1-0.6 Agrobacterium is infected 3-10min, puts aseptic filter paper and blots bacterium liquid;
Cultivate altogether: the embryoid after infecting changes on the callus of induce medium that adds the 1-50mg/L silver nitrate, and its pH value is 5.2, cultivates altogether 3-6 days for 20-28 ℃;
Recover to cultivate: the embryoid after cultivating altogether changes over to and adds the 1-50mg/L silver nitrate and suppress Agrobacterium and grow on the antibiotic callus of induce medium, and its pH value is 5.8,23-28 ℃ of dark the cultivation 3-10 days;
Callus of induce: the embryoid after will recovering to cultivate is cut into the embryo piece of 0.2-0.5 * 0.2-0.5cm, change over to then and added the 1-50mg/L silver nitrate, on the antibiotic of inhibition Agrobacterium growth and the callus of induce medium of corresponding selective agent, its pH value is 5.8,23-28 ℃ of dark the cultivation 15-40 days;
Differentiation: change the embryo piece of callusization over to the antibiotic of interpolation inhibition Agrobacterium growth and the differential medium of corresponding selective agent, its pH value is 5.8,23-28 ℃ of dark the cultivation 30-70 days;
(c) transform the propagation of embryoid: the embryoid of GUS stained positive is cut into the embryo piece of 0.2-0.5 * 0.2-0.5cm, the described callus of induce of repeating step (b), atomization, but remove the silver nitrate of the used medium of callus of induce; This process is capable of circulation, obtains enough ripe cotyledon shape embryoids until transformant; After circulation 1-3 time, antibacterial antibiotic can be removed;
(d) plant regeneration of conversion embryoid: forward the cotyledon shape embryoid of maturation to the medium of emerging, its pH value is 5.8, and 23-28 ℃ of illumination cultivation emerged in 20-60 days;
(e) transplanting of transformed plant: (1) bud grafting: as scion, seedling or tissue cultivating seedling be as stock with the transformed plant of regeneration, by bud grafting, separate and tie up, pack a series of processes and realize transplanting from the laboratory to the land for growing field crops; (2) husky bed is transplanted: move into husky bed after the transformed plant of regeneration is cleaned medium, control temperature, humidity, disease are lifted canopy gradually behind the two weeks, and transformed plant was extracted sprouting out, grown new root in one month, treated to pack after blade grows to stationary phase.
2. a kind of bamboo grows high-efficiency genetic transforming method that takes place based on secondary body embryo according to claim 1 is characterized in that: the employed genetic transformation acceptor of described step (a) is the embryoid that acquisition takes place by the secondary body embryo of flower pesticide, inner integument and tree root explant and body embryo thereof.
3. a kind of bamboo grows high-efficiency genetic transforming method that takes place based on secondary body embryo according to claim 1, it is characterized in that: the callus of induce medium of described step (b) is to be basal medium with the MS medium, and with the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, the content furnishing of epsom salt is potassium dihydrogen phosphate 350-500mg/L, anhydrous calcium chloride 150-400mg/L, four water manganese sulphate 10-40mg/L, epsom salt 400-600mg/L, and add 2,4-D 0.5-3mg/L, KT 0.5-3mg/L, NAA 0.5-3mg/L, Phytagel 2-3g/L, sucrose 50-90g/L and Sucus Cocois 40-100ml; The active ingredient of described differential medium is for being minimal medium with the MS medium, and the content of the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, epsom salt is adjusted into potassium dihydrogen phosphate 350-500mg/L, anhydrous calcium chloride 150-400mg/L, four water manganese sulphate 10-40mg/L, epsom salt 400-600mg/L, and add 6-BA 0.5-3mg/L, KT 0.5-3mg/L, NAA 0.1-2mg/L, GA31-5mg/L, Phytagel 2-3g/L, sucrose 50-90g/L, activated carbon 0.5-3g/L and Sucus Cocois 40-100ml.
4. a kind of bamboo grows high-efficiency genetic transforming method that takes place based on secondary body embryo according to claim 1, it is characterized in that: described step (d) is emerged the active ingredient of medium for being minimal medium with the MS medium, and with the potassium dihydrogen phosphate in the MS medium, anhydrous calcium chloride, four water manganese sulphates, the content of epsom salt is adjusted into potassium dihydrogen phosphate 350-500mg/L, anhydrous calcium chloride 150-400mg/L, four water manganese sulphate 10-40mg/L, epsom salt 400-600mg/L, and add KT 0.1-1.0mg/L, GA3 0.5-3mg/L, IAA 0.5-4mg/L, Phytagel 2-4g/L, sucrose 30-70g/L, activated carbon 0.5-3g/L and Sucus Cocois 40-100ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110198304 CN102293153B (en) | 2011-07-07 | 2011-07-07 | High efficiency genetic transformation method for rubber trees based on secondary embryogenesis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110198304 CN102293153B (en) | 2011-07-07 | 2011-07-07 | High efficiency genetic transformation method for rubber trees based on secondary embryogenesis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102293153A true CN102293153A (en) | 2011-12-28 |
| CN102293153B CN102293153B (en) | 2013-10-16 |
Family
ID=45354041
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110198304 Expired - Fee Related CN102293153B (en) | 2011-07-07 | 2011-07-07 | High efficiency genetic transformation method for rubber trees based on secondary embryogenesis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102293153B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102696390A (en) * | 2012-07-03 | 2012-10-03 | 广东农垦热带作物科学研究所 | Method for promoting budding of seedling budlings of Brazil rubber trees |
| CN117178897A (en) * | 2023-10-26 | 2023-12-08 | 中国热带农业科学院橡胶研究所 | Method for rejuvenating mature somatic embryos of rubber trees and regenerating plants |
| CN117487847A (en) * | 2023-10-31 | 2024-02-02 | 中国热带农业科学院橡胶研究所 | Method for obtaining homozygous gene editing plant of rubber tree |
-
2011
- 2011-07-07 CN CN 201110198304 patent/CN102293153B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| AROKIARAJ P,等: "Agrobacterium-mediated transformation of Hevea anther calli and their regeneration into plantlets", 《J NAT RUBB RES》 * |
| 华玉伟等,: "橡胶树次生体胚发生及其再生植株遗传稳定性的分子检测", 《热带作物学报》 * |
| 黄天带，等: "根癌农杆菌介导的橡胶树花药愈伤组织遗传转化体系的建立", 《作物学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102696390A (en) * | 2012-07-03 | 2012-10-03 | 广东农垦热带作物科学研究所 | Method for promoting budding of seedling budlings of Brazil rubber trees |
| CN102696390B (en) * | 2012-07-03 | 2013-10-23 | 广东农垦热带作物科学研究所 | Method for promoting budding of seedling budlings of Brazil rubber trees |
| CN117178897A (en) * | 2023-10-26 | 2023-12-08 | 中国热带农业科学院橡胶研究所 | Method for rejuvenating mature somatic embryos of rubber trees and regenerating plants |
| CN117178897B (en) * | 2023-10-26 | 2024-05-07 | 中国热带农业科学院橡胶研究所 | Method for rejuvenating mature somatic embryos of rubber trees and regenerating plants |
| CN117487847A (en) * | 2023-10-31 | 2024-02-02 | 中国热带农业科学院橡胶研究所 | Method for obtaining homozygous gene editing plant of rubber tree |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102293153B (en) | 2013-10-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101647393B (en) | Fast tissue culture reproducing method of actinidia eriantha | |
| CN102217540B (en) | Quick propagation method for lycoris chinensis | |
| CN102301952B (en) | Method for breeding chamomile | |
| CN100581352C (en) | Method for high frequency plant regenerating of tallow tree tissue culture adventitious bud | |
| CN104642109B (en) | A kind of is the method that outer implant builds Bulbus Allii micro-propagation with bulb sheet | |
| CN103461135A (en) | Method for propagating hemerocallis hybridus | |
| CN110679482A (en) | Chrysanthemum multocida detoxification culture medium with high stem tip induction rate and tissue culture quality and method | |
| CN108770690B (en) | Method for establishing efficient and stable regeneration system by using dendrocalamus malabaricus bud tips | |
| CN101779598A (en) | Method for building high-efficiency regeneration system of superior corn self-bred line agriculture line 531 | |
| WO2019153690A1 (en) | High-frequency somatic embryo regeneration growth medium without germplasm genotype restriction and application thereof | |
| CN103155862B (en) | Sinocalamus latiflorus flower pesticide inductor embryo the method obtaining regeneration plant | |
| CN102676574A (en) | Agrobacterium mediated method for obtaining transgenic plant of hevea brasiliensis | |
| CN101775408A (en) | Efficient genetic transformation method for embryogenic callus of cotyledon embryo of rubber tree | |
| CN102283128B (en) | Method for overcoming vitrification phenomenon of sisal hemp tissue culture seedlings | |
| CN101810144B (en) | Rapid breeding method of senecio cruentus | |
| CN103828716B (en) | The method for tissue culture of maiden China pink | |
| CN105028212A (en) | Establishment method of efficient regeneration system of mniochloa abersend | |
| CN102293153B (en) | High efficiency genetic transformation method for rubber trees based on secondary embryogenesis | |
| CN102511391A (en) | Hyacinthus orientalis L. in-vitro rapid propagation method | |
| CN102499075A (en) | Method for preparation of soybean composite explants and method for preparation of rapidly transgenic soybean plants by using the same | |
| CN101836589B (en) | Method of rapid propagation of populus | |
| CN109220809B (en) | Koelreuteria paniculata somatic embryogenesis and plant regeneration culture method | |
| CN101699992B (en) | Method of wheat test-tube plantlet blade repeated regeneration | |
| CN102283131A (en) | Method for establishing system for inducing and regenerating sisal hemp stem tip calluses | |
| CN101933457A (en) | Tissue culture and rapid propagation technology for wine bamboo |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131016 Termination date: 20210707 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |