CN102293153B - 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
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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 rubber tree genetic transforming method based on secondary embryogenesis.
Background technology
Natural rubber has the incomparable elasticity of synthetic rubber, ductility and thermal conductivity, is important industrial raw material, is mainly derived from Para rubber tree (Hevea brasiliensis Muell.Arg.).By the conventional cross-breeding means, cultivating rubber tree new variety needs 20～30 years.Transgenic technology is shortening Rubber Tree Breeding cycle, the highly effective approach of quickening rearing new variety.
The acceptor that is used at present the rubber tree Agrobacterium-mediated Transformation 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 regenerative power 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, efficient rubber tree genetic conversion system is most important for setting up.Hua Yuwei etc. (2010) have set up rubber tree secondary body embryo generation system, and this system is comprised of three processes: 1) inducing of elementary body embryo: flower pesticide or inner integument evoked callus, and then induce elementary somatic embryo to occur; 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 then evoked callus is induced secondary somatic embryo Somatic Embryogenesis again, and this process can circulate; 3) plant regeneration: the cotyledon shape embryoid of selecting maturation changes the substratum of emerging over to and induces plantlet regeneration.Use this system, a skilled labor can produce 8000 strains in 1 year from root clone plant.The foundation that is established as the rubber tree efficient genetic trasformation system 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 time and the recruitment of peeling off stamen or inducing, screening stable propagation friable embryogenic callus; 3 secondary embryogenesis have very strong regenerative power, and each workman can produce 8000 strain tissue cultured 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, save the time of conversion, renewal cultivation gripping callus, greatly improve conversion rate.
The rubber tree transgenic line Reproduction methods of having reported has three kinds: 1 transforms the rear inductor embryo of friable embryogenic callus propagation occurs and plant regeneration; 2 transfer-gen plant stem with bud micro cuttages are realized propagation; 3 take transfer-gen plant as scion, expands numerous by bud grafting.Callus after mode 1 propagation occurs through suspension culture inductor embryo, and vitrification phenomenon is serious, and the transfer-gen plant of regeneration deformity, and all not as contrast, application prospect is pessimistic for growth potential, output; Mode 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 take embryoid as explant, occurs by the loop body embryo, and the realization embryo is finally realized the method for body embryo plant propagation to the circulation propagation of embryo.This regeneration system embryoid induction rate is high, the normal chick embryo ratio is high, and complete three kinds of Reproduction methods different from the past, 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 regeneration system embryoid induction rate, the difficult problem that seedling rate is low.
The present invention is receptor system with the Secondary somatic embryos artificial body for generating, sets up a kind of efficient rubber tree genetic transforming method of having reported that is different from.
Summary of the invention
The purpose of this invention is to provide a kind of rubber tree high-efficiency genetic transforming method based on secondary embryogenesis, it occurs as the basis with Secondary somatic embryos, the somatocyte embryoid is that Agrobacterium is infected acceptor, by inducing secondary embryogenesis, efficient acquisition transforms embryoid and transformed plant, and verifying for the transgenosis of rubber tree transgenic breeding and functional gene provides efficient genetic transforming method.
The present invention is that the technical scheme that its technical problem of solution adopts is:
A kind of rubber tree high-efficiency genetic transforming method based on secondary embryogenesis is characterized in that: comprise the acquisition of genetic transformation acceptor-somatocyte embryoid, the genetic transformation of somatocyte embryoid, the propagation that transforms embryoid, the plant regeneration that transforms embryoid and the transplanting five steps of transformed plant:
(a) acquisition of genetic transformation acceptor-somatocyte embryoid: select stalwartness, embryoid that vitality is vigorous, it is separated as transformation receptor from original substratum;
(b) genetic transformation of somatocyte embryoid: its process comprise infect, altogether cultivation, renewal 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 calli induction media that adds the 1-50mg/L Silver Nitrate, and its pH value is 5.2,20-28 ℃ and cultivated altogether 3-6 days;
Renewal cultivation: the embryoid after the common cultivation changed over to add the 1-50mg/L Silver Nitrate and suppress Agrobacterium and grow on the antibiotic calli induction media 23-28 ℃ of dark the cultivation 3-10 days;
Callus of induce: the embryo piece that the embryoid behind the renewal cultivation is cut into 0.2-0.5 * 0.2-0.5cm, then change over to and added the 1-50mg/L Silver Nitrate, on the microbiotic of inhibition Agrobacterium growth and the calli induction media 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 callus over to the microbiotic of interpolation inhibition Agrobacterium growth and the division culture 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 callus of induce process used medium; This process is capable of circulation, until transformant obtains enough ripe cotyledon shape embryoids; After circulation 1-3 time, antibacterial microbiotic can be removed;
(d) plant regeneration of conversion embryoid: forward the cotyledon shape embryoid of maturation to the substratum of emerging, its pH value is 5.8,23-28 ℃ of illumination cultivation, emerges in 20-60 days;
(e) transplanting of transformed plant: (1) bud grafting: with regeneration transformed plant as scion, the transplanting of a series of process implementations from the laboratory to the land for growing field crops tied up, packed to seedling or tissue cultured seedling as stock by bud grafting, solution; (2) husky bed is transplanted: move into husky bed after the transformed plant of regeneration is cleaned substratum, 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, packed after blade grows to stationary phase.
The employed genetic transformation acceptor of described step (a) is the embryoid by the secondary embryogenesis acquisition of the generation of the explant body embryos such as flower pesticide, inner integument and tree root and body embryo thereof.
The calli induction media of described step (b) is as basic medium take the MS substratum, and be potassium primary phosphate 350-500mg/L, Calcium Chloride Powder Anhydrous 150-400mg/L, four water manganous sulfate 10-40mg/L, magnesium sulfate heptahydrate 400-600mg/L with the content furnishing of the potassium primary phosphate in the MS substratum, Calcium Chloride Powder Anhydrous, four water manganous sulfates, magnesium sulfate heptahydrate, 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 effective constituent of the division culture medium of described step (b) is take the MS substratum as minimum medium, and with the potassium primary phosphate in the MS substratum, Calcium Chloride Powder Anhydrous, four water manganous sulfates, the content of magnesium sulfate heptahydrate is adjusted into potassium primary phosphate 350-500mg/L, Calcium Chloride Powder Anhydrous 150-400mg/L, four water manganous sulfate 10-40mg/L, magnesium sulfate heptahydrate 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 effective constituent of substratum for take the MS substratum as minimum medium, and the content of the potassium primary phosphate in the MS substratum, Calcium Chloride Powder Anhydrous, four water manganous sulfates, magnesium sulfate heptahydrate is adjusted into potassium primary phosphate 350-500mg/L, Calcium Chloride Powder Anhydrous 150-400mg/L, four water manganous sulfate 10-40mg/L, magnesium sulfate heptahydrate 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 from 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 at any time the conversion needs; B, save time and the recruitment of peeling off stamen or inducing, screening stable propagation friable embryogenic callus; C, body embryo have very strong regenerative power, and each workman can produce 8000 strain tissue cultured 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, save the time of conversion, renewal cultivation gripping callus, greatly improve conversion rate.
2, the present invention adopts the transformant Reproduction methods and reports all different in the past, 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 regeneration system embryoid induction rate, the difficult problem that seedling rate is low.
3, all very healthy and strong through plant root, stem, the leaf of secondary body embryo generation regeneration, thin and weak fully different from the plant of long-term subculture callus regeneration, and the vitrifying seedling of some amount 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.Transfer-gen plant is healthy and strong, has improved greatly transplanting survival rate, thereby has further improved transformation efficiency.
4, the transfer-gen plant of regeneration has the root system next by radicle growth after secondary body embryo is bred, 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 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:
The below infects take agrobacterium tumefaciens EHA105 that the invention will be further described as example, need to prove that Molecular Detection and the former method of preparation, transformed plant of engineering bacteria is as broad as long.
(a) acquisition of genetic transformation acceptor-somatocyte embryoid: select the vigorous embryoid of stalwartness, vitality by flower pesticide, inner integument, tree root cultivation, secondary embryogenesis or the differentiation of other body embryogenesis paths, it is separated as transformation receptor from original substratum.Incubation time is excessively of a specified duration, and the embryoid of vitrifying, albefaction, brownization is not suitable for as transformation receptor.
(b) genetic transformation of somatocyte 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: culture temperature is 25 ℃ altogether, and substratum is that pH value transfers to 5.2 altogether, adds the calli induction media of 5mg/L Silver Nitrate; Renewal cultivation: the embryoid after transforming changed over to add 5mg/L Silver Nitrate and the 500mg/L spy fourth (ticarcillin: the calli induction media (PH5.8) of Clavulanic Potassium=15:1), 25 ℃ of darkroom renewal cultivations 10 days that vanishes; Callus of induce: the embryoid behind the renewal cultivation is cut into the embryo piece of 0.2 * 0.2cm, then changes over to and added the 5mg/L Silver Nitrate, the vanish calli induction media (PH5.8) of fourth and 50mg/L kantlex of 500mg/L spy, 25 ℃ of dark cultivations 20 days.Differentiation: kanamycin-resistant callus tissue changed over to add the vanish division culture medium (PH5.8) of fourth and 50mg/L kantlex of 500mg/L spy, 25 ℃ of dark cultivations 50 days obtain ripe embryoid.Described calli induction media is as basic medium take the MS substratum, and be potassium primary phosphate 350mg/L, Calcium Chloride Powder Anhydrous 200mg/L, four water manganous sulfate 20mg/L, magnesium sulfate heptahydrate 400mg/L with the content furnishing of the potassium primary phosphate in the MS substratum, Calcium Chloride Powder Anhydrous, four water manganous sulfates, magnesium sulfate heptahydrate, and add 2,4-D 2mg/L, KT 1mg/L, NAA 1.5mg/L, Phytagel 2g/L, sucrose 80g/L, Sucus Cocois 100ml; The effective constituent of described division culture medium is take the MS substratum as minimum medium, and the content of the potassium primary phosphate in the MS substratum, Calcium Chloride Powder Anhydrous, four water manganous sulfates, magnesium sulfate heptahydrate is adjusted into potassium primary phosphate 350mg/L, Calcium Chloride Powder Anhydrous 200mg/L, four water manganous sulfate 20mg/L, magnesium sulfate heptahydrate 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 callus of induce process used medium.This process is capable of circulation, until transformant obtains enough ripe cotyledon shape embryoids.After circulation 1-3 time, antibacterial microbiotic can be removed.
(d) plant regeneration of conversion embryoid: forward the cotyledon shape embryoid of maturation to the substratum of emerging (PH5.8), 25 ℃ of illumination cultivation were emerged in 20-60 days.Emerge the effective constituent of substratum for take the MS substratum as minimum medium, and the content of the potassium primary phosphate in the MS substratum, Calcium Chloride Powder Anhydrous, four water manganous sulfates, magnesium sulfate heptahydrate is adjusted into potassium primary phosphate 350mg/L, Calcium Chloride Powder Anhydrous 200mg/L, four water manganous sulfate 20mg/L, magnesium sulfate heptahydrate 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: with regeneration transformed plant as scion, the transplanting of a series of process implementations from the laboratory to the land for growing field crops tied up, packed to seedling or tissue cultured seedling as stock by bud grafting, solution; (2) husky bed is transplanted: move into husky bed after the transformed plant of regeneration is cleaned substratum, 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, packed after blade grows to stationary phase.
Embodiment 2:
The below infects take another agrobacterium tumefaciens lba4404 that the invention will be further described as example, and 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-somatocyte embryoid: select the vigorous embryoid of stalwartness, vitality by flower pesticide, inner integument, tree root cultivation, secondary embryogenesis or the differentiation of other body embryogenesis paths, it is separated as transformation receptor from original substratum.Incubation time is excessively of a specified duration, and the embryoid of vitrifying, albefaction, brownization is not suitable for as transformation receptor.
(b) genetic transformation of somatocyte 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: culture temperature is 20 ℃ altogether, and substratum is that pH value transfers to 5.2 altogether, adds the calli induction media of 10mg/L Silver Nitrate; Renewal cultivation: the embryoid after transforming changed over to add 10mg/L Silver Nitrate and the 300mg/L spy fourth (ticarcillin: calli induction media Clavulanic Potassium=15: 1) (PH5.8), 25 ℃ of darkroom renewal cultivations 5 days that vanishes; Callus of induce: the embryoid behind the renewal cultivation is cut into the embryo piece of 0.2 * 0.2cm, then changes over to and added the 5mg/L Silver Nitrate, the vanish calli induction media (PH5.8) of fourth and 5mg/L Totomycin of 300mg/L spy, 25 ℃ of dark cultivations 20 days.Differentiation: kanamycin-resistant callus tissue changed over to add the vanish division culture medium (PH5.8) of fourth and 5mg/L Totomycin of 300mg/L spy, 25 ℃ of dark cultivations 60 days obtain ripe embryoid.Described calli induction media is as basic medium take the MS substratum, and be potassium primary phosphate 500mg/L, Calcium Chloride Powder Anhydrous 400mg/L, four water manganous sulfate 20mg/L, magnesium sulfate heptahydrate 400mg/L with the content furnishing of the potassium primary phosphate in the MS substratum, Calcium Chloride Powder Anhydrous, four water manganous sulfates, magnesium sulfate heptahydrate, and add 2,4-D 1mg/L, KT1mg/L, NAA 1mg/L, Phytagel 2g/L, sucrose 60g/L, Sucus Cocois 40ml; The effective constituent of described division culture medium is take the MS substratum as minimum medium, and be potassium primary phosphate 500mg/L, Calcium Chloride Powder Anhydrous 400mg/L, four water manganous sulfate 20mg/L, magnesium sulfate heptahydrate 400mg/L with the content furnishing of the potassium primary phosphate in the MS substratum, Calcium Chloride Powder Anhydrous, four water manganous sulfates, magnesium sulfate heptahydrate, 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.
(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 callus of induce process used medium.This process is capable of circulation, until transformant obtains enough ripe cotyledon shape embryoids.After circulation 1-3 time, antibacterial microbiotic can be removed.
(d) plant regeneration of conversion embryoid: forward the cotyledon shape embryoid of maturation to the substratum of emerging (PH5.8), 25 ℃ of illumination cultivation were emerged in 20-60 days.Emerge the effective constituent of substratum for take the MS substratum as minimum medium, and be potassium primary phosphate 500mg/L, Calcium Chloride Powder Anhydrous 400mg/L, four water manganous sulfate 20mg/L, magnesium sulfate heptahydrate 400mg/L with the content furnishing of the potassium primary phosphate in the MS substratum, Calcium Chloride Powder Anhydrous, four water manganous sulfates, magnesium sulfate heptahydrate,, 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: with regeneration transformed plant as scion, the transplanting of a series of process implementations from the laboratory to the land for growing field crops tied up, packed to seedling or tissue cultured seedling as stock by bud grafting, solution; (2) husky bed is transplanted: move into husky bed after the transformed plant of regeneration is cleaned substratum, 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, packed after blade grows to stationary phase.
Claims (4)
1. rubber tree genetic transforming method based on secondary embryogenesis is characterized in that: comprise the acquisition of genetic transformation acceptor-somatocyte embryoid, the genetic transformation of somatocyte embryoid, the propagation that transforms embryoid, the plant regeneration that transforms embryoid and the transplanting five steps of transformed plant:
(a) acquisition of genetic transformation acceptor-somatocyte embryoid: select stalwartness, embryoid that vitality is vigorous, it is separated as transformation receptor from original substratum;
(b) genetic transformation of somatocyte embryoid: its process comprise infect, altogether cultivation, renewal cultivation, more
Hinder the Induction and differentiation process;
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 calli induction media that adds 5mg/L or 10mg/L Silver Nitrate, and the pH value is 5.2,20-28 ℃ and cultivated altogether 3-6 days;
Renewal cultivation: the embryoid after cultivating altogether changes over to and adds 5mg/L or 10mg/L Silver Nitrate and suppress Agrobacterium and grow on the antibiotic calli induction media, and pH value is 5.8,23-28 ℃ of dark cultivation 3-10 days;
Callus of induce: the embryo piece that the embryoid behind the renewal cultivation is cut into (0.2～0.5) cm * (0.2～0.5) cm, then change over to and added 5mg/L or 10mg/L Silver Nitrate, add on the microbiotic that suppresses the Agrobacterium growth and the calli induction media that adds kantlex or Totomycin, the pH value is 5.8,23-28 ℃ of dark the cultivation 15-40 days;
Differentiation: change the embryo piece of callus over to the microbiotic that adds the growth of inhibition Agrobacterium and the division culture medium that adds kantlex or Totomycin, the pH value is 5.8,23-28 ℃ of dark cultivation 30-70 days;
(c) propagation of conversion embryoid: the embryo piece that the embryoid of GUS stained positive is cut into (0.2～0.5) cm * (0.2～0.5) cm, the described callus of induce of repeating step (b), atomization, but the Silver Nitrate of removal callus of induce used medium; This process circulation is until transformant obtains enough ripe cotyledon shape embryoids; Circulate after 3 times, remove antibacterial microbiotic;
(d) plant regeneration of conversion embryoid: forward the cotyledon shape embryoid of maturation to the substratum of emerging, the pH value is 5.8,23-28 ℃ of illumination cultivation, emerges in 20-60 days;
(e) transplanting of transformed plant: (1) bud grafting: with regeneration transformed plant as scion, the transplanting of a series of process implementations from the laboratory to the land for growing field crops tied up, packed to seedling or tissue cultured seedling as stock by bud grafting, solution; (2) husky bed is transplanted: move into husky bed after the transformed plant of regeneration is cleaned substratum, 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, packed after blade grows to stationary phase.
2. a kind of rubber tree genetic transforming method based on secondary embryogenesis according to claim 1 is characterized in that: the employed genetic transformation acceptor of described step (a) is the embryoid that the secondary embryogenesis by flower pesticide, inner integument and tree root explant and body embryo thereof obtains.
3. a kind of rubber tree genetic transforming method based on secondary embryogenesis according to claim 1, it is characterized in that: the calli induction media of described step (b) is as basic medium take the MS substratum, and with the potassium primary phosphate in the MS substratum, Calcium Chloride Powder Anhydrous, four water manganous sulfates, the content furnishing of magnesium sulfate heptahydrate is potassium primary phosphate 350-500mg/L, Calcium Chloride Powder Anhydrous 150-400mg/L, four water manganous sulfate 10-40mg/L, magnesium sulfate heptahydrate 400mg/L, and add 2,4-D0.5-3mg/L, KT0.5-3mg/L, NAA0.5-3mg/L, Phytagel2-3g/L, sucrose 50-90g/L and Sucus Cocois 40-100ml; The effective constituent of described division culture medium is take the MS substratum as minimum medium, and the content of the potassium primary phosphate in the MS substratum, Calcium Chloride Powder Anhydrous, four water manganous sulfates, magnesium sulfate heptahydrate is adjusted into potassium primary phosphate 350-500mg/L, Calcium Chloride Powder Anhydrous 150-400mg/L, four water manganous sulfate 10-40mg/L, magnesium sulfate heptahydrate 400mg/L, and add 6-BA0.5-3mg/L, KT0.5-3mg/L, NAA0.1-2mg/L, GA
31-5mg/L, Phytagel2-3g/L, sucrose 50-90g/L, activated carbon 0.5-3g/L and Sucus Cocois 40-100ml.
4. a kind of rubber tree genetic transforming method based on secondary embryogenesis according to claim 1, it is characterized in that: described step (d) is emerged the effective constituent of substratum for take the MS substratum as minimum medium, and the content of the potassium primary phosphate in the MS substratum, Calcium Chloride Powder Anhydrous, four water manganous sulfates, magnesium sulfate heptahydrate is adjusted into potassium primary phosphate 350-500mg/L, Calcium Chloride Powder Anhydrous 150-400mg/L, four water manganous sulfate 10-40mg/L, magnesium sulfate heptahydrate 400mg/L, and add KT0.1-1.0mg/L, GA
30.5-3mg/L, IAA0.3mg/L or 0.5mg/L, Phytagel2-4g/L, sucrose 30-70g/L, activated carbon 0.5-3g/L and Sucus Cocois 40-100ml.
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