CN102154364A - Method for agrobacterium tumefaciens-mediated genetic transformation of sugarcane - Google Patents
Method for agrobacterium tumefaciens-mediated genetic transformation of sugarcane Download PDFInfo
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Abstract
The invention discloses a method for agrobacterium tumefaciens-mediated genetic transformation of sugarcane. The method comprises the steps: leading agrobacterium tumefaciens liquor carrying plant expression vectors to infect embryogenic callus of the sugarcane; and selecting and proliferating resistant calli to induce the resistant calli to obtain the resistant buds of the sugarcane, wherein matrix attachment region sequences are connected at two sides of a gene expression box of the plant expression vector. According to the method, MARs (metal artifact reduction) sequences are built at two sides of the gene expression box of the plant expression vector, thus greatly improving the exogenous gene transformation efficiency and seedling rate of the sugarcane, and being simple in process flow and low in cost.
Description
Technical field
The invention belongs to the plant transgenic technology field, be specifically related to a kind of Agrobacterium tumefaciens mediated sugarcane genetic transforming method.
Background technology
Sugarcane is main sugar crop, and high yield, high sugar, high anti-and high economic benefit are the final purposes of cultivation sugarcane, also are the major objectives of cane breeding.But main economic characters of sugarcane (as cane yield, sucrose part and growth characteristics etc.) and resistance belong to quantitative character more, controlled by minor-polygene, affected by environment big, linkage group is many, and therefore the whole good characters that want that by the conventional hybridization breeding parent is had integrate very difficulty.Especially be difficult in and introduce resistant gene in the sugarcane improved seeds targetedly.Modern cultivation sugarcane is compound heterozygosis offspring between a plurality of kinds, be height allopolyploid (chromosome number is up to 2n=36～170), the difficulty of blooming, conventional hybridization breeding difficulty is big, cycle long (the sugarcane traditional breeding method is bred from the sexual hybridization to the breeding and needed the chosen process in 10～13 years approximately), and the high yield and high quality strain of newly breeding also may be because the defective of a certain proterties be difficult to apply as the disease resistance difference.Genetically engineered can the directed change sugarcane some proterties, particularly aspect inherited character improvement such as resistance breeding such as sugarcane drought resisting, cold-resistant, disease-resistant worm and high photosynthetic, high sugar.Therefore, by transgenic technology raising sugarcane resistance and sugar part will be the important new way of high yield, high sugar, high anti-cane breeding.
Because it is the polyploid genome of sugarcane complexity is compared with other crops, less in sugarcane genetically modified research application facet progress.The Study on Genetic Transformation reported first of sugarcane starts from 1987, the method for transformation of successfully cultivating at present transgenic sugarcane has 3 kinds of protoplastis electroporation, particle bombardment and agrobacterium-mediated transformations etc., the acceptor that is used for the sugarcane conversion mainly contains embryo callus, shoot apical meristem, suspension cell and protoplastis.Although protoplastis is the desirable acceptor of foreign DNA in the genetic transformation process, can obtain the callus cell of exogenous origin gene integrator and stably express, the sugarcane plant and just being confined on indivedual genotype but the sugarcane protoplastis is difficult to regenerate, therefore this method for transformation is not the effective ways that obtain the transgenic sugarcane plant.Select for use the target tissue with high frequency regeneration ability and the cell in young age can shorten culturing process, prevent somatic variation, explant size homogeneous, volume helps transforming for a short time.The cultivation of present most of genetically engineered sugarcanes all is to adopt particle bombardment.Particle bombardment is with intact cell and is organized as acceptor, metallic particles such as the same tungsten of exogenous DNA solution, gold are incubated jointly, make DNA be adsorbed in surface of metal particles, under vacuum condition, quicken metallic particles bombardment cell or tissue then, make it directly to import recipient cell or tissue, after screening, survive, express the gene that imports.This method genotype specificity easy and simple to handle, no, and can use several plasmids simultaneously.Arencibia in 1998 etc. adopt engineering bacteria LBA4404 and EHA101, have successfully carried out agriculture bacillus mediated sugarcane genetic transformation, average transformation efficiency 0.194%～1.115%.In the same year, Elliott uses engineering strain AGL0, and transformation efficiency is 5.13%.After this all carried out agriculture bacillus mediated sugarcane Study on Genetic Transformation both at home and abroad in succession.At present transgenic sugarcane exists that transformation efficiency is low, gene expression amount is low or problem such as reticent, genetic stability difference, with regard to its reason be one be the foreign gene that transforms of particle bombardment because multiple copied inserts, random integrations etc. make that the reticent phenomenon of transgenosis in sugarcane is very serious; Particle bombardment conversion mosaic ratio is big, and the transgenosis copy number is many, and genetic stability is relatively poor; The 2nd, influence the factor that agrobacterium tumefaciens transforms sugarcane, be specifically related to structure (comprising promotor and selective marker, MAR sequence etc.), acceptor material type and the pre-treatment of sugarcane genotype, high-efficiency plant expression vector, suitable Agrobacterium strain system, cell concentration, Vir gene activation, immerged time, effective removal etc. of incubation time and Agrobacterium altogether.Agriculture bacillus mediated sugarcane transformation technology because its equipment requirements is simple, expense is low, and usually only can be integrated foreign gene single or the minority copy, thereby, seldom have and methylate and gene silencing phenomenon (Silencing) takes place.Because the allopolyploidy of sugarcane genetic background and height heterozygosity, foundation is efficient, repeatedly the agriculture bacillus mediated sugarcane genetic transformation technology of stable expression of exogenous gene is still the technology of present most critical.In addition, transgenosis is at random being subjected to the integration on the Autosome, there are very big-difference in genetically modified genetic stability and expression activity thereof in different transformant, if do not have a large amount of transfer-gen plant, just can not obtain that inheritance stability, transgenosis proterties are good, the unspoilt transformed variety of other original economical characters or strain system for screening.
Cross problems such as low and gene silencing at present transgenic sugarcane exogenous gene expression amount, the invention of various high efficient expression starters and expression vector is the effective ways that address this problem with utilizing.Wherein (Matrix attachment regions, application MARs) has been subjected to widely paying close attention in the nuclear matrix land.MARs is a kind of good gene expression regulation element, and utilizing MARs is effective ways that overcome the foreign gene inactivation.Form LB-MAR-selectable marker gene-target gene-MAR-RB by add nuclear matrix binding sequence MAR at the target gene two ends, LB-selectable marker gene-MAR-target gene-MAR-RB expression structure, after inserting transgenic plant karyomit(e), form independently ring texture, have the gene expression dose of stabilizing and increasing, part alleviates because position effect and the transgene silencing that methylates and cause.
Summary of the invention
In order to address the above problem, the invention provides a kind of Agrobacterium tumefaciens mediated sugarcane genetic transforming method.
Agrobacterium tumefaciens mediated sugarcane genetic transforming method provided by the invention, comprise that the agrobacterium liquid that will carry plant expression vector infects the embryo callus of sugarcane, by screening and propagation resistant calli, and the induction of resistance callus obtains the sugarcane resistant buds, wherein, the expression casette both sides of described plant expression vector are connected with caryoplasm land sequence.
Wherein, described agrobacterium strains can be any bacterial strain that is used for Plant Transformation well known in the art, is preferably EHA105 or LBA4404.
Wherein, described sugarcane embryo callus is the II type embryo callus of the continuous succeeding transfer culture of blade inductive I type embryo callus 1～5 generation acquisition, be preferably the II type embryo callus of cultivating for 2～3 generations, described II type embryo callus be rich in that gloss, color are yellowish, good dispersity and loose embryo callus.
Wherein, described blade inductive I type embryo callus obtains as follows:
The sugarcane young leaflet tablet of getting apical growing point 5～10cm is as explant, become the thick thin slice of 0.1～0.2cm to be inoculated on the embryonic callus induction substratum its crosscut, 26 ℃～28 ℃ dark cultivations were covered with the fine and close I type embryo callus tissue of white in 20～40 days to blade, wherein said inducing culture is to be minimum medium with MS, and add 2 of 2～3mg/L, 4-D.
According to the present invention, described embryo callus succeeding transfer culture is 26 ℃～28 ℃ dark cultivations, and subculture was 1 time in 20～30 days, and subculture medium is to be minimum medium with MS, and adds 2 of 0.5～1mg/L, 4-D.
According to the present invention, described screening and propagation resistant calli are transferred in the callus screening proliferated culture medium for the embryo callus after will infecting, 26 ℃～28 ℃ dark cultivations, choose after 20～25 days that regenerated is faint yellow, the callus of good dispersity carries out subculture, continuous 1～5 generation of subculture, 2～3 generations of preferred subculture, wherein, described callus screening proliferated culture medium is to be minimum medium with MS, and add 2 of 1mg/L, the Pyocianil (Carb) of the Totomycin of 4-D and 50mg/L (Hyg) and 300～500mg/L.
According to the present invention, the resistant calli that obtains is seeded to the callus subculture medium to be recovered to cultivate 3～5 days, be forwarded to the resistant buds division culture medium again, 26 ℃～28 ℃ illumination cultivation, induced bundle is sprouted, wherein said resistant buds division culture medium is to be minimum medium with MS, and adds the Totomycin (Hyg) of NAA, 50mg/L of 6-BA, 0.05～0.1mg/L of 1.0～2.0mg/L and the Pyocianil (Carb) of 300～500mg/L.
According to the present invention, Agrobacterium tumefaciens mediated sugarcane genetic transformation provided by the invention also comprises peels off into 2-3 strain bud/clump with the bud of growing thickly of 1～2cm, move to the strong seedling culture base and carry out 2～3 weeks of strong seedling culture, 26 ℃～28 ℃ illumination cultivation, when treating that the resistance seedling grows to 3～4cm, change the root media root induction over to, 26 ℃～28 ℃ illumination cultivation, wherein, described strong seedling culture base is to be minimum medium with MS, and add the 6-BA of 0.5～1.0mg/L, 0.01 the NAA of～0.05mg/L, the Totomycin of 30mg/L and the Pyocianil of 100～300mg/L (Carb), described resistance seedling rooting substratum is to be minimum medium with MS, wherein macroelement reduces by half, and adds 1.0～4.0mg/LNAA, the Pyocianil of the Totomycin of 30mg/L (Hyg) and 100～200mg/L.
Substratum NOS of the present invention all is meant solid medium, and all contains the sucrose of 30g/L and the agar of 8g/L.
The present invention carries out 1～5 generation of continuous succeeding transfer culture by sugarcane I type embryo callus is inserted in the embryo callus shoot proliferation substratum, in 2～3 generations of preferred subculture, obtain to be rich in gloss in a large number, color is yellowish, the loose II type embryo callus of good dispersity.With II type embryo callus is transformation receptor, overcome the wretched insufficiency of dedifferentiation callus in conversion, and further improved transformation efficiency, and be transformation receptor with II type embryonal connective tissue, be not subjected to the restriction of natural condition such as plant strain growth period, season and weather, improved efficient.
Resistance embryo callus multiplication capacity is strong, on the basis that does not influence the variation of embryo callus bud differentiation capability and plant, select suitable subculture number, can increase the quantity of resistance embryo callus greatly, increase the quantity of induction of resistance seedling, provide screening for obtaining a large amount of transfer-gen plants.
The present invention is with the both sides of MARs sequence construct to the plant expression vector expression cassette, the transgenosis of MAR sequence mediation, single resistant calli anti-Hyg resistant plant of 4.18 strains of on average regenerating, the transgenosis of no MAR sequence mediation, single resistant calli anti-Hyg resistant plant of 3.0 strains of on average regenerating, the average regeneration plant number that transforms the kanamycin-resistant callus tissue that obtains has improved 39.33%.
Technical process of the present invention is simple, and cost is low, and MAR sequence mediation desinsection fusion gene has improved sugarcane foreign gene transformation efficiency and seedling rate to the sugarcane genetic transformation, has very big scientific research and using value.
Description of drawings
Fig. 1 is the T-DNA structural representation of plant expression vector.Wherein, I, pC2MAR-Hyg-AVA; II, pC13-AVA.
Fig. 2 is the sugarcane resistant calli.
Fig. 3 induces for sugarcane resistance seedling.
Fig. 4 is a sugarcane resistance seedling.
Fig. 5 is the transgenic sugarcane plant.
Fig. 6 is that transgenic sugarcane plant PCR detects.Wherein, 1:DL-2000; 2: non-transgenic plant; 3～5: the transgenic sugarcane that contains the MAR sequence; 6～8: the transgenic sugarcane that does not contain the MAR sequence.
Fig. 7 transgenic sugarcane plant PCR-Southern detects.1: wild-type; 2～5: the transgenic sugarcane that contains the MAR sequence; 6～8: the transgenic sugarcane that does not contain the MAR sequence; 9: plasmid pC2MAR-AVAPCR product.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The preparation of embodiment 1 embryo callus
Select No. 25 plant end pins of sugar cane breed platform sugar of robust growth, with 70% ethanol disinfection surface, get its young tender leaf tissue apart from about the about 10cm of apical growing point as explant, become the thick thin slice of 0.1-0.2cm to be inoculated in embryonic callus induction substratum MS+2 its crosscut, on the 4-D 2mg/L, 26 ℃～28 ℃ dark cultivations 40 days are covered with the fine and close callus of white on the blade, promptly get I type embryo callus.
Picking just inserts embryo callus substratum MS+2 for embryo callus, and carry out shoot proliferation in the 4-D1mg/L and cultivate, 26 ℃～28 ℃ dark cultivations, subculture was 1 time in 20～30 days; These embryo callus further induce a large amount of is rich in that gloss, color are yellowish, the loose II type embryo callus of good dispersity.
The preparation of embodiment 2 embryo callus
Select No. 25 plant end pins of sugar cane breed platform sugar of robust growth, with 70% ethanol disinfection surface, get its young tender leaf tissue apart from about the about 10cm of apical growing point as explant, become the thick thin slice of 0.1～0.2cm to be inoculated in embryonic callus induction substratum MS+2 its crosscut, on the 4-D 3mg/L, 26 ℃～28 ℃ dark cultivations 20 days are covered with the fine and close callus of white on the blade, promptly get I type embryo callus.
Picking just inserts embryo callus substratum MS+2 for callus, and carry out shoot proliferation in the 4-D 1mg/L and cultivate, 26 ℃～28 ℃ dark cultivations, subculture was 1 time in 20～30 days; These embryo callus further induce a large amount of is rich in that gloss, color are yellowish, the loose II type embryo callus of good dispersity.
The callus succeeding transfer culture stage: Totomycin (Hyg) is provided with 6 gradient concentrations (0,10.0,20.0,30.0,40.0,50.0mg/L).Choose II type embryo callus (the about 1cm of size of vigorous embodiment 1 preparation of growth
2) be experiment material, be seeded in the shoot proliferation substratum MS+2 that contains Totomycin, 4-D1.0mg/L substratum on, under 26 ℃～28 ℃ dark conditions, cultivate fresh culture of switching in per 20 days, 5 culture dish of every kind of concentration inoculation, every ware connects 10 block of material, observed the single test result in per 10 days, record callus growth situation, statistics after 40 days.Determine the Hyg mass concentration according to the callus Growth situation.
The differentiation adventitious buds cultivation stage: Totomycin (Hyg) is provided with 6 gradient concentrations (0,10.0,20.0,30.0,40.0,50.0mg/L).Choose II type embryo callus (the about 1cm of size of eugonic embodiment 1 preparation
2) be experiment material, be seeded on the bud division culture medium MS+6-BA2.0mg/L+NAA0.1mg/L substratum that contains Totomycin, at 26 ℃～28 ℃, 14 hours every days, the 2000Lx illumination condition was cultivated down, fresh culture of switching in per 20 days, and every kind of concentration is inoculated 5 bottles, every bottle 10 block of material, observed the single test result in per 10 days, record bud differentiation situation after 40 days, statistics after 40 days.Determine the Hyg mass concentration according to the differentiation of calli situation.
The root culture stage: Totomycin (Hyg) is provided with 6 gradient concentrations (0,10.0,20.0,30.0,40.0,50.0mg/L).Choose the growth vigorous, growing way is consistent, the differentiation seedling of plant height 3～4cm, be seeded on root media 1/2MS (macroelement the decrement)+NAA4.0mg/L that contains Totomycin, at 26 ℃～28 ℃, 14 hours every days, 2000～4000Lx illumination condition was cultivated down, every kind of concentration is inoculated 5 bottles, every bottle 10 strain differentiation seedling, per 10 days observation single test results, write down the situation of taking root, statistics after 40 days.According to bud seedling root of hair situation determine the to take root Hyg mass concentration of screening.
The result shows that the Hyg concentration that callus succeeding transfer culture and differentiation adventitious buds stage use is 50mg/L; The Hyg mass concentration of adventitious bud rooting screening is 30mg/L.
The structure of embodiment 4 plant expression vectors
PCAMBIA1200, pBI121, pGA1611, pCMABIA1300, pBI101.2, because of clone's Herba amaranthi ascendentis agglutinin gene (AVA) among the group DNA, sequence is shown in SEQ ID No.5.The PCR product that obtains is reclaimed through glue, be connected on the pGEM-3zf/T carrier, obtain recombinant plasmid pAVA
HindIII and KpnI enzyme are cut (the T4DNA polysaccharase is mended flat) plant expression vector pGA1611 (containing the Ubi promotor), be connected with the Herba amaranthi ascendentis agglutinin gene AVA fragment of cutting (the T4DNA polysaccharase is mended flat) pAVA acquisition with HindIII with the EcoRI enzyme, obtain plant expression vector pGAAVA.BamHI digested plasmid pGAAVA mends and puts down, and cuts with the SacI enzyme again, obtains the Ubi-AVA fragment.PBI101.2 cuts with the HindIII enzyme, mends and puts down, and cuts with the SacI enzyme again, obtains to remove the linear carrier of 35S promoter and gus gene, and both link to each other, and obtain plant expression vector pBIAVA.With EcoRI and HindIII double digestion pBIAVA and pC2MARHyg, the small segment that reclaims is connected with linear carrier, transformed competence colibacillus cell intestinal bacteria competence DH5 α cell, the final acquisition contained the pest-resistant fusion gene high-efficiency plant expression vector pC2MAR-Hyg-AVA that forward repeats MAR, shown in Fig. 1-I.
With EcoRI and HindIII double digestion pCMABIA1300 and these two kinds of plasmids of pBIAVA, glue reclaims linear carrier and target gene fragment (Ubi-AVA-TNOS), the directed connection and conversion JM109, obtains not contain the recombinant plasmid pC13-AVA of MAR sequence.
The plant expression carrier plasmid that builds is transformed Agrobacterium EHA105, obtain positive colony by screening on the YEP flat board of the Rifampin (Rif) that contains 100mg/L Totomycin (Hyg) and 80mg/L.
The genetic transformation that embodiment 5 is agriculture bacillus mediated
Utilizing agrobacterium-mediated transformation, is that acceptor material carries out genetic transformation with the embryo callus of shoot proliferation.
The single bacterium colony of EHA105/pC2MAR-Hyg-AVA that picking embodiment 4 makes up and EHA105/pC13-AVA Agrobacterium is inoculated into respectively in the 10mL YEP liquid nutrient medium, and 28 ℃, the 200rpm shaking culture is to logarithmic phase.Getting 1mL bacterium liquid puts into 50mL and contains identical antibiotic YEP substratum, when Agrobacterium bacterium liquid is cultured to OD and is 0.5-0.6, be resuspended in the MR liquid nutrient medium that equal-volume contains 200 μ mol/L Syringylethanones (AS), because of clone's Herba amaranthi ascendentis agglutinin gene (AVA) among the group DNA, sequence is shown in SEQ ID No.5.The PCR product that obtains is reclaimed through glue, be connected on the pGEM-3zf/T carrier, obtain recombinant plasmid pAVA
HindIII and KpnI enzyme are cut (the T4DNA polysaccharase is mended flat) plant expression vector pGA1611 (containing the Ubi promotor), be connected with the Herba amaranthi ascendentis agglutinin gene AVA fragment of cutting (the T4DNA polysaccharase is mended flat) pAVA acquisition with HindIII with the EcoRI enzyme, obtain plant expression vector pGAAVA.BamHI digested plasmid pGAAVA mends and puts down, and cuts with the SacI enzyme again, obtains the Ubi-AVA fragment.PBI101.2 cuts with the HindIII enzyme, mends and puts down, and cuts with the SacI enzyme again, obtains to remove the linear carrier of 35S promoter and gus gene, and both link to each other, and obtain plant expression vector pBIAVA.With EcoRI and HindIII double digestion pBIAVA and pC2MARHyg, the small segment that reclaims is connected with linear carrier, transformed competence colibacillus cell intestinal bacteria competence DH5 α cell, the final acquisition contained the pest-resistant fusion gene high-efficiency plant expression vector pC2MAR-Hyg-AVA that forward repeats MAR, shown in Fig. 1-I.
With EcoRI and HindIII double digestion pCMABIA1300 and these two kinds of plasmids of pBIAVA, glue reclaims linear carrier and target gene fragment (Ubi-AVA-TNOS), the directed connection and conversion JM109, obtains not contain the recombinant plasmid pC13-AVA of MAR sequence.
The plant expression carrier plasmid that builds is transformed Agrobacterium EHA105, obtain positive colony by screening on the YEP flat board of the Rifampin (Rif) that contains 100mg/L Totomycin (Hyg) and 80mg/L.
The genetic transformation that embodiment 5 is agriculture bacillus mediated
Utilizing agrobacterium-mediated transformation, is that acceptor material carries out genetic transformation with the embryo callus of shoot proliferation.
The single bacterium colony of EHA105/pC2MAR-Hyg-AVA that picking embodiment 4 makes up and EHA105/pC13-AVA Agrobacterium is inoculated into respectively in the 10mL YEP liquid nutrient medium, and 28 ℃, the 200rpm shaking culture is to logarithmic phase.Getting 1mL bacterium liquid puts into 50mL and contains identical antibiotic YEP substratum, when Agrobacterium bacterium liquid is cultured to OD and is 0.5-0.6, be resuspended in the MR liquid nutrient medium that equal-volume contains 200 μ mol/L Syringylethanones (AS) 28 ℃, 200rpm cultivates 2h, induces bacterium Vir expression of gene.Microbiotic uses Streptomycin sulphate (Str) 25 μ g/L, Rif 25 μ g/L.
Be chosen on the embryo callus shoot proliferation substratum eugonic sugarcane embryo callus and be transferred on the fresh shoot proliferation substratum and cultivate 4d, the callus after the activation is as converting material.With tweezers its folder is broken into 1mm
2Size places Bechtop blowing 30-60min, makes the tissue block surface be the drying shrinkage state.Go in the culture dish, add EHA105/pC2MAR-Hyg-AVA and EHA105/pC13-AVA Agrobacterium bacterium liquid respectively, contaminate 45min with AS (final concentration is 200 μ mol/L) activation 2h, during slightly vibrate.Be transferred to antibiotic-free after with aseptic filter paper bacterium liquid being blotted and contain on the MS substratum of 100 μ mol/L AS, 23 ℃ of dark are cultivated 4d altogether.Being transferred to add has in the resistance shoot proliferation screening culture medium of 500mg/L Pyocianil (Carb) and 50mg/L Totomycin (Hyg) again, under 26 ℃～28 ℃ dark conditions, cultivate 1 fresh screening culture medium induction of resistance embryo callus (Fig. 2) of per 2 weeks switching.Described resistance embryo callus shoot proliferation substratum is to be minimum medium with the shoot proliferation substratum, and adds Totomycin Hyg (50mg/L), Pyocianil Carb (500mg/L).Described MR liquid nutrient medium is to be minimum medium with the MS substratum, macroelement decrement wherein, and add 2,4-D (1.0mg/L), AS (200 μ mol/L), fructose (10mmol/L), glucose (10mmol/L), sucrose (30g/L), pH5.3.Described altogether substratum is to be minimum medium with the shoot proliferation substratum, and adds AS (100 μ mol/L).
The shoot proliferation culturing process of resistance embryo callus: through transforming and screening process, regeneration particulate state resistance embryo callus yellowish, good dispersity in the embryo callus of brownization.The resistance embryo callus is inserted on the resistance embryo callus shoot proliferation substratum, under 26 ℃～28 ℃ dark conditions, cultivates, fresh culture of switching in per 20 days, carry out 2 generation shoot proliferation, to increase the quantity of resistance embryo callus.Through resistance subculture screening process, can further kill non-transformed cell, improve transformation efficiency, can obtain a large amount of resistance embryo callus simultaneously.Described resistance embryo callus shoot proliferation substratum is to be minimum medium with the shoot proliferation substratum, and adds Totomycin Hyg (50mg/L), Pyocianil Carb (500mg/L).
The culturing process of the resistant calli differentiation and seedling emergence behind the subculture: the resistance embryo callus subculture behind the shoot proliferation is gone to recover to cultivate 4d on the shoot proliferation substratum of antibiotic-free, changing the resistant buds division culture medium then over to induces and emerges, 26 ℃～28 ℃, sprout (Fig. 3) induced in 2000～4000Lx illumination every day in 14 hours.Described resistant buds division culture medium is to be minimum medium with the MS substratum, and adds 6-BA (2.0mg/L), NAA (0.1mg/L), sucrose (30g/L), Totomycin Hyg (50mg/L), Pyocianil Carb (500mg/L).
The culturing process of resistance seedling rooting: through screening, differentiation, when treating that the resistance seedling grows to 2cm, the clump bud is peeled off into simple bud move to and carry out in the strong seedling culture base 2 weeks of strong seedling culture (Fig. 4), 26 ℃～28 ℃, every day, 2000～4000Lx illumination was 14 hours.When treating that the resistance seedling grows to 3cm, change the root media root induction over to, 26 ℃～28 ℃, 2000～4000Lx illumination every day root induction in 14 hours.Grow approximately about 10 roots, open bottle cap and move to outdoor hardening, behind the 2d seedling is taken out, clean the substratum that adheres to flowing water, be placed in the greenhouse of (25 ℃) of 80% relative air humidity and cultivate, treat that seedling survives the back and divides single-strain planting in the dish that nutrition soil is housed, obtain to transform sugarcane plant (Fig. 5).Described strong seedling culture base is to be minimum medium with MS, and adds 1.0mg/L 6-BA, 0.05mg/L NAA, sucrose (30g/L), 300mg/L Carb, 30mg/L Hyg; Described resistance seedling rooting substratum is to be minimum medium with MS, wherein macroelement decrement, and interpolation 4.0mg/L NAA, 30mg/L Hyg, 200mg/L Carb.
To the EHA105/pC2MAR-Hyg-AVA and the EHA105/pC13-AVA resistance seedling of embodiment 5 preparations, the extraction and the PCR that carry out genomic dna respectively detect, and are contrast with the non-transgenic seedling.The primer that wherein is used for the PCR detection is shown in SEQ ID No.2 and SEQ ID No.3.The PCR of partial resistance seedling detects as shown in Figure 6.(special primer of AVA gene (5 '-GGAAGATCTACCATGGCGGGATTACCAGTG-3 ') (SEQ ID No.6) and (5 '-AGCGTCGACTTAGTTGTTGGATCCCAATTC-3 ') (SEQ ID No.7) carry out pcr amplification, and dna fragmentation length is about 1900bp between two primers.With non-conversion sugarcane plant in contrast.
DNA is a test sample with PCR male transformed plant, reclaims the PCR product, and point sample is on nylon membrane respectively.The probe of hybridization analysis is by the AVA gene fragment preparation of/pC13-AVA specific amplified, and the DIG High Prime DNA Labeling and Detection Starter Kit II test kit of Roche company is adopted in probe mark and hybridization.Design non-transformed plant in contrast.
Transformed 200 II type embryo callus with EHA105/pC2MAR-Hyg-AVA, 92 strain resistance seedlings have been obtained, 85 strain PCR positive plants wherein, PCR-Southern hybridization positive plant is 83 strains, transformation efficiency is 38% (transformation efficiency=detection positive plant quantity/inoculation callus quantity), and screening rate is 90.22% (screening rate=detection positive plant quantity/resistant plant number); Transformed 200 II type embryo callus with EHA105/pC13-AVA, 42 strain resistance seedlings have been obtained, 36 strain PCR positive plants wherein, PCR-Southern hybridization positive plant is 27 strains, transformation efficiency is 13.5% (transformation efficiency=detection positive plant quantity/inoculation callus quantity), and screening rate is 64.29% (screening rate=detection positive plant quantity/resistant plant number).The PCR-Southern results of hybridization of the positive seedling of part PCR as shown in Figure 7.
The pC2MAR-Hyg-AVA plasmid of MAR sequence mediation transgene expression obtains 22 gene lines altogether; the pC13-AVA plasmid of no MAR sequence mediation transgene expression obtains 14 gene lines, MAR sequence mediation transgene expression make the acquisition quantity of transgenic lines in the conversion process mediate than no MAR sequence transgene expression raising 57.14%.Single resistant calli of MAR sequence mediation transgene expression anti-Hyg resistant plant of 4.18 strains of on average regenerating, single resistant calli of transgene expression of no MAR sequence mediation anti-Hyg resistant plant of 3.0 strains of on average regenerating, the average regeneration plant number that transforms the kanamycin-resistant callus tissue that obtains has improved 39.33%.Most of resistant plant form is normal, and plant type is more strong, and root of hair is also normal, and root system is more sturdy, long.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. Agrobacterium tumefaciens mediated sugarcane high-efficiency genetic transforming method, it is characterized in that, comprise that the agrobacterium liquid that will carry plant expression vector infects the embryo callus of sugarcane, by screening and propagation resistant calli, and the induction of resistance callus obtains the sugarcane resistant buds, wherein, the expression casette both sides of described plant expression vector are connected with caryoplasm land sequence.
2. method according to claim 1 is characterized in that, described sugarcane embryo callus is the II type embryo callus of the continuous succeeding transfer culture of blade inductive I type embryo callus 1～5 generation acquisition.
3. method according to claim 2 is characterized in that, described sugarcane embryo callus is the II type embryo callus of the continuous succeeding transfer culture of blade inductive I type callus 2～3 generations acquisition.
4. according to claim 2 or 3 described methods, it is characterized in that described blade inductive I type embryo callus obtains as follows:
The sugarcane young leaflet tablet of getting apical growing point 5～10cm is as explant, become the thick thin slice of 0.1～0.2cm to be inoculated on the embryonic callus induction substratum its crosscut, 26 ℃～28 ℃ dark cultivations were covered with the fine and close I type embryo callus of white in 20-40 days to blade, wherein said inducing culture is to be minimum medium with MS, and add 2 of 2～3mg/L, 4-D.
5. according to claim 2 or 3 described methods, it is characterized in that described succeeding transfer culture is 26 ℃～28 ℃ dark cultivations, subculture was 1 time in 20～30 days, and subculture medium is to be minimum medium with MS, and adds 2 of 0.5～1mg/L, 4-D.
6. method according to claim 1, it is characterized in that, described screening and propagation resistant calli are transferred in the callus screening proliferated culture medium for the embryo callus after will infecting, 26 ℃～28 ℃ dark cultivations, choose after 20～25 days that regenerated is faint yellow, the callus of good dispersity carries out shoot proliferation, continuous 1～5 generation of subculture, wherein, described callus screening proliferated culture medium is to be minimum medium with MS, and add 2 of 1mg/L, the Totomycin of 4-D and 50mg/L and the Pyocianil of 300～500mg/L.
7. method according to claim 6 is characterized in that, the algebraically of described continuous subculture was 2～3 generations.
8. method according to claim 1, it is characterized in that, resistant calli is seeded to subculture medium to be recovered to cultivate 3～5 days, be forwarded to the resistant buds division culture medium again, 26 ℃～28 ℃ illumination cultivation, induced bundle is sprouted, and wherein said resistant buds division culture medium is to be minimum medium with MS, and adds the Totomycin of NAA, 50mg/L of 6-BA, 0.05～0.1mg/L of 1.0～2.0mg/L and the Pyocianil of 300～500mg/L.
9. method according to claim 1, it is characterized in that, also comprise the bud of growing thickly of 1～2cm is peeled off into 2-3 strain bud/clump, move to the strong seedling culture base and carry out 2～3 weeks of strong seedling culture, 26 ℃～28 ℃, illumination cultivation, when treating that the resistance seedling grows to 3～4cm, change the root media root induction over to, 26 ℃～28 ℃ illumination cultivation, wherein, described strong seedling culture base is to be minimum medium with MS, and adds the 6-BA of 0.5～1.0mg/L, 0.01 the NAA of～0.05mg/L, the Totomycin of 30mg/L and the Pyocianil of 100～300mg/L, described resistance seedling rooting substratum is to be minimum medium with MS, wherein macroelement reduces by half, and adds 1.0～4.0mg/LNAA, the Totomycin of 30mg/L and the Pyocianil of 100～200mg/L.
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