CN101948868B - Genetic transformation method taking sweet sorghum young ear or young ear induced callus as explant - Google Patents
Genetic transformation method taking sweet sorghum young ear or young ear induced callus as explant Download PDFInfo
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Abstract
The invention discloses a genetic transformation method taking sweet sorghum young ear or young ear induced callus as an explant, and belongs to the technical field of plant gene engineering. The sweet sorghum young ear or the young ear induced callus is taken as a transformation receptor, exogenous genes are transformed by adopting a direct transformation method, the young ear or the young ear callus after bombardment is subjected to recovery culture and screening culture to obtain resistant callus, and the resistant callus is subjected to successive differentiated culture, germination, rooting and transplant to obtain regenerative transformation plants. The sweet sorghum plants are transformed into transgenic insect-resistant plants by using the established genetic transformation system. The method provides technical support for researches of improving the quality and stress resistance of the sweet sorghum by using biological technology, and lays a good function for accelerating the foundation research of the sweet sorghum.
Description
Technical field
The invention belongs to the plant gene engineering technology field, particularly relate to a kind of genetic transforming method that is explant with sweet sorghum children's fringe or young fringe evoked callus.
Background technology
Sweet sorghum is the mutation of common Chinese sorghum, and the growth water loss only is 1/3 of a corn, and per mu yield can be up to 5～10 tons, and its stalk juice is the desirable biomass material that alcohol and culturing yeast are made in fermentation.With the novel process of food crop such as sweet sorghum replacement corn as raw material production alcohol, can not only save food, reduce cost, effectively drive the fermentation industry, revitalize the Alcohol Production industry and can advance renewable energy source cause and Developing of Animal Industry.Developing sweet sorghum industry key is the seed selection and efficient cultivation of sweet sorghum new variety.Traditional breeding technology is being brought into play vital role aspect the output of sweet sorghum, quality, the characteristic improvement such as disease-resistant, pest-resistant, but under the situation about on producing, kind, quality requirements being improved day by day, because of being subjected to the restriction of existing resource, depend merely on traditional breeding method and be difficult to obtain important breakthrough.Utilizing transgenic technology improvement sorghum variety to cause widely pays close attention to.Plant transgenic technology depends on usually that plant is quick, the foundation of high-frequency regeneration system.Yet sweet sorghum is the same with common Chinese sorghum, obtains relatively difficulty of regeneration plant by tissue culture.At present also seldom at the research of sweet sorghum tissue culture.
Though before the whole regeneration plant of acquisition, had some reports (MacKinnon et al., 1986, Plant Cell Rep.5:349-351 from mature embryo and the immature embryo callus induction of sweet sorghum; Ma et al., 1987, Theor App Genet.73:389-394; Rao and Kavi Kishor, 1989, Curr Sci.58:692-693), but regrettably can be from the explant induction embryo callus subculture to the plant that regenerates only limit to indivedual several kinds (Smith and Bhaskaran, 1986, Springer-Verlag, New York, pp.220-223; Rao et al., 1995, Plant Cell Rep.15:72-75).Along with the attention of people to the sweet sorghum using value, the research of sweet sorghum tissue culture aspect has also obtained development fast in recent years.By different genotype kind and explant, different medium component (comprising sucrose concentration and hormone combination etc.), different culture condition are carried out systematic study to the influence of inducing, breeding and breaking up regenerative power of callus, setting up effective tissue culture regeneration system for the sweet sorghum kind provides with reference to (Zhao et al., 2008, Chinese Bull Bot.25:465-468; Zhao et al., 2010, African Journal of Biotechnology.9 (16): 2367-2374).But above research is to be explant with mature embryo of sweet sorghum and immature embryo mostly, and being that the research of explant is still rarely found with young fringe so far has a report.
The various countries scientist is being devoted to the research of related fields in recent years, compares other crop, sweet sorghum tissue culture plant regeneration difficulty, and be subjected to genotypic restriction, so the sweet sorghum Study on Genetic Transformation is made slow progress still rarely found at present successfully report.It is reported, the researchist of U.S. University of Queensland in 2009 has cultivated the transgenic sweet sorghum plant that the first in the world content of lignin reduces, they import in the sweet sorghum kind at will the encode dna fragmentation of caffeoyl coenzyme A-O-methyltransgerase (CCoAOMT) and coffic acid-O-methyltransgerase (COMT), obtained success by content and the component that changes xylogen, the research achievement has been applied for patent (Pub.No.US 2010/0058496A1).Relevant report is not seen in other research as yet.
Therefore set up the genetic conversion system of sweet sorghum, cultivate the sweet sorghum new variety, make it satisfy the demand of actual production better by transgenic technology.The research and development of transformation system will be opened up new approach for the development and use of green energy resource biofuel and biomaterial.
Summary of the invention
The purpose of this invention is to provide a kind of tissue culture regeneration system and genetic transforming method and with sweet sorghum children fringe material with the tissue culture regeneration system and the genetic transforming method of sweet sorghum children fringe inductive callus, to be used for the genetically modified research of sweet sorghum.
A kind of genetic transforming method that is explant with sweet sorghum children's fringe or young fringe evoked callus, comprise following sequential steps: 1) choosing the sweet sorghum boot leaf phase is about 2-5cm children fringe, thinly slice, it is inoculated in the inducing culture cultivates, described inducing culture is: MS basic medium+2,4-D 2mg/L+ZT 2.0mg/L+PVP 10mg/L+ xitix 10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+7.2g/L agar, pH 5.8; Form callus; 2) choosing good young fringe of growth conditions or young fringe callus lines is inoculated in height and oozes in the substratum and cultivate, described height oozes substratum: MS basic medium+2,4-D 2mg/L+ZT 2.0mg/L+KT 0.2mg/L+PVP 10mg/L+ xitix 10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+7.2g/L agar+0.4M N.F,USP MANNITOL, pH 5.8; 3) directly conversion method changes foreign gene over to, continues to ooze overnight incubation in the substratum at height; 4) change recovery cultivation in the subculture medium over to, described subculture medium is: MS basic medium+2,4-D 2mg/L+ZT 2.0mg/L+KT 0.2mg/L+PVP 10mg/L+ xitix 10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+7.2g/L agar, pH 5.8; 5) change on the screening culture medium and screen, described screening culture medium is: MS subculture medium+add suitable selective agent, and pH 5.8; 6) kanamycin-resistant callus tissue that obtains is transferred to be cultured in division culture medium emerge, described division culture medium is: MS basic medium+ZT2.0mg/L+KT 0.2mg/L+PVP 8-10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+8g/L agar, and pH 5.8; 7) seedling is moved into turn out root in the root media, described root media is: 1/2MS basic medium+inositol 500mg/L+NAA 400mg/L+IBA 2mg/L+PVP 8-10mg/L+30g/L sucrose+8g/L agar, and pH 5.8; 8) transplant to greenhouse flowerpot or land for growing field crops.
Described young spike length degree is 2-3cm, and the about 3mm of thin slice is long.
Described step 2) cultivation, every ware 30-40 piece callus.
Described direct conversion method is a particle bombardment.
Described particle bombardment is that the golden bullet that adopts parcel to contain the plasmid DNA bronze formation of foreign gene bombards explant to be transformed.
Described packaging method is: the bronze suspension (50mg/ml) of getting 25 μ l diameters, 1 μ m is put into 1.5mL eppendorf pipe, adds the plasmid DNA solution (1 μ g/ μ l) that 2.5 μ l contain foreign gene, adds 25 μ l 2.5mol/LCaCl behind the mixing more respectively
2Solution and 10 μ l 0.1mol/L spermidine solution leave standstill 10min on ice, centrifugal 3sec (1000rpm), and abandoning supernatant adds 50 μ l dehydrated alcohols, gets final product after fully suspending.
Described foreign gene is cry1Ah, and described selective agent is the 0.8mM glyphosate isopropyl amine salt.
Described recovery incubation time is a week.
Described screening time was two weeks.
Culture condition in the described division culture medium is: cultivate under 28 ℃ of conditions in the illumination of 16 hours photoperiods and 8 hours dark cycle, per two all subcultures once.
Before described step 7) changed over to, the sweet sorghum plant was about the 2-3cm height.
Before described step 8) changed over to, the sweet sorghum plant was in the 3-4 leaf phase.
Described sweet sorghum kind is " BABUSH " and " MN-3025 ".
The present invention is by relatively finding the influence of sweet sorghum tissue culture factors such as plant hormone proportioning, medium component, different genotype, add zeatin 2.0mg/L in the substratum and have important effect, can increase substantially the more rate that lures for inducing of young fringe callus; An amount of 8-10mg/L PVP and the 10mg/L xitix of adding in substratum can alleviate browning effectively simultaneously, promotes to lure the more raising of rate.The present invention has set up sweet sorghum children fringe callus culture regeneration system, and foreign gene cry1Ah is transformed young fringe callus by direct conversion method (embodiments of the invention are particle bombardment) success, obtain transgenic regenerated plant, show after testing, foreign gene has obtained the expression of distinct program, illustrates that regeneration system of the present invention can be used for genetic transformation.Though the present invention does not directly transform young fringe, those skilled in the art adopt to transform identical method and the step of young fringe callus lines, can realize equally.
The present invention utilize sweet sorghum children fringe for explant when condition is suitable, can obtain higher callus induction rate, still, the embryo proterties condition and the regenerative power difference of the callus in different genotype source are bigger.Experimental result shows that young fringe is to set up the more satisfactory a kind of explant of sweet sorghum regeneration system, can be used for further Study on Transformation.The still rarely found at present report of sweet sorghum transgenic research, the present invention lays a good foundation for carrying out the sweet sorghum transgenic research from now in a deep going way by the foundation of sweet sorghum particle bombardment transformation system.
Description of drawings:
Fig. 1 is used for sweet sorghum particle bombardment plant transformed expression vector expression casette structural representation
The regeneration of Fig. 2 sweet sorghum particle bombardment transformed plant, insect-resistance detection and Herbicid resistant blade are coated with and dye experiment.
A gets boot leaf phase children fringe and is inoculated in callus of induce substratum (CI); B children fringe is induced the generation callus; Be inoculated in height after the c bombardment callus and ooze substratum; D screens callus with the 0.8mM glyphosate isopropyl amine salt; The growth of sprouting of e callus; F seedling root culture; G and h T0 are for transformed plant intermediate house (seedling with become seedling); I and j T0 give birth to for sweet sorghum transformed plant Pyrausta nubilalis (Hubern). and survey the result: resistant transgenic plant (i) and non-transgenic adjoining tree (j); K glyphosate resistance experimental result: resistant plant (two blades in the left side) and non-transgenic adjoining tree (two blades in the right).
Fig. 3 part T0 is for the PCR detected result of sweet sorghum transformed plant.
A:cry1Ah gene primer amplification.1:DNA standard molecular weight λ/Eco130I (λ/E) (bp): 19329,7743,6223,4254,3472,2690,1882,1489,925,421; 2:CK
+, the pMUHEGM plasmid is done the positive control of template; 3: blank; 4: unconverted plant; 5-13: transformed plant;
B:2mG2-epsps gene primer amplification.1:DNA standard molecular weight λ/Eco1301 (λ/E); The 2:pMUHEGM plasmid is done the positive control of template; 3: unconverted plant; 4: blank; 5-13: transformed plant.
Fig. 4 part T0 is for transformed plant RT-PCR result.
1, DNA standard molecular weight DL 2000 (bp): 100,250,500,750,1000,2000; 2,4,6, be template with transformed plant cDNA; 3,5,7, be template with the total RNA of transformed plant; 8, be template with the total RNA of unconverted plant; 9, CK
+, be template with the pMUHEGM plasmid.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
Below the sweet sorghum of related different genotype show all and originated that the public can freely obtain from applicant's experiment.
The foundation of embodiment 1 sweet sorghum children fringe tissue culture regeneration system
1.1 being used for the young fringe material of sweet sorghum tissue culture selects and preparation
The present invention gets the young fringe that the boot leaf phase is about 2～5cm, carries out surface sterilization 3min with 70% alcohol, and aseptic water washing repeatedly; In super clean bench, be cut into uniform thin slice by knife and be inoculated in inducing culture [MS basic medium+2,4-D 2mg/L+ZT (zeatin) 2.0mg/L+PVP (polyvinylpyrrolidone) 10mg/L+ xitix 10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+7.2g/L agar, pH 5.8] place 20～21 ℃ of dark 3～4 weeks of cultivation, make it to begin dedifferentiation, form callus; Select the callus succeeding transfer culture [inducing culture+KT (kinetin) 0.2mg/L] that growth is rapid, quality is crisp, color is vivid afterwards, per 7～10 days subcultures once.Select the good callus of growth conditions and be connected to division culture medium [MS basic medium+ZT 2.0mg/L+KT 0.2mg/L+PVP 8-10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+8g/L agar, pH 5.8] go up 26-28 ℃ of light 2～3 weeks of cultivation, the seedling that afterwards callus regeneration is gone out moves into root media [1/2MS basic medium+inositol 500mg/L+NAA 400mg/L+IBA 2mg/L+PVP 8-10mg/L+30g/L sucrose+7.2g/L agar, pH 5.8] in, (height of seedling 3～5cm when sending out roots, root long 2～3cm), the flush away substratum, transfer to and practice seedling cultivation 7-15d, intermediate house or land for growing field crops then in the little culturing pot that the sterilization vermiculite is housed.
1.2. select the young fringe evoked callus of suitable developmental stage
The young fringe of different development stage (being as the criterion with young spike length degree) is inoculated on the young fringe substratum, and there is difference in its callus of induce ability.Children's spike length degree during less than 1.0cm the callus induction rate very low, only be 16.7%; Children's spike length degree be 1.0～2.0cm period lure more that rate increases, can reach 43.8%; When young spike length degree during at 2.0～3.0cm, lure more that rate reaches the highest, be 61.7%.After young spike length degree is greater than 5.0cm, along with the prolongation of its developmental stage, the increase of young spike length degree, the inductivity of callus descend gradually (table 1).This experimental result shows, sweet sorghum children spike length degree is that young fringe has bigger influence at different development stage to inducing of callus, therefore, with sweet sorghum children fringe be explant material when setting up tissue culture regeneration system or genetic transformation, should noting selecting the young fringe of the developmental stage that suits.
The young spike length degree of table 1 is to the influence of callus induction
1.3. different genotype is to the influence of young fringe callus induction
Choose 7 kinds of different sweet sorghum genotype children fringe materials, have 617 outer planting physical efficiencys and induce callus in 866 explants of inoculation, on average luring the rate of healing is 71.25%, and brownization mortality ratio is 28.75%.More rate is widely different but lure between different genotype, wherein lures the rate of healing the highest with " BABUSH ", can reach 84.23%, secondly is " MN-3025 ", and luring more, rate is 79.26%; " sweet No. three of product " lure the rate of healing very low, only are 31.11% (table 2).Illustrate that genotype is an important factor that influences sweet sorghum children fringe tissue culture, therefore should note selecting genotype in actually operating.
Table 2 different genotype is to the influence of sweet sorghum children fringe callus induction
Annotate :/--expression does not induce callus
1.4. the regenerative power of different genotype children fringe callus relatively
As shown in table 3, different genotype differentiation of calli regenerative power shows evident difference.In the young fringe genotype of 7 kinds of differences for examination, young fringe callus differentiation rate is obviously different.In 368 young fringe callus of inoculation, have 228 callus and can break up regeneration and emerge, average differentiation rate is 61.96%, brownization mortality ratio is 38.04%.Wherein the differentiation rate of " BABUSH " is the highest, can reach 94.44%, and its brownization mortality ratio is minimum, be 5.56%, and the differentiation rate of " sweet No. three of product " is minimum, only has 26.67%, and brownization mortality ratio is then up to 73.33%.Between each genotype of this presentation of results callus differentiation rate difference clearly, genotype also is to influence one of its regenerative power factor of crucial importance.
Table 3 different genotype is to the influence of callus differentiation rate
Annotate: a: the callus lines of representing the energy differentiation and seedling emergence; B: represent the callus of differentiation and seedling emergence to account for the percentage of the callus sum that is transferred on the division culture medium.
Conclusion: the present invention has remarkable influence (p=0.000＜0.001) by relatively finding different sweet sorghum children spike length degree to the inductivity of callus, therefore, with sweet sorghum children fringe be explant material when setting up tissue culture regeneration system or genetic transformation, selecting the young fringe of developmental stage of 2-3cm comparatively suitable.Callus induction ability and the differentiation regenerative power of discovering different genotype children fringe material show evident difference: on average luring the rate of healing is 71.25%, and average differentiation rate is 61.96%.Through more therefrom filtering out two sweet sorghum kind " BABUSH " and " MN-3025 ", can be used for further Study on Genetic Transformation with high frequency regeneration ability.
The present invention is by relatively finding the influence of sweet sorghum tissue culture factors such as plant hormone proportioning, medium component, different genotype, add zeatin 2.0mg/L in the substratum and have important effect, can increase substantially the more rate that lures for inducing of young fringe callus; An amount of 8-10mg/L PVP and the 10mg/L xitix of adding in substratum can alleviate browning effectively simultaneously, promotes to lure the more raising of rate.The present invention has set up sweet sorghum children fringe callus culture regeneration system: wherein callus inducing medium is: MS basic medium+2,4-D 2mg/L+ZT 2.0mg/L+ caseinhydrolysate 500mg/L+PVP10mg/L+ xitix 10mg/L+30g/L sucrose+7.2g/L agar, pH 5.8; Subculture medium is: inducing culture+KT 0.2mg/L; Division culture medium is: MS+ZT 2.0mg/L+KT 0.2mg/L+PVP 8-10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+8g/L agar 8, and pH 5.8; Root media is: 1/2MS+ inositol 500mg/L+NAA 400mg/L+IBA 2mg/L+PVP 8-10mg/L+30g/L sucrose+8g/L agar, pH5.8.
2.1 the used substratum of particle bombardment:
Callus inducing medium: MS+2,4-D 2mg/L+ZT 2.0mg/L+PVP 10mg/L+ xitix 10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+7.2g/L agar, pH 5.8;
Callus subculture medium: MS+2,4-D 2mg/L+ZT 2.0mg/L+KT 0.2mg/L+PVP 10mg/L+ xitix 10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+7.2g/L agar, pH5.8;
Height oozes substratum: subculture medium+0.4M N.F,USP MANNITOL;
Screening culture medium: subculture medium+suitable selective agent (deciding as the case may be);
Callus division culture medium: MS+ZT 2.0mg/L+KT 0.2mg/L+PVP 8-10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+8g/L agar, pH5.8;
Root media: 1/2MS+ inositol 500mg/L+NAA 400mg/L+IBA 2mg/L+PVP 8-10mg/L+30g/L sucrose+8g/L agar, pH5.8.
2.2 method for transformation:
Choose the young fringe material (2-3cm) of suitable developmental stage, be cut into the thin slice of big or small homogeneous (about 3mm), be inoculated in callus induction on the inducing culture.4hr before transforming selects and induces the good callus lines of growth conditions of generation to transfer to the height that contains 0.4M N.F,USP MANNITOL by young fringe to ooze on the substratum, and 30～40 callus of every culture dish are placed in the about 2.5cm scope of culture dish mid-diameter.With plasmid DNA (plant expression vector that contains goal gene) parcel diameter 1.0 μ m bronzes, the packaging method of gold bullet is as follows: the bronze suspension (50mg/ml) of getting 25 μ l diameters, 1 μ m is put into 1.5mL eppendorf pipe, adds 2.5 μ l plasmid DNA solutions (1 μ g/ μ l).Add 25 μ l 2.5mol/L CaCl behind the mixing more respectively
2Solution and 10 μ l 0.1mol/L spermidine solution leave standstill 10min on ice, of short duration centrifugal 3sec (1000rpm), and abandoning supernatant adds 50 μ l dehydrated alcohols, can go up sample after fully suspending.Adopt PDS-1000/He particle gun (production of Bio-Rad company), according to the described program of particle gun specification sheets acceptor material is bombarded, the bombardment parameter is: Gap distance:20mm; Little missile-borne body flying distance: 10mm; Little bullet flying distance: 7cm; Can split film pressure: 1100psi; Vacuum tightness: 25inches Hg.Every ware material bombardment 1-2 time, it is 12.5 μ l/ time that gold plays consumption.
The screening of transformed calli and the regeneration of plant: the young fringe callus after will bombarding continues to ooze 26 ℃ of dark overnight incubation on the substratum at height, transfers to then on the inducing culture, recovers to cultivate a week; Callus is transferred on the screening culture medium, screened for two weeks, discard the dead callus of brownization, select color and luster callus normal, that growth conditions is good to be kanamycin-resistant callus tissue; Kanamycin-resistant callus tissue is moved on the division culture medium that does not have the screening pressure, cultivate under photoperiod 16h illumination/8h dark, 28 ℃ of conditions, per two all subcultures once; With the callus cutting of green bud takes place, be beneficial to the growth of main bud; When plantlet length to 2～when 3cm was high, (containing the MS root media) continuation root culture in the vial was advanced in transfer; When treating that 3～4 leaf phases of seedling and root system are flourishing, flush away agar moves in the small flower, moves into hot-house culture; The big flowerpot of two weeks back immigration is until blossoming and bearing fruit.
2.3 sweet sorghum changes the acquisition of cry1Ah gene regeneration plant
Select the good young fringe callus of growth conditions and be used for the particle gun conversion, used plant expression vector pMUHEGM (preserve in the applicant laboratory, and the public can freely obtain from the applicant) as shown in Figure 1.Callus after the particle gun bombardment, through the screening of 2 all 0.8mM glyphosate isopropyl amine salts, most of callus is browning gradually, short texture, it is dead then to stop growing; But on minority callus surface, begin to occur the growth district of projection, be milk yellow, structure is fine and close, is resistant calli.After the screening kanamycin-resistant callus tissue that obtained through regenerate, take root, after strong sprout, vermiculite transition cultivation intermediate house.After the regeneration plant to be transformed heading, bagging, pollination self, solid naturally, gather in the crops T0 for seed (accompanying drawing 2, a-h).
2.3.1 the Molecular Identification of sweet sorghum transformed plant
2.3.1.1T0 the PCR for the sweet sorghum transformed plant detects
Utilize particle bombardment that carrier pMUHEGM is transformed sweet sorghum children fringe callus,, obtain T0 altogether for transformed plant 83 strains through the screening of 0.8mM glyphosate isopropyl amine salt.Whether be integrated in the sweet sorghum genome for detecting foreign gene, the 83 strain transformed plants that obtain are extracted total DNA, carried out pcr analysis, the primer: cry1Ah: upstream primer is: GCCCAAGCTGC CAACCTCCAC TTGTCTTTGC
Downstream primer is: GGTGCTCTTA TGATGCTGAC ACTGCTACTA GAGCC2mG2-epsps: upstream primer is: CCGTGACC GTTACACCAC CTAACTTCCC
Downstream primer is: TTGAATGCAG CGAGAACCGC TAGAGTCGGG
It is as follows that PCR detects amplification condition: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 1min, 58 ℃ of annealing 40sec, 72 ℃ are extended 50sec, amplification cycles number: 30～35; Last 72 ℃ are extended 10min.Accompanying drawing 3 is respectively the pcr analysis result of part transfer-gen plant cry1Ah gene and 2mG2-epsps gene.It is 865bp that the result has 16 strain transformed plants to amplify the band of two target gene fragment sizes: cry1Ah simultaneously, and 2mG2-epsps is 909bp.In the total DNA of unconverted adjoining tree, fail to amplify corresponding DNA fragments, illustrate that foreign gene has been integrated in the transgenic sweet sorghum genome.
2.3.1.2T0 the RT-PCR for the sweet sorghum transformed plant detects
Selected part PCR positive plant and non-transgenic adjoining tree are extracted respectively after the total RNA reverse transcription of blade and detect cry1Ah gene transcription situation with the RT-PCR method.The result shows, the adjoining tree target stripe that do not increase, and the transfer-gen plant size that all can increase is the target fragment of 865bp, showing that the cry1Ah gene imports to can normal transcription (accompanying drawing 4) in the sweet sorghum genome.
2.4 the biological activity assay of sweet sorghum transformed plant
Treating to transform seedling by sweet sorghum, to grow into 6～8 leaves be lobus cardiacus during mid-term, and the Pyrausta nubilalis (Hubern). newly hatched larvae is connected in the lobus cardiacus, and every strain connects 40～60 of Pyrausta nubilalis (Hubern). newly hatched larvaes, and as negative control, it is other to connect worm 1～2 all " Invest, Then Investigate " food leaf-size classes with non-transformed plant.The result shows, after manually connecing worm, performance has certain difference to the insect-resistance of Pyrausta nubilalis (Hubern). between the transfer-gen plant individual plant: the non-transgenic plant or the insect-resistant transgenic plant not stung food by Pyrausta nubilalis (Hubern). serious, the blade worm channel is big and the worm channel number is many, has had influence on the normal growth of plant.Screen 3 strains at T0 altogether in for transformed plant and show insect-resistance preferably, do not have or have only on a small quantity, the worm channel of very little Pyrausta nubilalis (Hubern). harm, all the other plant show as sense worm or high sense (accompanying drawing 2, i and j), to analyze reason may be that the expressing quantity of cry1Ah gene in sweet sorghum is low causes.Resistant plant accounts for changes 3.61% of cry1Ah gene plant sum.
2.5 the Herbicid resistant of sweet sorghum transformed plant detects
Treat that sweet sorghum transforms growth of seedling to 4～6 leaf phases, (farming reaches RoundUP with 0.2% glyphosate medicament
) carry out blade and smear experiment: in that line serves as a mark from blade tip 5cm place, dip in the weedicide soup of getting suitable concentration with cotton swab and spread upon the line position equably, one week back statistics sweet sorghum blade victimization state.After glyphosate is used 8 days as a result, symptom of chemical damage such as leaf chlorosis, wilting, brownization appear in contrast and the non-transgenic sweet sorghum seedling leaf place of smearing, pass in time, the poisoning degree is constantly deepened, the small part plant shows the phenomenon of deformity and cessation of growth cessation, in addition some transformed plant blade is to showing the symptom of significantly being injured, and showing has preferably resistance (see accompanying drawing 2, k) to glyphosate.Spray glyphosate and observed statistics on the 14th day, screen 14 strain glyphosate resistances altogether and transform seedling, account for 16.87% of total seedling number.
Claims (10)
1. one kind is the explant genetic transforming method with sweet sorghum children's fringe or young fringe evoked callus, comprise following sequential steps: 1) choose long 2-5cm children of sweet sorghum boot leaf phase fringe, thinly slice, it is inoculated in the inducing culture cultivates, described inducing culture is: MS basic medium+2,4-D 2mg/L+ZT 2.0mg/L+PVP 10mg/L+ xitix 10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+7.2g/L agar, pH 5.8; Form callus; 2) choosing good young fringe of growth conditions or young fringe callus lines is inoculated in height and oozes in the substratum and cultivate, described height oozes substratum: MS+2,4-D 2mg/L+ZT 2.0mg/L+KT 0.2mg/L+PVP 10mg/L+ xitix 10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+7.2g/L agar+0.4M N.F,USP MANNITOL, pH 5.8; 3) directly conversion method changes foreign gene over to, continues to ooze overnight incubation in the substratum at height; 4) change recovery cultivation in the callus inducing medium over to, described callus inducing medium is: MS+2,4-D 2mg/L+ZT 2.0mg/L+PVP 10mg/L+ xitix 10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+7.2g/L agar, pH 5.8; 5) change on the screening culture medium again and screen, described screening culture medium is: MS+2,4-D 2mg/L+ZT 2.0mg/L+KT 0.2mg/L+PVP 10mg/L+ xitix 10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+7.2g/L agar+suitable selective agent, pH 5.8; 6) kanamycin-resistant callus tissue that obtains is transferred to be cultured in division culture medium emerge, described division culture medium is: MS basic medium+ZT 2.0mg/L+KT 0.2mg/L+PVP 8-10mg/L+ caseinhydrolysate 500mg/L+30g/L sucrose+8g/L agar, pH5.8; 7) seedling is moved into turn out root in the root media, described root media is: 1/2MS basic medium+inositol 500mg/L+NAA 400mg/L+IBA 2mg/L+PVP 8-10mg/L+30g/L sucrose+8g/L agar, and pH 5.8; 8) transplant to greenhouse flowerpot or land for growing field crops.
2. genetic transforming method according to claim 1, described young spike length degree is 2-3cm, the about 3mm of thin slice is long, described step 2) in cultivation, every ware 30-40 piece callus.
3. genetic transforming method according to claim 1, described direct conversion method is a particle bombardment.
4. genetic transforming method according to claim 3, described particle bombardment are that the bronze that adopts parcel to contain the plasmid DNA of foreign gene bombards acceptor to be transformed.
5. genetic transforming method according to claim 4, described packaging method is: the bronze suspension concentration of getting 25 μ l diameters, 1 μ m is that 50mg/ml puts into 1.5mL eppendorf pipe, adding the plasmid DNA solution concentration that 2.5 μ l contain foreign gene is 1 μ g/ μ l, adds 25 μ l 2.5mol/L CaCl behind the mixing more respectively
2Solution and 10 μ l 0.1mol/L spermidine solution leave standstill 10min on ice, and centrifugal 3sec rotating speed is 1000rpm, and abandoning supernatant adds 50 μ l dehydrated alcohols, get final product after fully suspending.
6. genetic transforming method according to claim 5, described foreign gene are Bt killing gene cry1Ah, and described selective agent is the 0.8mM glyphosate isopropyl amine salt.
7. genetic transforming method according to claim 1, described recovery incubation time is a week, described screening time was two weeks.
8. genetic transforming method according to claim 1, culture condition in the described division culture medium is: cultivate under 28 ℃ of conditions in the illumination of 16 hours photoperiods and 8 hours dark cycle, per two all subcultures once, before described step 7) moved into, the sweet sorghum plant was the 2-3cm height.
9. genetic transforming method according to claim 1, before described step 8) was transplanted, the sweet sorghum plant was in the 3-4 leaf phase.
10. genetic transforming method according to claim 1, described sweet sorghum kind are " BABUSH " and " MN-3025 ".
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| CN106613978B (en) * | 2016-12-23 | 2018-08-17 | 山西省农业科学院高粱研究所 | A kind of sorghum body cell suspension culture method and application |
| CN109609545A (en) * | 2019-01-24 | 2019-04-12 | 安徽科技学院 | Sorghum mature seed transgenic method |
| CN116584391A (en) * | 2023-06-09 | 2023-08-15 | 辽宁省农业科学院 | Induction method of mature sorghum seed somatic embryo |
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Non-Patent Citations (3)
| Title |
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| Liming Zhao, et al.Optimization of callus induction and plant regeneration from germinating seeds of sweet sorghum (Sorghum bicolor Moench).《African Journal of Biotechnology》.2010,第9卷(第16期),2367-2374. * |
| 徐丹,等.甜高粱离体再生体系的建立和组织结构变化的观察.《植物生理学通讯》.2009,第45卷(第8期),771-774. * |
| 赵利铭，等.甜高粱再生体系的建立.《植物学通报》.2008,第25卷(第4期),465-468. * |
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