CN1429904A

CN1429904A - Method for gene conversion of corn

Info

Publication number: CN1429904A
Application number: CN02158758A
Authority: CN
Inventors: 王国英; 张�荣
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2002-12-26
Filing date: 2002-12-26
Publication date: 2003-07-16

Abstract

A process for gene conversion of corn includes such steps as immersing the embryo or callus of corn in the agrobacterium D-inf solution containing acetyl syringone, waving for 30-60 seconds, laying aside for 5-60 min, adding it to D-As culture medium, culturing at 22-26 deg.c for 2-7 days in dark condition, washing with aseptic water containing thisporomycin, immersing for 15-60 min, culturing in the culture medium containing thisporomycin for 2-7 days, low-pressure selective culture, high-pressure selective culture, inducing embryoid, and differentiating in differential culture medium to obtain regenerated transgenic plant.

Description

A kind of method of corn being carried out gene transformation

Technical field

The present invention relates in the bioengineering field plant be carried out the method for gene transformation, particularly relate to the method for corn being carried out gene transformation.

Background technology

Plant genetic engineering is a directed genetic recombination material on molecular level, improvement plant trait, the molecular breeding technology of cultivation good quality and high output crop new variety.At the end of the seventies, confirmed that from molecular level soil Agrobacterium (Agrobacterium tumefacieus) after infecting vegetable cell, not only can be inserted into its section of DNA by in the genome of infected cell, and can entail the offspring first.Afterwards, plant genetic engineering research has obtained a series of important breakthroughs continuously.From obtaining the first strain transfer-gen plant till now less than in the vicennial time, transgenic plant have expanded to 35 genus that comprise cash crop, food crop, vegetables, flowers, medicinal plant, fruit, trees and herbage etc., more than 120 species.Some have all realized commercialization through crop such as tomato, rape, tobacco, summer squash, flax, corn, soybean and the cotton of genetically engineered improvement.Several important steps in the plant genetic engineering: the modification of the separation of goal gene and evaluation, gene and the structure of expression vector, goal gene have all been obtained very big progress to the screening of the conversion of vegetable cell, transformant and evaluation etc., formed sophisticated experiment flow, and novel method, new technology continue to bring out.

Aspect the research of plant acceptor, the explant that experiment confirm can be used as acceptor has: blade, petiole, cotyledon, cotyledon petiole, hypocotyl, stem, shoot apical meristem, stem tuber, bud, root, pollen, zygotic embryo, ovary, ovule, somatocyte, embryo callus, suspended culture cell, protoplastis, mature seed etc.Cell or tissue as transformation receptor should possess two conditions: (1) can accept foreign gene, and by gene recombination or other approach foreign gene is stably inserted in the plant chromosome group; (2) have regenerative power, can form new plant materials.Certainly, may be different for the required explant kind of different plants, on the same species, different explants has different changing effects.The most frequently used blade of dicotyledons, cotyledon, protoplastis; Monocotyledons is used immature embryo, callus, protoplastis more.It is difficult generally speaking to obtain the Regeneration in Vitro plant for cereal crop, sets up high-frequency plant regeneration system, is the basis of realizing efficient genetic transformation.

The genetic transformation of plant (genetic transformation) is meant means such as utilizing biology and physical chemistry, with the technology of exogenous gene transfered plant cell with the acquisition transgenic plant.The plant genetic Study on Transformation not only has important significance for theories, and is with a wide range of applications.On the one hand,, some foreign genes can be used as molecular biological research tool by being imported plant, research expression of gene, regulation and control, 26S Proteasome Structure and Function, and on molecular level, help to resolve the plant biochemistry process.On the other hand, genetic transformation can be broken the species boundary, on purpose realizes between the species or the interchange of the inner genetic material of species, is a new way of genetic modification of plants.

The research that plant genetic transforms starts from the end of the seventies, and the achievement in research of agrobacterium tumefaciens and Ti-plasmids has promoted its process, and the dicotyledons of Agrobacterium-mediated Transformation reaches 120 kinds.Some early stage experiments show that Agrobacterium is difficult to infect cereal crop, so the Study on Transformation of cereal crop is later relatively.After separation of cereal plants protoplastis and culture system foundation, adopt means such as PEG method, electric shock processing, begun the genetic transformation of cereal crop.

Agriculture bacillus mediated conversion method is that foreign DNA is cloned into the T-DNA district, and Agrobacterium contacts with vegetable cell, utilizes the natural metastasis of Agrobacterium that foreign gene is imported in the recipient cell.The factor that influences the agrobacterium tumefaciens genetic transformation comprises: 1, different agrobacterium tumefaciens bacterial strains have the host range that is not quite similar, and there is difference in the transformation efficiencies of different plants.2, bacterial concentration.3, the different explants of the different varieties of same species, same kind is different for Agrobacterium tumefaciens mediated genetic transformation reaction, and different transformation efficiencies is arranged.4, agrobacterium tumefaciens and Ti-plasmids thereof are transformed, can expand the host range of agrobacterium tumefaciens and improve transformation efficiency.5, the application of inductive substance and suitable pH value.

Over past ten years, because the appearance of a series of new transgenic methods such as particle gun blast technique, ultrasonication, both overcome host's restriction of agrobacterium mediation converted, the protoplastis that has got around cumbersome complexity is again cultivated, and makes many plant transformed that were difficult to originally transform success in succession.

The principal element that influences the particle bombardment transformation frequency is the physiologic factor of bombardment parameter and acceptor.The bombardment parameter of particle gun comprises range, the vacuum tightness in the cartridge chamber, the purity of bombarding number of times, DNA and the concentration of the movement velocity of little bullet, little bullet, the concentration of DNA precipitation agent etc.The movement velocity of little bullet is an important factor that influences transformation frequency, and little bullet of friction-motion speed is variant to the penetration power of tissue and cell.The range of little bullet refers to the baffle plate of particle gun and the distance between target cell, and it also is an important factor that influences transformation frequency.In plant tissue, have only some specific cell to have regenerative power, therefore can preferentially transform these cells by regulating range.Though the vacuum tightness in transformation frequency and the cartridge chamber is proportionate, target cell has certain tolerance to vacuum tightness, so the vacuum tightness in the cartridge chamber also has certain restriction.The bombardment number of times is also influential to transformation frequency, and test finds that the transient expression activity bombard cat and GUS once more all increases to some extent, increases that often to cause bombarding cell injury serious but bombard number of times again, can reduce transformation frequency on the contrary sometimes.The purity of DNA and concentration all influence transformation frequency.The purity of DNA is high more, and changing effect is good more; But the concentration of DNA is high more, makes little bullet adhesion become big condensing and transformation frequency is reduced easily more, and the concentration of the DNA that generally adopts is 1ug/ul at present.Calcium chloride and spermidine are often made the precipitation agent of DNA, make DNA attached to surface of metal particles, and the concentration of precipitation agent is influential to transformation frequency.People such as Klein (1998) report, the optimal concentration of calcium chloride is at 1.9M-2.4M, and the optimal concentration of spermidine is 7.69mM-76.9mM.

The advantage of particle bombardment is no host's restriction, and acceptor type is extensive, and operation rapidly, simply can transform plastid effectively.Extensively, the injection of metal particle is wide owing to draw materials, and the transformation frequency height is so be with a wide range of applications.Shortcoming be the dna fragmentation that transforms when too big dna fragmentation rupture easily, foreign gene inserts genome with multiple copied easily, easily causes the foreign gene silence.In addition, the whole transformation period of particle bombardment is longer, and along with long succeeding transfer culture, the possibility of somatic variation increases, and the embryo generating ability of callus descends.Simultaneously, this method consumption manpower is also big.

Summary of the invention

The purpose of this invention is to provide a kind of method of corn being carried out gene transformation.

For achieving the above object, the present invention is by the following technical solutions: a kind of method that corn is carried out gene transformation may further comprise the steps:

1) maize immature embryos or callus are immersed in the Agrobacterium D-inf solution that carries foreign gene that adds 50-200 μ M Syringylethanone, vibrate 30-60 second, and place the 5-60 branch and plant, taking-up is put on the D-AS substratum, cultivates altogether 2-7 days under 22-26 ℃ of dark condition;

2) move into and contain in the sterilized water of 150-250mg/L Reflin cultivating rataria after 2-7 days or callus altogether, wash 1-3 time, immersion 15-60 minute in the sterilized water that contains the 150-250mg/L Reflin for the last time;

3) taking out rataria or callus put to the callus of induce substratum that contains the 150-250mg/L Reflin 2-7 days;

4) low pressure is selected, and transformation receptor is moved on the screening culture medium that is added with the lower concentration selective agent cultivated for 2 weeks;

5) high pressure is selected, and transformation receptor is moved on the screening culture medium that is added with the high density selective agent cultivated for 3 weeks, carries out the 2-3 wheel altogether;

6) the resistance tissue of choosing is forwarded on the embryoid induction substratum, induce the formation embryoid; Break up forming being transferred on the division culture medium again of embryoid, obtain transgenic plant.

In order to make the better effects if of conversion, before transforming, with particle gun explant is handled earlier with Agrobacterium.

When explant was rataria, with empty little missile-borne body bombardment, at this moment sample vacuum chamber degree 25-27mmHg can split film pressure 1100-1350psi, little bullet flying distance 3-9cm.Rataria causes wound through the particle gun bombardment, infects with Agrobacterium again, and the kanamycin-resistant callus tissue rate average out to 40% of generation, transformation efficiency is significantly improved.When explant was callus, by the micropellet bombardment of Agrobacterium, at this moment sample vacuum chamber degree 25-27mmHg can split film pressure 1100-1350psi, little bullet flying distance 3-9cm with bag.The preparation method of described little bullet is: get bronze suspension 50 μ l, put into the 1.5ml centrifuge tube, add 1ml Agrobacterium solution, vibration is 5 minutes on the vibrator, 4000 rev/mins centrifugal 1 minute, remove supernatant liquor, repeat 3 times; The sterilized water that little missile-borne body and function 16 μ l contain 50-200 μ M Syringylethanone is resuspended, the each shooting with 4 μ l.

Utilize method of the present invention, foreign gene successfully can be changed in the corn tissue, all significant for the theoretical investigation and the genetic breeding practice of corn gene engineering aspect.

Description of drawings

Fig. 1 is the hpt gene PCR analytical electrophoresis figure of regeneration plant.

Fig. 2 is the bar gene PCR analytical electrophoresis figure of regeneration plant.

Fig. 3 is the hpt gene Southern blotting analytical results figure of regeneration plant.

Fig. 4 is the bar gene Southern blotting analytical results figure of regeneration plant.

Embodiment

Material and method:

1, corn material

Corn inbred line is combined 3 (Z3), combines 31 (Z31), neat 31 (Q31), Lay 1029 (L1029), P9-10, is obtained white (H), A188, B73, the wherein F of A188 and B73 ₂In generation, be designated as F

2, bacterial strain, plasmid

As shown in table 1, plasmid pTOK233, pAt5 are super binary vector, and clone has VirB, VirC, VirD gene on it.Plasmid p1301, p3301, pMG8 are common binary vector.The gus gene that contains intron on the T-DNA of plasmid pTOK233, pAt5, p3301, p1301, it is only expressed at vegetable cell, and does not express in bacterium, can detect the plant transformed cell.The GFMcryIA gene is arranged on the plasmid pMG225; The hpt gene is arranged on the plasmid pMH; The bar gene is arranged on the plasmid pDM302.

Table 1, the used Agrobacterium bacterial classification of the present invention and plasmid thereof

Bacterial strain bacterial classification plasmid carries gene microbiotic (mg/l)

LBA4404 pTOK233 GUS?HPT?NPT?II Hpt：50

LBA4404 pAt5 GUS?CryIB?Bar?NPT?II Kan：100

EHA105 p1301 GUS?HPT?NPT?II Kan：50

LBA4404 p3301 GUS?Bar?NPT?II Kan：100

EHA105 pMG8 Bar?NPT?II Kan：100?Str：25

3, biochemical reagents

Totomycin is available from BOEHRINGER MANNHEIM company, Reflin is available from Sigma company, weedicide is available from Japanese Mingzhi company, restriction enzyme, random primer test kit are available from promega company, Syringylethanone (AS) is available from Fluka company, isotropic substance is available from inferior brightness biomedical engineering company, and other reagent such as X-Gluc and medicine are available from relevant producer and company both at home and abroad.

The preparation of selective agent:

Totomycin (50mg/L) is stored in 4 ℃ after 0.22 μ m millipore filtration;

Reflin (250mg/L) is stored in-20 ℃ after 0.22 μ m millipore filtration;

It is 10mg/L that weedicide is made into PPT effective constituent concentration with redistilled water, after the 0.22 μ m millipore filtration, is stored in room temperature.

4, instrument

Pcr amplification instrument (GeneAmp PCR System 9700); PDS-1000/He type particle gun

5, substratum

(1) selects culture medium preparation

The D substratum is cooled to about 50 ℃ behind autoclaving, adds Totomycin and Reflin or weedicide and Reflin and is made into certain density selection substratum.

(2) other culture medium preparation is shown in table 2-table 4:

Table 2, Agrobacterium YEB substratum and YEP substratum

Composition YEB composition YEP

Extractum carnis 5g/L NaCl 5g/L

Tryptones 5g/L Tryptones 10g/L

Sucrose 5g/L yeast extract 10g/L

Yeast extract 5g/L pH 7.0

Sal epsom 0.5g/L solid medium adds agar 15g/L

Table 3, Agrobacterium AB substratum: pH7.2

The constituent concentration constituent concentration

KH ₂PO ₄ 3g/L Cacl ₂·H ₂O 0.012g/L

NaHP ₄·2H ₂O 1g/L FeSO ₄·7H ₂O 0.0025g/L

NH ₄Cl 1g/L glucose 5g/L

MgSO ₄·7H ₂O 0.3g/L KCl 0.15g/L

Solid medium adds agar 15g/L

Table 4, maize immature embryos, callus culture, conversion, inducing embryoid body reach

Plant division culture medium purposes substratum plant hormone microbiotic, (mg/L) height oozes pre-cultivation D+0.4M N.F,USP MANNITOL Dicamba:15-30 μ M and infects D-inf Dicamba:15-30 μ M and cultivate D-AS Dicamba:15-30 μ M callus of induce D Dicamba:15-30 μ M Hpt:15 altogether, (PPT:5) cef:250

N6 2，4-D：2mg/L

Select D1.5 Dicamba:15-30 μ M Hpt:20 (PPT:10) cef:250

Embryoid induction D1.5; DM 6-BA:5mg/L cef:100

Differentiation RM cef:100

D-inf solution: N6 macroelement, B5 trace element, RTV organism, molysite, sucrose 68 grams per liters, glucose 36 grams per liters, proline(Pro) 0.7 grams per liter, caseinhydrolysate 0.5 grams per liter, Dicamba:15-30 μ M, pH5.2.

D substratum: N6 macroelement, B5 trace element, RTV organism, sucrose 30 grams per liters, L-proline(Pro) 0.7 grams per liter, caseinhydrolysate 0.5 grams per liter, agar 8 grams per liters, Dicamba:15-30 μ M, Silver Nitrate 100 μ M, pH5.8.

6, pcr amplification primer

1) the hpt gene PCR amplimer 0.48kb fragment that can increase, its sequence is:

5 ' primer: 5 ' GGC GAA GAA TCT CGT GCT TTC A 3 '

3 ' primer: 5 ' CAG GAC ATT GTT GGA GCC GAA A 3 '

2) the bar gene PCR amplimer 0.46kb fragment that can increase, its sequence is:

5 ' primer: 5 ' GCG GTC TGC ACC ATC GTC A 3 '

3 ' primer: 5 ' GTA CCG GCA GGC TGA AGT CCA 3 '

Embodiment 1, Agrobacterium-mediated Transformation maize immature embryos

1, the preparation of plant acceptor material:

Corn examination material after planting need carry out controlled pollination by test, to obtain corresponding self-mating system material or cross-fertilize seed.Pollination back 6-13 days is got corn children fringe and peel off bract on Bechtop, takes out rataria.

2, the cultivation of agrobacterium strains

(1) store method of bacterial strain

Single bacterium colony with transfering loop picking Agrobacterium lines on the corresponding antibiotic AB solid medium of interpolation, cultivates 3 days in 28 ℃ incubator, and the refrigerator that changes 4 ℃ over to is preserved, and subculture once every other month.

(2) preparation of bacterium liquid

The single bacterium colony of the Agrobacterium of picking subculture after 3 days, in being added with corresponding antibiotic liquid YEB substratum, carry out shaking culture with 250rpm, be cultured to the logarithmic growth after date, bacterium liquid is placed centrifuge tube at 28 ℃, centrifugal 5min and collect thalline under 5000rpm, the thalline of collecting is washed with the D-inf liquid nutrient medium that adds 100 μ M AS,, at last Agrobacterium is suspended among the D-inf that adds 100 μ M AS to remove remaining YEB substratum, the about 0.3-0.4 of OD value, standby.Or cultivated 2-3 days with the AB solid medium, collect thalline, the thalline of collecting is washed with the D-inf liquid nutrient medium that adds 100 μ M AS, at last Agrobacterium is suspended among the D-inf that adds 100 μ M AS, the about 0.3-0.4 of OD value, placement can be used for transforming half an hour.

3, infection and cultivation altogether

Rataria is placed on height oozed on the substratum 3-4 hour, wash once, immerse again in the D-inf Agrobacterium bacterium liquid that adds 100 μ M with D-inf, with vibrator vibration 30 seconds, and place 5 fens kinds, take out and blot with sterilization filter paper, be put on the D-AS substratum, cultivate 3 days altogether 25 ℃ of dark, and establish contrast.

4, the recovery of Agrobacterium removing and transformation receptor

The rataria of cultivating altogether after 3 days is moved in the sterilized water of the Reflin that contains 250mg/L, wash 1-3 time, immersion 30 minutes in the sterilized water of the Reflin that contains 250mg/L for the last time.Take out vegetable material, blot, put a week to the callus of induce substratum of the Reflin that contains 250mg/L with aseptic filter paper.

5, low pressure is selected

The transformation receptor immigration is added with on the screening culture medium LSD1.5 that contains 10mg/L Totomycin (PPT5mg/L), and low selection is depressed and was cultivated for 2 weeks.

6, high pressure is selected

Carry out 2-3 wheel 20mg/L Totomycin (PPT10mg/L) high pressure and select every the wheel for 3 weeks.Each subculture notes eliminating the callus that is brown and water stainization, and broken with the tweezers folder the normal callus of growth, separately selects to cultivate.

7, embryoid induces

The resistant calli of choosing is forwarded on the inducing embryoid body substratum, and embryoid can appear in 3 weeks.

8, regeneration

Break up forming being transferred on the division culture medium again of embryoid, culture condition is 28 ℃, illumination every day 3000Lx light intensity illumination 16 hours, and it is born again to have seedling soon.

9, seedling hardening

During regenerated plantlet length to 3 slice leaf, seedling can be divided and be transplanted in the Cans, and at indoor cultivation.

10, regrowth is transplanted for the first time

After treating that seedling grows young leaves and root, seedling is taken out from Cans, tap water washes down substratum, transplants in the small flower that is mixed with nutrition soil and vermiculite (1: 3), and initial 3-5 days, seedling should hide plastics film, to keep humidity

11, transplant the second time of regrowth

When maize seedling grows 2-3 sheet young leaves again, it can be moved in land for growing field crops or the big flowerpot.The resistant plant number that obtains is as shown in table 5.

The most of forms of regeneration plant of self-mating system Z3, Z31, P9-10 and cross-fertilize seed F are normal, and female tassel is normal, can selfing obtain seed.The regeneration plant of self-mating system Q31 is shorter, and many plant plant heights are less than 20 centimetres, and most of plant do not have tassel and produce, and the top also grows filigree, need get pollen in addition and be its pollination.

The transient expression of gus gene detects among embodiment 2, the embodiment 1

With the explant of cultivating altogether after 3 days, add in the X-Gluc substrate solution that has prepared, 37 ℃ of insulations, spend the night.Naked eyes or surperficial and inner at stereoscope observation explant.Chlorenchyma need be with FAA solution fixedly more than the 1h, and uses 70%, 90%, 100% ethanol decolorization successively, observes its expression rate of statistics then, and expression rate is as shown in table 6:

From the result of table 6 as can be seen, the rataria of range gene type with contain super binary vector pTOK233, At5 and cultivate 3 days altogether after, all detected the transient expression of gus gene.GUS transient expression rate from 20% to 68.7%, average out to 52.3%.Illustrate that Agrobacterium can infect corn, T-DNA enters in the maize cell.

Table 5, the resistance regeneration plant number that obtains

Kind plasmid plant number

Z3 pTOK233 32

Z31 pTOK233 25

Q31 pTOK233 23

P9-10 pTOK233 11

F pTOK233 26

Z3 pAt5 22

Z31 pAt5 25

P9-10 pAt5 3

F pAt5 11

Amount to 178

Table 6, Agrobacterium are infected the GUS transient expression rate of maize immature embryos

Kind plasmid inoculation number GUS+ ratio %

Z3 PTOK233 10 4 40

Z31 PTOK233 40 13 42.5

Z31 At5 10 2 20

Q31 PTOK233 50 28 56

Q31 At5 20 15 75

P9-10 PTOK233 32 22 68.7

F pTOK233 10 6 60

Average 172 90 52.3

The screening of resistant calli among embodiment 3, the embodiment 1

Put to the screening culture medium that contains PPT5mg/L through high pressure rataria of cultivating and the rataria that the Agrobacterium that contains plasmid At5 is cultivated altogether, beginning two weeks of screening and culturing, forward screening and culturing on the screening culture medium that contains PPT10mg/L then to, per three all subcultures are once.Screening and culturing to two month has some callus growth conditions good, is resistant calli.The kanamycin-resistant callus tissue rate that statistics produces, as shown in table 7.

The kanamycin-resistant callus tissue rate that table 7, Agrobacterium-mediated Transformation rataria produce

Kind plasmid inoculation number kanamycin-resistant callus tissue percentage (%)

Z3 pTOK233 108 24 22

pAt5 117 26 22

Z31 pTOK233 179 44 26

pAt5 248 44 18

Q31 pTOK233 286 126 44

At5 157 27 17

P9-10 pTOK233 183 61 44

pAt5 121 37 31

F pTOK233 10 4 40

pAt5 61 33 54

Average 1,470 426 29

After two months, the kanamycin-resistant callus tissue rate of generation reaches 17% to 54%.Two kinds of plasmids transform the kanamycin-resistant callus tissue rate average out to 29% that rataria produces.The kanamycin-resistant callus tissue rate that F produces is the highest, and is II type callus.Plasmid pTOK233 transforms the kanamycin-resistant callus tissue rate average out to 33.8% that produces, and plasmid pAt5 transforms the kanamycin-resistant callus tissue rate average out to 23.7% that produces.

The Molecular Detection of embodiment 4, Agrobacterium-mediated Transformation maize immature embryos regeneration plant

One, the Molecular Detection in RO generation

1, PCR detects

Extract RO for the regeneration plant leaf DNA, plant 53 strains that picked at random plasmid pTOK233 is changeed, primer with the hpt gene carries out pcr amplification, it is the PCR positive that 48 strains are arranged, amplify with plasmid over against according to onesize fragment (about 480bp), and unconverted plant does not amplify corresponding fragment.The PCR result of part plant as shown in Figure 1, M: λ/HindIII+EcoRI marker wherein; Ck+:Plasmid pTOK233; CK-:Untransformed, except that swimming lane 13, all amplified with plasmid over against according to onesize fragment.

Plant 35 strains that picked at random plasmid At5 is changeed are carried out pcr amplification with the primer of bar gene, and it is the PCR positive that 28 strains are arranged, amplify with plasmid over against according to onesize fragment (about 460bp), and unconverted plant does not amplify corresponding fragment.The PCR result of part plant as shown in Figure 2, Lane M:100bp Ladder wherein; CK+:Plasmid At5 CK-:Untransformed control; Lane1-12:transgenic plants; Except that swimming lane 11, all amplified with plasmid over against according to onesize fragment.

2, Southern detects

Extract RO for the regeneration plant leaf DNA, after the BamHI enzyme is cut, the hpt gene fragment that cuts back to close 1.0kb with plasmid MH enzyme is a probe, the plant that changes the pTOK233 plasmid is carried out Southern blotting, the part plant has obtained the signal band, detected the hpt gene, the result as shown in Figure 3, the 1-4 swimming lane is a transfer-gen plant among the figure.

To changeing the plant of At5 plasmid, after the BamHI enzyme is cut, the bar gene fragment that cuts back to close 0.6kb with plasmid pDM302 enzyme is a probe, carry out Southern blotting, the part plant has obtained the signal band, has detected the bar gene, and the result as shown in Figure 4, swimming lane 1-8 is transgenic plant among the figure, illustrates that foreign gene has been integrated in the corn gene group.

Two, the offspring of transformed plant detects

1, transformed plant offspring's Molecular Detection

Transfer-gen plant is carried out pollination self, and plant that can not pollination self is got the pollen pollination with other transformed plants or the non-transformed plant of its homologous genes type.Get offspring's part planting seed in the greenhouse, extract the total DNA of blade and carry out PCR and Southern detection.Change the plant offspring of pTOK233 plasmid, wherein 6 R1 have detected PCR male plant for strain system, and wherein 5 R1 are that Southern blotting has These positive bands for strain.Illustrate that transgenosis can arrive the offspring by genetic stability.The R1 that is numbered 28 transfer-gen plants detects the hpt gene for 14 strain Southern in the 21 strain plant.

The PCR that changes the plant offspring of At5 plasmid finds that the plant of 7 R1 strain systems has detected PCR male plant; 2 R2 strains system, strain is that the 10 strain PCR of A2-1 are all positive, strain is that 15 strains are the PCR positive in 17 strains of A5-1.Southern blotting result confirms that 7 R1 strain systems and 2 R2 are in the strain system These positive bands plant being arranged all.Illustrate transgenosis genetic stability to R1, R2 generation.

2, transformed plant offspring's resistance detects

Think that genetically modified seed Z31 does contrast, the R1 that gets the plant that changes the hpt gene for seed 20-2 and 28-6,28-5,28-12,28-13 sterilization after, screening is germinateed in substratum that contains Totomycin or nutrient solution, after two weeks, observe germination, statistics germination grain number and root are long, the results are shown in Table 8.

The hygromycin resistance of table 8, transgenic progeny

The total seed number chitting piece percentage % of seed code name average root long (cm)

Z31(CK-) 1 28 4 14.3

20--2 1 28 19 67.9

28--6 1 30 30 100

28--5 0.5 30 30 100

28--12 3 17 6 36.0

28--13 1 17 2 11.8

Most of germination of transgenic seed (CK-) is not suppressed, and has only 14.3% seed germination, and the average root length of seeds germinated is 1 centimetre.The offspring of transfer-gen plant is most of germinates normally, also is affected but grow, and average root is grown up many more than 1 centimetre.

3, transformed plant offspring's resistance detects

The R1 of the plant of Pignus pignoris grain At5 is as shown in table 9 for the germination result of seed A2-3, A2-5, A5-6 antiweed PPT for seed A6-4, A6-12 and R2:

Total seed number chitting piece percentage %Z31 (CK-) 3 27 13 48.1A2-3 10 29 27 93.1A2-5 8 29 25 86.3A5-6 9 27 26 96.3A6-4 8 25 25 100A6-12 5 27 22 81.5 of the PPT resistance seed code name average root of table 9, transgenic progeny long (cm)

Most of germination of transgenic seed (CK-) is not suppressed, and has only 48.1% seed germination, and the average root length of chitting piece is 3 centimetres.The most of growth of the offspring of transfer-gen plant is normal, and average root is grown up many about 9 centimetres.

Embodiment 5, Agrobacterium-mediated Transformation maize calli

Nascent callus: with the rataria scultellum up, be inoculated on the callus substratum, 20-40 rataria of inoculation in every culture dish, 28 ℃ of dark 2-3 that cultivate can induce nascent callus after week.

Callus: after inducing nascent callus, every 2-3 week subculture once keeps the good callus of growth conditions.

Method according to embodiment 1 is that acceptor transforms it with the Agrobacterium that has foreign gene with the callus, and the result shows that nascent callus is as transformation receptor, and the kanamycin-resistant callus tissue rate that obtains is 8% to 46.4%, average generation kanamycin-resistant callus tissue rate 22.0%.

Embodiment 6, the auxiliary Agrobacterium-mediated Transformation maize immature embryos of particle gun

1,, comprises and to split film, carrier film, stop that net etc. sprays or soak sterilization with the sample chamber and the parts thereof of 70% ethanol with PDS-1000/He type particle gun.

2, (diameter 1.0 μ m Bio-Rad), place the 1.5ml centrifuge tube, add one night of 1ml soaked in absolute ethyl alcohol, and vibration suspends for several times, centrifugal collection bronze particulate deposits to take by weighing the 60mg bronze.Use aseptic water washing 3-4 time then, suspending again with the 1ml sterilized water at last promptly becomes particulate carrier.

3, the preparation of empty little missile-borne body:

Get bronze suspension 50ul, put into aseptic 1.5ml centrifuge tube, can be for bombardment 5 rifles.

4, getting the little bullet solution for preparing is applied on the carrier film quickly and evenly, treat after the volatilization of water or ethanol DNA faced down and pack in the carrier anchor, load onto then and can split film, put into the sample chamber ooze the maize immature embryos of handling 4 hours on the substratum of newly peeling off at height.

5, according to PDS-1000/He type particle gun operational guidance, vacuumize bombardment.Sample vacuum chamber degree 27mmHg can split film pressure 1100psi, little bullet flying distance 9cm, and every ware bombardment is once.

6, material is taken out in the bombardment back, seals, and places in 28 ℃ of culturing room.Height ooze on the substratum place 20 hours after, cultivated altogether 3 days according to method and the Agrobacterium of embodiment 1 again, be put into the enterprising row filter of screening culture medium then and cultivate, the kanamycin-resistant callus tissue number of statistics generation after 2 months, as shown in table 10:

Table 10, the auxiliary Agrobacterium-mediated Transformation of particle gun

Plasmid kind inoculation number kanamycin-resistant callus tissue percentage (%)

pTOK233 Z3 90 39 43.3

pAt5 Z31 90 34 37.7

Average 180 73 40

As can be seen from Table 10, rataria causes wound through the particle gun bombardment, infect with Agrobacterium again, and the kanamycin-resistant callus tissue rate average out to 40% of generation, transformation efficiency is significantly improved.Kanamycin-resistant callus tissue has obtained regeneration plant through differentiation.

Embodiment 7, the auxiliary Agrobacterium-mediated Transformation maize calli of particle gun

1, the callus of corn inbred line Z3, Z31, Q31, P9-10 be placed on height ooze on the substratum handle 4 hours standby.

2,, comprise and to split film, carrier film, stop that net etc. sprays or soak sterilization with the sample chamber and the parts thereof of 70% ethanol with PDS-1000/He type particle gun.

3, (diameter 1.0 μ m Bio-Rad), place the 1.5ml centrifuge tube, add one night of 1ml soaked in absolute ethyl alcohol, and vibration suspends for several times, centrifugal collection bronze particulate deposits to take by weighing the 60mg bronze.Use aseptic water washing 3-4 time then, suspending again with the 1ml sterilized water at last promptly becomes particulate carrier.

4, the particulate carrier bag is by Agrobacterium: get bronze suspension 50 μ l, put into aseptic 1.5ml centrifuge tube, add 1mL Agrobacterium solution, vibration is 5 minutes on the vibrator, 4000 rev/mins centrifugal 1 minute, remove supernatant liquor, repeat 3 times.The sterilized water that little missile-borne body and function 16 μ l contain 100 μ M Syringylethanones is resuspended, the each shooting with 4 μ l.

5, get the little bullet solution for preparing and be applied to quickly and evenly on the carrier film, treat after the volatilization of water or ethanol DNA faced down and pack in the carrier anchor, load onto then and can split film, the sample that the 1st step prepared is put into the sample chamber.

6, according to PDS-1000/He type particle gun operational guidance, vacuumize bombardment.Sample vacuum chamber degree 27mmHg can split film pressure 1100psi, little bullet flying distance 9cm, and every ware bombardment is once.

7, material is taken out in the bombardment back, seals, and places in 28 ℃ of culturing room, oozes on the substratum at height and places 20 hours, transfers on the D-AS substratum, looks the growth of Agrobacterium situation, is standard can't see the Agrobacterium bacterium colony, prolongs incubation time altogether as far as possible.

8, cultivate 3-7 days altogether.Washing goes to transfer to the enterprising row filter cultivation of screening culture medium behind the bacterium, and the kanamycin-resistant callus tissue number that statistics produces is as shown in table 11.

Table 11, the auxiliary Agrobacterium-mediated Transformation of particle gun produce the kanamycin-resistant callus tissue rate

pTOK233 Z3 110 9 8.2

p3301 Q31 110 17 15.5

p3301 Z31 110 13 11.8

pAt5 P9-10 60 7 11.7

Average 390 46 11.8

Particle gun directly is injected into Agrobacterium in the plant callus, produces kanamycin-resistant callus tissue rate average out to 11.8%, and is lower than the transformation efficiency of Agrobacterium-mediated Transformation rataria, but directly penetrates the transformation efficiency height of plasmid than particle gun.

Claims

1, a kind of method that corn is carried out gene transformation may further comprise the steps:

1) maize immature embryos or callus immersion interpolation 50-200 μ M are carried in the Agrobacterium D-inf solution of foreign gene, vibrate 30-60 second, and place the 5-60 branch and plant, taking-up is blotted bacterium liquid and is put on the D-AS substratum, cultivates altogether 2-7 days under 22-26 ℃ of dark condition;

2) rataria after cultivating altogether or callus immigration are contained in the sterilized water of 150-250mg/L Reflin, wash 1-3 time, in the sterilized water that contains the 150-250mg/L Reflin, soaked 15-60 minute for the last time;

3) take out rataria or callus and put 2-7 to the callus of induce substratum that contains the 150-250mg/L Reflin;

6) resistant calli of choosing is forwarded on the embryoid induction substratum, induce the formation embryoid; Break up forming being transferred on the division culture medium again of embryoid, obtain transgenic plant.

2, the method that corn is carried out gene transformation according to claim 1 is characterized in that: before transforming with Agrobacterium, with particle gun explant is handled earlier.

3, the method that corn is carried out gene transformation according to claim 2 is characterized in that: with the rataria of empty little missile-borne body bombardment corn, at this moment sample vacuum chamber degree 25-27mmHg can split film pressure 1100psi, little bullet flying distance 3-9cm.

4, method of corn being carried out gene transformation according to claim 2, it is characterized in that: with wrapping by the micropellet bombardment maize calli of Agrobacterium, at this moment sample vacuum chamber degree 25-27mmHg can split film pressure 1100-1350psi, little bullet flying distance 3-9cm.

5, method of corn being carried out gene transformation according to claim 4, it is characterized in that: the preparation method of described little bullet is: get bronze suspension 50 μ l, put into the 1.5ml centrifuge tube, add 1ml Agrobacterium solution, vibration is 5 minutes on the vibrator, 4000 rev/mins centrifugal 1 minute, remove supernatant liquor, repeat 3 times; The sterilized water that little missile-borne body and function 16 μ l contain 50-200 μ M Syringylethanone is resuspended, the each shooting with 4 μ l.

6, according to claim 1 or 2 or 3 or 4 or 5 described methods of corn being carried out gene transformation, it is characterized in that: before in maize immature embryos or callus immersion Agrobacterium bacterium liquid, rataria or callus are placed on height ooze and cultivated 1-4 hour on the substratum or be put in the D-inf solution 0.1-3 hour, wash once with D-inf liquid then.

7, a kind of method that corn is carried out gene transformation is to bombard with the rataria or the callus of particle gun to corn, and sample vacuum chamber degree 25-27mmHg can split film pressure 1100-1350psi, little bullet flying distance 3-9cm, and every ware bombardment is once.

8, method of corn being carried out gene transformation according to claim 7, it is characterized in that: the preparation method of described little bullet is: get bronze suspension 50 μ l, put into the 1.5ml centrifuge tube, add 1ml Agrobacterium solution, vibration is 5 minutes on the vibrator, 4000 rev/mins centrifugal 1 minute, remove supernatant liquor, repeat 3 times; The sterilized water that little missile-borne body and function 16 μ l contain 50-200 μ M Syringylethanone is resuspended, the each shooting with 4 μ l.

9, method of corn being carried out gene transformation according to claim 7, it is characterized in that: the preparation method of described little bullet is: get bronze suspension 50ul, put into the 1.5ml centrifuge tube, add 10ul carrier DNA solution, mix, add 50 μ lCaCl then ₂Solution is put in after mixing and leaves standstill 5-10min on ice, supernatant discarded, and each cleans once with 100 μ l dehydrated alcohols and 100ul70% ethanol, discards ethanol after centrifugal, is suspended at last in the 50 μ l00% ethanol, and once preparation can be for bombardment 6 rifles.