CN102533848A - High-efficiency genetic transformation method using soybean Jilin No.35 embryonic tip as explant - Google Patents

Abstract

The invention relates to a high-efficiency genetic transformation method using a soybean Jilin No.35 embryonic tip as an explant and belongs to the technical field of plant transgenosis. The method comprises the following steps of: selecting Jilin No.35 soybean seeds with integrated seed coat and without diseases; performing ultrasonic treatment on the explant; transferring the infected embryonic tip explant into a co-culture medium; co-cultivating; inoculating into a rooting medium; and transplanting to a nutrition pot after the root grows strong. According to the method, the regeneration cycle is shortened. Through the adoption of a strong and effect infection method, browning degree and necrosis degree during co-cultivation are reduced, and conversion rate is increased.

Description

A kind of high-efficiency genetic transforming method that is explant with No. 35 embryo points in soybean Jilin

Technical field

The invention belongs to the plant transgenic technology field, being specifically related to soybean embryo point is explant, screens high regeneration rate and to the extremely sensitive genotype of Agrobacterium, cooperates mode of infection efficiently, has improved the transformation efficiency of Agrobacterium.

Background technology

Soybean is important oil crops and fodder crop; Utilizing transgenic technology to improve soybean quality is the important directions of current breeding; Though genetically engineered soybean is at global implant mass; But its genetic transformation is still one of difficult point, and therefore, setting up efficient, a stable regeneration system is to solve the low foundation stone of soybean heredity transformation efficiency.The research that soyabean tissue cultivates can be traced back to the sixties in 20th century, just obtains tremendous breakthrough up to the eighties, and explants such as the protoplastis through soybean, hypocotyl, little true leaf, rataria, cotyledonary node, embryo point have obtained regeneration plant in succession.

At present, strong, the problems of having set up such as shoot regeneration frequency is low, poor repeatability of still existence group of soybean heredity transformation system training operation skill property have seriously restricted and have utilized molecular biology method improvement inherited character.So setting up easy, efficient, a stable regeneration system is that soybean heredity transforms successful basis, also is the critical problem that numerous investigators need solve.

The soybean embryo point regeneration system that grew up in recent years has been widely used in the soybean heredity transformation technology because of the advantages such as convenience, the regeneration period is short, easy and simple to handle, brownization rate is low of drawing materials.But there are some fatal shortcomings equally in embryo point regeneration system, and is strong like the genotype dependency, transformation efficiency is low etc.

Therefore, screening is fit to the genotype of embryo point regeneration system, takes mode of infection efficiently, and sets up and be specific to this genotypic genetic conversion system, improves transformation efficiency, and the development that soybean heredity is transformed has great practice significance.

Summary of the invention

The object of the present invention is to provide a kind of high-efficiency genetic transforming method that is explant with No. 35 embryo points in soybean Jilin.

The technical scheme that the present invention takes is to comprise the following steps:

(1) choose No. 35 soybean seeds in the complete disease-free Jilin of kind of skin, tap water is rinsed the back well and is handled 50s with 75% alcohol, and aseptic water washing 3～5 times then is soaked in the sterilized water 6h and cuts the explant embryo sharp;

(2) explant is carried out ultrasonication 6s earlier, infect 15min with the auxiliary Agrobacterium of vacuum pump afterwards, and in infecting liquid, add 0.03% Silwet L-77;

(3) point of the embryo after infecting explant is transferred in the common substratum, altogether the additional 3.0 mg ﹒ L of substratum ^-16-BA is sprouted and 3.0 mg ﹒ L with induced bundle ^-1Silver Nitrate is to suppress brownization and necrosis;

(4) cultivate altogether to move after 3 days and be connected in the bud elongation medium of growing thickly, elongation medium is added 0.5 mg ﹒ L ^-16-BA and 0.2 mg ﹒ L ^-1IBA, this stage is added selective agent Basta 0.6 mg ﹒ L ^-1

When (5) treating regeneration plant length to 4.7 ~ 5.3cm, insert root media, root media as carbon source, is selected 1/2 MSB and additional 0.5 mg ﹒ L with sucrose for use ^-1IBA treats to transplant to Nursery after the root growing way stalwartness;

(6) transplant back extraction leaf DNA and do the PCR detection, and adopt bar gene test strip to carry out Protein Detection.

Soybean varieties used in the present invention, by Jilin University's plant germplasm resource with utilize the research department to provide.

The present invention is an explant with soybean embryo point; At first; To ten genotype: Jilin 35, Jilin 47, safety 8, east farming 42, the flat stem of the U.S., No. 1, Jinlin University beans, No. 2, Jinlin University beans, Jinlin University 113, close rich 43 and Sui Nong 10 and 5 6-BA, 0,0.2,0.5,1.0,3.0 and 5.0 mg ﹒ L ^-1The bud rate that lures of level has been carried out the dual factors test, then ten kinds is carried out the transient expression of gus gene, measures fluorescent value, and comprehensive above-mentioned two test-results filter out high regeneration rate and the kind Jilin 35 responsive to Agrobacterium.Then; With No. 35 embryo points in Jilin is acceptor material, carries out chlorine, mercuric chloride and three kinds of seed sterilization methods of alcohol relatively, is soaked in 6h in the sterilized water after the sterilization; Cutting explant embryo point directly carries out Agrobacterium and infects; Adopt static, shaking table, three kinds of modes of infection of vacuum pump, 10min, 15min, three times of infection of 20min and ultrasonication combination, and in resuspended liquid, add 0.03% and 0.06% Silwet L-77.Adopt 3.0 mg ﹒ L altogether between during cultivation ^-1The 6-BA induced bundle is sprouted, and adds different concns proline(Pro) and Silver Nitrate (0,1.0,2.0,3.0,4.0 and 5.0 mg ﹒ L in substratum ^-1), to suppress brownization, lure the bud stage not add any hormone and additional substances, the bud elongation medium of growing thickly is provided with 3 kinds of 6-BA concentration (0.0,0.2 and 0.5 mg ﹒ L ^-1) and 3 kinds of IBA concentration (0.0,0.2 and 0.5 mg ﹒ L ^-1), 1/2 MSB, 1/4 MSB, sucrose, glucose and 3 kinds of IBA concentration (0.2,0.5 and 1.0mg ﹒ L is set in the root media ^-1) totally 12 kinds of combinations.To close rich 35, eastern agricultural 42 is contrast, adopt conventional genetic transforming method, and above-mentioned genetic transforming method is adopted in Jilin 35, and extraction leaf DNA in transplanting back is done the PCR detection, and adopts bar gene test strip to carry out Protein Detection, compares transformation efficiency between the three at last.

Beneficial effect of the present invention: this invention provides a kind of high efficiency method for transformation for the soybean heredity transformation technology.Compare with the soybean embryo point genetic conversion system of routine; The present invention has shortened the regeneration period; Adopt strong effectively mode of infection, reduced brownization and degree of necrosis between common during cultivation, improved transformation efficiency; The high-efficiency genetic transforming method that to have set up with No. 35 embryo points in Jilin be explant transforms problems such as difficulty, regeneration rate are low and has bigger practice significance solving soybean heredity.

Below in conjunction with the practical implementation method the present invention is elaborated.

Description of drawings

Fig. 1 is that the bud of growing thickly of different genotype soybean embryo point lures bud rate figure;

Fig. 2 is the active detected result figure of different genotype soybean embryo point GUS;

Fig. 3 is the active detection figure of different mode of infection GUS;

Fig. 4 is the influence figure of different concns Silwet L-77 to infecting, A among the figure: do not infect B: vacuum infestation 20min C: vacuum infestation 15min D: add 0.03% Silwet L-77 vacuum infestation 20min E in the resuspended liquid: add 0.03% Silwet L-77 vacuum infestation 15min F in the resuspended liquid: add 0.06% Silwet L-77 vacuum infestation 20min G in the resuspended liquid: add 0.06% Silwet L-77 vacuum infestation 15min in the resuspended liquid:

Fig. 5 is proline(Pro) and the Silver Nitrate figure that influences to brownization;

Fig. 6 is that 6-BA and IBA influence figure to embryo point regenerated;

Fig. 7 is inorganic salt, carbohydrate and the IBA figure that influences to taking root, A:1/2 MSB+ sucrose+0.2mg ﹒ L ^-1IBA; B:1/2 MSB+ sucrose+0.5mg ﹒ L ^-1IBA; C:1/2 MSB+ sucrose+1.0mg ﹒ L ^-1IBA; D:1/4 MSB+ sucrose+0.2mg ﹒ L ^-1IBA; E:1/4 MSB+ sucrose+0.5mg ﹒ L ^-1IBA; F:1/4 MSB+ sucrose+1.0mg ﹒ L ^-1IBA; G:1/2 MSB+ glucose+0.2mg ﹒ L ^-1IBA; H:1/2 MSB+ glucose+0.5mg ﹒ L ^-1IBA; I:1/2 MSB+ glucose+1.0mg ﹒ L ^-1IBA; J:1/4 MSB+ glucose+0.2mg ﹒ L ^-1IBA; K; 1/4 MSB+ glucose+0.5mg ﹒ L ^-1IBA; L; 1/4 MSB+ glucose+1.0mg ﹒ L ^-1IBA;

Fig. 8 is soybean Jilin 35 an embryos point genetic transformation schema, A: vacuum infestation B: cultivate C altogether: lure bud D: the E of taking root: transplant F: the G of blooming: bear pods;

Fig. 9 is the PCR detection figure of regeneration plant; M:marker, 1: negative control, 2: positive control, 3-19 part test sample DNA;

Figure 10 is the Protein Detection figure of regeneration plant.

Embodiment

(1) choose No. 35 soybean seeds in the complete disease-free Jilin of kind of skin, tap water is rinsed the back well and is handled 50s with 75% alcohol, and aseptic water washing 3～5 times then is soaked in the sterilized water 6h and cuts the explant embryo sharp;

(2) explant is carried out ultrasonication 6s earlier, infect 15min with the auxiliary Agrobacterium of vacuum pump afterwards, and in infecting liquid, add 0.03% Silwet L-77;

(3) point of the embryo after infecting explant is transferred in the common substratum, altogether the additional 3.0 mg ﹒ L of substratum ^-16-BA is sprouted and 3.0 mg ﹒ L with induced bundle ^-1Silver Nitrate is to suppress brownization and necrosis;

(4) cultivate altogether to move after 3 days and be connected in the bud elongation medium of growing thickly, elongation medium is added 0.5 mg ﹒ L ^-16-BA and 0.2 mg ﹒ L ^-1IBA, this stage is added selective agent Basta 0.6 mg ﹒ L ^-1

When (5) treating regeneration plant length to 4.7 ~ 5.3cm, insert root media, root media as carbon source, is selected 1/2 MSB and additional 0.5 mg ﹒ L with sucrose for use ^-1IBA treats to transplant to Nursery after the root growing way stalwartness;

(6) transplant back extraction leaf DNA and do the PCR detection, and adopt bar gene test strip to carry out Protein Detection.

Soybean varieties used in the present invention, by Jilin University's plant germplasm resource with utilize the research department to provide.

Below in conjunction with specific embodiment improve one the explanation the present invention.

Embodiment 1: the bud of growing thickly of different genotype soybean embryo point lures the bud rate

With Jilin 35, Jilin 47, safety beans 8, east farming 42, the flat stem of the U.S., No. 1, Jinlin University beans, No. 2, Jinlin University beans, No. 3, Jinlin University beans, close rich 43 and Sui Nong 10 be material, on inducing culture, add different concns 6-BA (0,0.2,0.5,1.0,3.0 and 5.0 mg ﹒ L respectively ^-1) (available from Beijing ancient cooking vessel state), light induction 5 d, on the switching MSB substratum (MS is a large amount of, MS is micro-, MS molysite and B5 organic), each handles 30 explants, 3 repetitions.Each genotype embryo point adventitious bud induction frequency of 14d investigation statistics soybean, result such as Fig. 1.

Can know that from Fig. 1 beans No. 2 integral body in Jinlin University lure the bud rate the highest in the experimental cultivar, be Jilin 35 secondly.On the whole, suitable 6-BA concentration mainly concentrates on 0.2～3.0 mg ﹒ L ^-1Between.

Embodiment 2: the active detected result of different genotype soybean embryo point GUS

The embryo point explant of above-mentioned ten kinds immerses respectively in the resuspended liquid of Agrobacterium EHA105 (available from Biovector) that changes pCAMBIA1301 (available from CAMBIA) plasmid vector over to; Vacuum infestation 15 min; The bacterium liquid that adheres on the flush away explant; Explant is transferred altogether in the substratum (1/2MS is a large amount of+and MS trace+MS molysite+B5 is organic+30 g/L sucrose+0.8g/L agar+100uMAS+5mg/L6-BA PH5.4), cultivate 3 d.

Choose not the about 100mg of brownization embryo point explant, the adding liquid nitrogen, grind into powder is with the powder 1.5m1 centrifuge tube of packing into; Add 600ul GUS and extract damping fluid, abundant mixing, 4 ℃, 12; 000rpm, centrifugal 10min gets supernatant and is sub-packed in 2 new centrifuge tubes, and 4 ℃ of preservations are subsequent use.Supernatant is the gus protein crude extract, and wherein a part of supernatant is with spectrophotometric instrumentation protein concentration, and another part supernatant adds GUS reaction substrate 4-MUG (biological available from the Changchun Delta), 37 ℃, react 10 min, and add reaction terminating liquid (0.2 mmol/L Na ₂CO ₃) termination reaction, under spectrophotofluorometer, measure fluorescent value.It is active to calculate GUS.

The result is as shown in Figure 2 for the GUS fluorescent quantitation; It is the highest to infect Jilin, back 35 GUS activity; Be higher than No. 2, Jinlin University beans and other soybean varieties far away, explain that Jilin 35 embryos point explant is the strongest to Agrobacterium susceptibility, because it lures the bud rate also higher relatively in embryo point transformation system; Therefore, Jilin 35 can be used as the good acceptor material of soybean embryo point genetic conversion system.

Embodiment 3: different sterilization methods are to the influence of embryo point bud ratio

Choose No. 35 soybean seeds in ripe Jilin of the complete no scab of kind of skin, no insect pest, no mould, adopt 3 kinds of seed sterilization methods.(1) disinfection by chlorine method: soybean seeds is put in the encloses container in the stink cupboard, mixes 96 ml, 5% Naclo and 4 ml Hcl (all available from Beijing ancient cooking vessel state), sterilization 6～10 h.(2) mercuric chloride sterilization: tap water flushing soybean seeds, 75% alcohol is handled 35 s, then with 0.1% Hgcl2,10 min that sterilize, aseptic water washing 3～5 times.(3) alcohol disinfecting method: tap water flushing soybean seeds, 75% alcohol is handled 50 s, aseptic water washing 3～5 times.Every kind of sterilization method is handled 100 seeds, 3 repetitions.

Result such as following table: adopt 3 kinds of methods that Jilin 35 mature seeds are carried out disinfection, contamination phenomenon all do not occur, but bud ratio is obviously different.Alcohol method disinfectant percentage of germination is up to 98.3%, and effect is superior to other two kinds of sterilization methods.The alcohol disinfecting method is less to the seed injury, can guarantee higher bud ratio, is a kind of easy, easy row, reliable disinfection way.

Embodiment 4: different mode of infection GUS are active to be detected

To carry pCAMBIA1301 Agrobacterium EHA105 transforms Jilin 35 embryo points; Static, shaking table and three kinds of modes of infection of vacuum pump are set altogether; 10min, 15min and three times of 20min handle, and additional UW and no ultrasonication amount to 18 kinds and infect combination.Infect the back according to the method in the example 2, measure protein concentration and fluorescent value, it is active to calculate GUS.

The result is as shown in Figure 3 for the GUS fluorescent quantitation, and the active integral planar average of GUS is higher than other two kinds of modes of infection behind the vacuum infestation, and ultrasonication generally is higher than no ultrasonication.Wherein, it is the highest to infect 15min GUS activity with the ultrasonication final vacuum.

Embodiment 5: the influence of different concns Silwet L-77 to infecting

To carry pCAMBIA1301 Agrobacterium EHA105 transforms No. 35 embryo points in Jilin; A blank and 6 kinds of processing are set, are respectively and do not infect, add to add in 0.03% Silwet L-77 (available from Beijing ancient cooking vessel state) vacuum infestation 15min, the resuspended liquid to add in 0.03% Silwet L-77 vacuum infestation 20min, the resuspended liquid among the vacuum infestation 15min, vacuum infestation 20min, resuspended liquid and add 0.06% Silwet L-77 vacuum infestation 20min in 0.06% Silwet L-77 vacuum infestation 15min and the resuspended liquid.Metainfective embryo point explant is forwarded in the common culture medium, and 22 ℃, dark culturing.With reference to the method for Jefferson etc., select to cultivate altogether 3 d not the embryo point of brownization be immersed in X-Gluc (the giving birth to the worker) staining fluid available from Shanghai, 37 ℃ of incubated overnight are observed the dyeing situation.

Transient expression result such as Fig. 4; The gus gene expression of results is widely different between six kinds of processing; Comparatively outstanding to add 0.03% Silwet L-77 performance in the resuspended liquid; Express simultaneously in tip of a root meristem zone and apical meristem district, vacuum infestation 20min expression effect is the most obvious, and whole explant is all dyed is blueness.

Comprehensive instance 4 infects 15min with the ultrasonication final vacuum, and additional 0.03% Silwet L-77 is that the best infects method in resuspended liquid.

Embodiment 6: proline(Pro) and Silver Nitrate are to the influence of brownization

With Jilin is for No. 35 material, and sterilization obtains explant, after infecting according to the method described above, outwells bacterium liquid, blots the bacterium liquid on embryo point surface with the filter paper after the sterilization, then explant is lain against in the common substratum that is covered with filter paper.Add different concns proline(Pro) (0,1.0,2.0,3.0,4.0 and 5.0 mg ﹒ L respectively in substratum and the resuspended liquid altogether ^-1) and Silver Nitrate (0,1.0,2.0,3.0,4.0 and 5.0 mg ﹒ L ^-1), under 22 ℃, dark condition, cultivate.Each handles 45 explants, and 5 d are cultivated in 3 repetitions, respectively brownization of embryo point situation under the investigation statistics different concns etcetera.

The result shows (Fig. 5), raises again after reaching minimum value along with brownization of the increase rate of proline(Pro) and silver nitrate concentration reduces gradually.Proline(Pro) and Silver Nitrate are all at 3.0 mg ﹒ L ^-1Brownization of embryo point rate is minimum under the concentration, is respectively 3.70% and 2.96%, shows that proline(Pro) and Silver Nitrate have remarkable inhibition effect to brownization rate.

Embodiment 7:6-BA and IBA are to the influence of embryo point regenerated

With Jilin is test materials No. 35, is soaked in after the sterilization in the sterilized water, cuts prophyll and cotyledon and obtains embryo point explant.Cultivated altogether 3 days after infecting according to instance 4 and 5, insert to lure in the bud substratum and cultivated 14 days, then be forwarded in the bud elongation medium of growing thickly.The bud elongation medium of growing thickly is provided with 3 kinds of 6-BA concentration (0.0,0.2 and 0.5 mg ﹒ L ^-1) and 3 kinds of IBA concentration (0.0,0.2 and 0.5 mg ﹒ L ^-1), totally 9 kinds of combinations, each handles 30 explants, 3 repetitions.20d investigation statistics the grow thickly upgrowth situation and the regeneration rate of bud, application software SPSS16.0 does the variance analysis and the test of significance of the shoot regeneration rate of growing thickly.

Regeneration rate (%)=grow thickly bud height is greater than 3 cm explant numbers/go out bud explant number * 100%

As shown in Figure 6,6-BA concentration is 0.0 mg ﹒ L ^-1The time, along with the increase regeneration rate of IBA concentration sharply descends; 6-BA concentration is 0 .2 mg ﹒ L ^-1The time, the regeneration rate downtrending slows down to a certain extent; 6-BA concentration is 0.5 mg ﹒ L ^-1The time, regeneration takes the lead in increasing then and descends.General trend shows that 6-BA has the greatest impact to regeneration rate, and use also can obtain higher regeneration rate separately, and IBA has only when its concentration is lower than 6-BA concentration, and the two is used and could improves regeneration rate, otherwise, then reduce regeneration rate.

The significant difference inspection show, regeneration rate there are differences between different hormone concentration and media.0.5 mg ﹒ L ^-16-BA and 0.2 mg ﹒ L ^-1IBA is used and 0.5 mg ﹒ L ^-16-BA uses separately, and regeneration rate is significantly higher than other combination, the two no significant difference, but the two when being used regeneration rate the highest.Take all factors into consideration 0.5 mg ﹒ L ^-16-BA and 0.2 mg ﹒ L ^-1IBA is best to the Jilin 35 bud elongation action effect of growing thickly.

Embodiment 8: inorganic salt, carbohydrate and the IBA influence to taking root

Obtain regeneration plant through aforesaid method, when treating its length, insert root media to the 5 cm left and right sides.1/2 MSB, 1/4 MSB, sucrose, glucose and 3 kinds of IBA concentration (0.2,0.5 and 1.0mg ﹒ L is set respectively in the root media ^-1) totally 12 kinds of combinations.30 explants of every kind of combination repeat for 3 times, cultivate every kind of combination of 3 all " Invest, Then Investigate "s situation of taking root.

Can know by Fig. 7; Sucrose in water ratio glucose is hestening rooting better; Glucose then be prone to produce callus and growth is slower, selects for use sucrose as under the carbon source material situation, and 1/2 MSB is more sturdy and quantity is many (A and D) than the root in the 1/4 MSB substratum; But along with IBA concentration increases, this trend fade away (C and F).

As carbon source, 1/2 MSB cooperates the IBA of high density comparatively suitable as root media with sucrose.

Embodiment 9: the comparison of closing transformation efficiency between rich 35, east farming 42 and 35 3 kinds in Jilin

, east farming 42 two kinds rich 35 to close are contrast; The embryo point genetic transforming method of the bright grade of will is defended in employing, and the genetic transforming method that adopt among the present invention in Jilin 35, idiographic flow such as Fig. 8; After treating the regeneration plant transplant survival; Extract the total DNA of blade with the CTAB method, the pcr amplification product of testing goal gene is about 1100bp (like Fig. 9).

Get a fritter blade in the 1.5ml centrifuge tube, grind about 30s, add the 500ul protein extract with spillikin; Continue to grind 30s; The bar gene test strip that will contain absorbent pad one end is inserted in the centrifuge tube, and observations behind the 10min is like Figure 10; The negative plant that has only a band contains the positive plant of two bands.

Statistics regeneration plant number, PCR and the test strip of closing explant number after infect in rich 35, east farming 42 and Jilin 35, transplant survival detects and is all male plant number respectively, calculates final transformation efficiency.

Result such as following table; Number can be known from table; The transformation efficiency in Jilin 35 is the highest, and the high-efficiency genetic transforming method that the higher transformation efficiency of being obtained is mainly given the credit to employing is the excellent combination of various factors of influence, and conversion results has been verified the high efficiency of Jilin 35 embryos point genetic transforming method fully.

Claims (1)

1. the high-efficiency genetic transforming method that is explant with No. 35 embryo points in soybean Jilin is characterized in that comprising the following steps:

(1) choose No. 35 soybean seeds in the complete disease-free Jilin of kind of skin, tap water is rinsed the back well and is handled 50s with 75% alcohol, and aseptic water washing 3～5 times then is soaked in the sterilized water 6h and cuts the explant embryo sharp;

(2) explant is carried out ultrasonication 6s earlier, infect 15min with the auxiliary Agrobacterium of vacuum pump afterwards, and in infecting liquid, add 0.03% Silwet L-77;

(3) point of the embryo after infecting explant is transferred in the common substratum, altogether the additional 3.0 mg ﹒ L of substratum ^-16-BA is sprouted and 3.0 mg ﹒ L with induced bundle ^-1Silver Nitrate is to suppress brownization and necrosis;

(4) cultivate altogether to move after 3 days and be connected in the bud elongation medium of growing thickly, elongation medium is added 0.5 mg ﹒ L ^-16-BA and 0.2 mg ﹒ L ^-1IBA, this stage is added selective agent Basta 0.6 mg ﹒ L ^-1

When (5) treating regeneration plant length to 4.7 ~ 5.3cm, insert root media, root media as carbon source, is selected 1/2 MSB and additional 0.5 mg ﹒ L with sucrose for use ^-1IBA treats to transplant to Nursery after the root growing way stalwartness;

(6) transplant back extraction leaf DNA and do the PCR detection, and adopt bar gene test strip to carry out Protein Detection.

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