CN1179636C - Soybean stem apex conversion ultrasonic wave helped exogenous gene introduction method - Google Patents
Soybean stem apex conversion ultrasonic wave helped exogenous gene introduction method Download PDFInfo
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- CN1179636C CN1179636C CNB021507759A CN02150775A CN1179636C CN 1179636 C CN1179636 C CN 1179636C CN B021507759 A CNB021507759 A CN B021507759A CN 02150775 A CN02150775 A CN 02150775A CN 1179636 C CN1179636 C CN 1179636C
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Abstract
The present invention relates to an exogenous gene introducing method for converting soybean stem tips through ultrasonic assistance. An ultrasonic assistance method is adopted in the process for converting soybean stem tips by using agrobacterium-mediated BT genes; ultrasonic invasion treatment is carried out when agrobacteria are infected after soybean seeds sprout and are precultured; the seeds produced by converted plants are obtained by co-cultivation, adventitious bud cultivation and converting bud root induction. The present invention adopts ultrasonic for making tiny wounds on explants for increasing the contact area of the agrobacteria and the explants, has a good effect in the process for introducing BT genes; the infection efficiency of the agrobacteria is obviously improved; and the germination quantity and the quantity of converted seedlings are effectively increased.
Description
Technical field:
The present invention relates to a kind of soybean stem apex and transform the auxiliary foreign gene introduction method of ultrasonic, be that a kind of ultrasonic that utilizes imports soybean for the foreign gene of supplementary means, thereby the improvement soybean genetic character method of operating, belong to biotechnology and modern agricultural technology field.
Background technology:
The success that plant gene transforms depends on whether have good receptor system, promptly whether has the stronger vegetable regeneration capacity and the conversion ratio of high frequency, (" plant genetic engineering philosophy and technique " Wang Guanlin chief editor, technology publishing house).Transgene method mainly is divided into two classes at present: 1 direct method, for example, and pollen tube passage method, microinjection, electric shocking method, PEG method and particle bombardment, 2 support methods, for example, agrobacterium-mediated transformation and phage make support methods etc.Wherein agrobacterium-mediated transformation occupies critical role in plant transgene.The possessor successively selects cotyledonary node for use to the greatest extent, and rataria etc. transform as explant and obtained success, but transformation efficiency not high (0.6%).Bechtold once inoculates the arabidopsis plant with the vacuum technology of infiltrating with Agrobacterium and has obtained mass mutation body (Bechtold 1993.life sciences 316:1194-99).But this technology is used on soybean and is not reported.Thereby the subject matter that exists at present is to improve the soybean transformation efficiency.
Summary of the invention:
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of soybean stem apex to transform the auxiliary foreign gene introduction method of ultrasonic, it is many that soybean is sprouted, and the regrowth ability is strong, improves the soybean conversion ratio.
For realizing such purpose, the present invention is in the process that agriculture bacillus mediated BT gene pairs soybean stem apex transforms, adopted the ultrasonic householder method, soya seeds is through sprouting, after pre-the cultivation, be aided with ultrasonic when carrying out agroinfection and infect processing, make seed in the certain hyperacoustic air effect of selection pressure process, the closed instantaneous pressure that forms of bubble group, continuously high pressure resembles a succession of little " blast " constantly impact material surface, cause many micro-damages, avoided the excessive damage that in the past explant was caused, also increased the contact area between Agrobacterium and the explant simultaneously, improved the efficiency of infection of Agrobacterium with the wound that lancinates.Cultivate and the root induction of conversion bud through cultivation altogether, indefinite bud again, obtain the seed that transformed plant produces.
Method of the present invention comprises following concrete steps:
1. getting soya seeds washes repeatedly with running water, be placed in 70% the alcohol and soaked 0.5～1 minute, change over to contain among the saturated liquor natrii hypochloritis and soaked 15 minutes, with aseptic water washing 4～5 times, then seed is placed sterile water to soak 18～24 hours, make seed germination.
2. peel off kind of a skin, remove cotyledon, under anatomical lens, remove two prophylls, expose the apical meristem district, therefrom take out the stem apex of about 0.5cm, place on MSB (organic principle of inorganic constituents+Gamborg (B5) medium of Murashige and Skoog (MS) medium)+6-BA 3.0mg/L medium, cultivated in advance 24 hours.
3. pre-incubated stem apex is taken out, place the triangular flask of 100ml, add the Agrobacterium bacterium liquid (OD600nm ≈ 0.5) for preparing, triangular flask is dipped in the central authorities that ultrasonic is washed device (power 500W, operating frequency 50kHz), and ultrasonic infects to be handled 15～20 minutes.
4. infect and finish, material is taken out, blot, put into MSB+6-BA 1.0mg/L+IBA (indolebutyric acid) 0.2mg/L (PH5.8) medium lucifuge and cultivated 3 days with aseptic filter paper.
5. change MSB+6-BA 0.5mg/L+cb (carbenicillin) 500mg/L (PH 5.8 liquid) continuous culture 3 days over to, renew bright medium every day.
6. change MSB+6-BA (6-benzyladenine) 0.5mg/L+cb 500mg/L+KM (kanamycin sulfate) 80mg/L (PH5.8 solid) over to, per 2 week switchings once 2～3cm occurs being elongated to indefinite bud.
7. the indefinite bud on the cutting-out explant and being placed on 1/2MSB (inorganic constituents in the MS medium reduces by half+in the B5 medium organic principle)+KM50mg/L (PH5.8) medium, transformed the bud root induction 15～20 days, and be transplanted to big basin to obtaining the seed that transformed plant produces.
The present invention adopts ultrasonic to make small wound on explant, increased the contact area between Agrobacterium and the explant, in the process that imports the BT gene, played good effect, compare with common agrobacterium mediation method, obviously improve the efficiency of infection of Agrobacterium, effectively raised the quantity of sprout quantity and conversion seedling.
Embodiment:
Below in conjunction with specific embodiment technical scheme of the present invention is further described.
Embodiment 1
The experiment kind is 43,60 of east farmings.
Get soya seeds and wash repeatedly, be placed in 70% the alcohol and soaked 0.5 minute, change over to contain among the saturated liquor natrii hypochloritis and soaked 15 minutes,, then seed is placed sterile water to soak 18 hours with aseptic water washing 4 times with running water.Peel off kind of a skin, remove cotyledon, under anatomical lens, remove two prophylls, expose the apical meristem district, therefrom take out the stem apex of about 0.5cm, place on the MSB+6-BA 3.0mg/L medium, cultivated in advance 24 hours, make seed germination.
Pre-incubated stem apex is taken out, place the triangular flask of 100ml, add the Agrobacterium bacterium liquid (OD for preparing
600nm≈ 0.5), triangular flask is dipped in the central authorities that ultrasonic is washed device (power 500W, operating frequency 50kHz), and ultrasonic infects to be handled 20 minutes.
Infect and finish, material is taken out, blot, put into MSB+6-BA 1.0mg/L+IBA0.2mg/L (PH 5.8) medium lucifuge and cultivated 3 days with aseptic filter paper.
Change MSB+6-BA 0.5mg/L+cb 500mg/L (PH5.8 liquid) continuous culture 3 days over to, renew bright medium every day.
Change MSB+6-BA 0.5mg/L+cb 500mg/L+KM80mg/L (PH5.8 solid) over to, per 2 week switchings once 2cm occurs being elongated to indefinite bud.
Indefinite bud on the cutting-out explant also is placed on 1/2MSB+KM50mg/L (PH5.8) medium, transforms the bud root induction 15 days, is transplanted to big basin to obtaining the seed that transformed plant produces.
Obtain to change soybean plant strain No. 43 34 strains in east of BT gene.
Embodiment 2
The experiment kind is 27,60 in Jilin.
Get soya seeds and wash repeatedly, be placed in 70% the alcohol and soaked 1 minute, change over to contain among the saturated liquor natrii hypochloritis and soaked 15 minutes,, then seed is placed sterile water to soak 24 hours with aseptic water washing 5 times with running water.Peel off kind of a skin, remove cotyledon, under anatomical lens, remove two prophylls, expose the apical meristem district, therefrom take out the stem apex of about 0.5cm, place on the MSB+6-BA 3.0mg/L medium, cultivated in advance 24 hours, make seed germination.
Pre-incubated stem apex is taken out, place the triangular flask of 100ml, add the Agrobacterium bacterium liquid (OD for preparing
600nm≈ 0.5), triangular flask is dipped in the central authorities that ultrasonic is washed device (power 500W, operating frequency 50kHz), and ultrasonic infects to be handled 15 minutes.
Infect and finish, material is taken out, blot, put into MSB+6-BA 1.0mg/L+IBA0.2mg/L (PH5.8) medium lucifuge and cultivated 3 days with aseptic filter paper.
Change MSB+6-BA 0.5mg/L+cb 500mg/L (PH5.8 liquid) continuous culture 3 days over to, renew bright medium every day.
Change MSB+6-BA 0.5mg/L+cb 500mg/L+KM80mg/L (PH5.8 solid) over to, per 2 week switchings once 3cm occurs being elongated to indefinite bud.
Indefinite bud on the cutting-out explant also is placed on 1/2MSB+KM50mg/L (PH5.8) medium, transforms the bud root induction 20 days, is transplanted to big basin to obtaining the seed that transformed plant produces.
Obtain to change No. 27 36 strains in soybean plant strain Jilin of BT gene.
Embodiment 3
The experiment kind is for closing rich 35,60.
Get soya seeds and wash repeatedly, be placed in 70% the alcohol and soaked 0.7 minute, change over to contain among the saturated liquor natrii hypochloritis and soaked 15 minutes,, then seed is placed sterile water to soak 19 hours with aseptic water washing 5 times with running water.Peel off kind of a skin, remove cotyledon, under anatomical lens, remove two prophylls, expose the apical meristem district, therefrom take out the stem apex of about 0.5cm, place on the MSB+6-BA3.0mg/L medium, cultivated in advance 24 hours, make seed germination.
Pre-incubated stem apex is taken out, place the triangular flask of 100ml, add the Agrobacterium bacterium liquid (OD for preparing
600nm≈ 0.5), triangular flask is dipped in the central authorities that ultrasonic is washed device (power 500W, operating frequency 50kHz), and ultrasonic infects to be handled 18 minutes.
Infect and finish, material is taken out, blot, put into MSB+6-BA 1.0mg/L+IBA0.2mg/L (PH5.8) medium lucifuge and cultivated 3 days with aseptic filter paper.
Change MSB+6-BA 0.5mg/L+cb 500mg/L (PH5.8 liquid) continuous culture 3 days over to, renew bright medium every day.
Change MSB+6-BA 0.5mg/L+cb500mg/L+KM80mg/L (PH5.8 solid) over to, per 2 week switchings once 2cm occurs being elongated to indefinite bud.
Indefinite bud on the cutting-out explant also is placed on 1/2MSB+KM50mg/L (PH5.8) medium, transforms the bud root induction 18 days, is transplanted to big basin to obtaining the seed that transformed plant produces.
The soybean plant strain that obtains commentaries on classics BT gene closes rich No. 35 37 strains.
Method of the present invention can be suitable for soybean changes range gene, can obtain the transformed soybean plant of upper frequency fast.
Claims (1)
1, a kind of soybean stem apex transforms the auxiliary foreign gene introduction method of ultrasonic, it is characterized in that comprising the steps:
1) is placed on after the soya seeds flushing in 70% the alcohol and soaked 0.5～1 minute, change over to contain among the saturated liquor natrii hypochloritis and soaked 15 minutes,, make seed germination with placing sterile water to soak 18～24 hours in seed behind the aseptic water washing;
2) remove kind of a skin, remove cotyledon, under anatomical lens, remove two prophylls, expose the apical meristem district, therefrom take out the stem apex of about 0.5cm, place on the MSB+6-BA 3.0mg/L medium, cultivated in advance 24 hours;
3) pre-incubated stem apex is taken out, place the triangular flask of 100ml, add the Agrobacterium bacterium liquid (OD for preparing
600nm≈ 0.5), triangular flask is dipped in the ultrasonic of power 500W, operating frequency 50kHz and washes in the device, and ultrasonic infects to be handled 15～20 minutes;
4) infect and finish, material is taken out, blot, put into MSB+6-BA 1.0mg/L+IBA0.2mg/L (PH5.8) medium lucifuge and cultivated 3 days with aseptic filter paper;
5) change MSB+6-BA 0.5mg/L+cb 500mg/L (PH5.8 liquid) continuous culture 3 days over to, renew bright medium every day;
6) change MSB+6-BA 0.5mg/L+cb 500mg/L+KM80mg/L (PH5.8 solid) over to, per 2 week switchings once 2～3cm occurs being elongated to indefinite bud;
7) downcut the indefinite bud on the explant and being placed on 1/2MSB+KM50mg/L (PH5.8) medium, transformed the bud root induction 15～20 days, be transplanted to big basin to obtaining the seed that transformed plant produces, wherein 1/2MSB is that inorganic constituents in the MS medium reduces by half+organic principle in the B5 medium.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101175557B (en) * | 2005-05-09 | 2010-09-08 | 欧雷恩诊断公司 | Sonication of a medium |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101011027B (en) * | 2007-02-07 | 2010-12-22 | 华中农业大学 | Method for converting cotton germ by agrobacterium with ultrasonic wave aid |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101175557B (en) * | 2005-05-09 | 2010-09-08 | 欧雷恩诊断公司 | Sonication of a medium |
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