CN1429480A

CN1429480A - Method of quickly obtaining transgenic cotton plant

Info

Publication number: CN1429480A
Application number: CN 01138378
Authority: CN
Inventors: 杨业华; 王学奎
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2001-12-31
Filing date: 2001-12-31
Publication date: 2003-07-16
Anticipated expiration: 2021-12-31
Also published as: CN1185925C

Abstract

A method for quickly obtaining transgenic cotton plant includes such steps as improving MSB5 culture medium by adding VB1, VB6, nicotinic acid and auximone, culturing the hypocotyl dissection of aseptic cotton, and direct induction. Its advantages are simple and feasible method and high transgenic efficiency (60-70%).

Description

Obtain the method for transgenic cotton plant fast

Technical field:

The invention belongs to the molecular biology of plants technical field, relevant with plant transgenic technology.

Technical background:

Cotton is the important crops that the agriculture bacillus mediated method for transformation of a kind of very difficult employing obtains transfer-gen plant, though the example that utilizes the Agrobacterium-mediated Transformation cotton has all been reported in many laboratories both at home and abroad, but because this crop is difficult to differentiate plant again by callus, so it is extremely low to transform successful frequency, even can not obtain transfer-gen plant at all.At present both at home and abroad the stem apex conversion method (J.H.Gould etc. 1999, Plant Mol.Bio.Rptr.16:3) of the method for Umbeck etc. (1987, Bio/Technology 263-266) and J.H.Gould etc. is adopted in each laboratory mostly.The former adopts by evoked callus, and inducing embryoid body forms then, induces the flow process of transfer-gen plant at last again.The greatest problem of this method is to transform required time long (between at least one year half), and transformation efficiency is low.Though the Shoot Tip Culture method of Gould can comparatively fast obtain transformant, transfer-gen plant chimera degree is very high, and transgenic seedling shared frequency in whole regeneration plant colony is very low, so it is also not really high to obtain the efficient of transfer-gen plant.

Goal of the invention:

The objective of the invention is to overcome the deficiency of prior art, set up a kind of method of quick acquisition transgenic cotton plant, utilize this method that transgenic cotton plant is carried out early stage Rapid identification, screen needed transgenic cotton plant.

Summary of the invention

The present invention is achieved through the following technical solutions:

A kind of breeding method of quick acquisition transgenic cotton plant, contain MS and B5 medium, described medium adopts the MSB5 medium of improvement, this medium is added with vitamin B1, B6, nicotinic acid and auxin, aseptic cotton hypocotyl segment is cultivated on described medium, generate rather than, obtain transgenic cotton plant by direct induced bud by the approach that inducing embryoid body generates.

Adopt following technical step:

(1) get the cotton hypocotyl, sterilization, or prepare aseptic hypocotyl segment, and create otch, the Agrobacterium that grows into logarithmic phase is infected 10-15min, blot bacterium liquid, trained altogether 2-3 days;

(2) the described material of step (1) is changed over to be added with 1mg/L nicotinic acid, the 20mg/L vitamin B1, the 2mg/L vitamin B6,30g/L glucose, cephalo thiophene hip (cefotaxim) and kanamycin or add cephalo thiophene hip and hygromycin (Hggromycin), 0.1mg/L 2,4-D selects to cultivate 6-8 week on the MSB5 medium of 0.1mg/L kinetin;

(3) by the green fluorescence protein gene (GFP) of material in the fluorescence stereomicroscope observation step (2) or the expression of gus gene;

(4) with the material that obtains in the step (3) at the improvement MSB that removes plant hormone ₅8-12 week is cultivated in regeneration on the medium;

(5) with the root induction to the root media of the resulting material transfer of step (4), transplant to the field then, or will

The material grafting that step (4) obtains is to the cotton seedling with 1-2 sheet true leaf;

(6) material that step (5) is obtained carries out the field evaluation.

With the 10-50mg/L kanamycin, or the 5U/L hygromycin joins and selects to express the Rapid identification transformant by GFP gene or gus gene in the fluorescence microscope transformant on the transformant medium.

Transgenic seedling transferred to be added with 0.1mg/L IAA, 0.1mg/L,, kinetin cultivates on the Hsu root media of 0.1mg/L gibberellic acid (GA3), and the seedling after taking root is transferred in the greenhouse soil of sterilization again and is cultivated.Perhaps,

With material that tissue culture obtained by grafting to the cotton seedling of 1-2 sheet true leaf and transfer to field cultivation.

0.1% mercuric chloride is adopted in seed disinfection in the said method, soaks cotton seed 8-10 minute after shelling, and uses 70% alcohol-pickled 3 minutes then, goes up aseptic germination at MS medium (with sucrose 20g/L), the preparation hypocotyl segment.The medium that is used for common training and selection is commercial product culture medium (MS), adds the vitamin ingredients of B5 medium: 1mg/L nicotinic acid, cobalamin 0mg/L, vitamin B6 2mg/L, glucose 30g/L.Be used to select the antibiotic of transformant, if kanamycin concentration is 10-50mg/L, if hygromycin, concentration is 5 international unit/L, during the transformant Rapid identification, if reporter gene is the GFP gene, can adopt the fluorophor stereomicroscope directly to observe; If gus gene then adopts conventional method to observe (Cell:A Laboratorymanual, D.L.Spector etc. 1997).Root media be Hsu (Stewart, J and Hsu, G., 1977, Planta137:113-117), mend IAA0.1mg/L, kinetin 0.1mg/L, gibberellic acid (GA3) 0.1mg/L; Seedling after taking root is transferred in the greenhouse soil of sterilization again and is cultivated, and avoids the plant evaporation in 7 days.If carry out grafting, choose the cotton seedling of tool 1-2 sheet true leaf, go to the top, keep cotyledon, therefrom rive with pocket knife, scion is trimmed to wedge inserts in the interface, tighten with cotton thread, to seal and be transplanted to again in the alms bowl after 7 days, wrapping is removed in 4 all backs.The invention effect:

Can in 4～6 months, obtain transgenic cotton plant according to the method described above, solve the difficult problem of differentiation in the cotton tissue incubation.Method of the present invention is easy and simple to handle, the transformation efficiency height, the seedling differentiation rate is greater than 90%, the efficient that obtains transformed plant is 60-70%, this is than (1987) such as Umbeck P., and the used method of Gould J.H.h and Magallances-Cedeno M. (1999) obtains about 3% and 5% transformation efficiency and exceeds nearly 20 times and 10 times respectively in the process of converting cotton.Therefore, solved cotton effectively and transformed the problem that the back forms the regeneration plant difficulty, be not subjected to the influence of cotton gene type, and the cycle has been shorter, for the transgenic research of cotton and other plant that is difficult to regenerate has been opened up bright prospects.

Description of drawings:

Fig. 1: be that the present invention observes the result that the GFP gene is expressed in the blade guard cell;

Fig. 2 and Fig. 3: be that the present invention observes the result that the GFP gene is expressed in the blade palisade tissue;

Fig. 4 is the cotton transgenic test-tube plantlet that the present invention cultivates;

Fig. 5 is the transfer-gen plant that the present invention cultivates.

Strain transfer-gen plant the present invention has obtained 400 from 6 different cotton varieties surplus, the acquisition of every approving and forwarding gene plant is not Above 6 months.

Contrast effect of the present invention is as shown in table 1:

The different method for transformation of table 1, different cultivars are to obtaining the comparison of transgenic cotton plant efficient

Claims

1, a kind of breeding method of quick acquisition transgenic cotton plant, contain MS and B5 medium, it is characterized in that, described medium adopts the MSB5 medium of improvement, this medium is added with vitamin B1, B6, nicotinic acid and auxin, aseptic cotton hypocotyl segment is cultivated on described medium, generated rather than, obtain transgenic cotton plant by the approach that inducing embryoid body generates by direct induced bud.

2, the breeding method of quick transgenic cotton plant according to claim 1 is characterized in that, adopts following technical step:

(2) the described material of step (1) is changed over to be added with 1mg/L nicotinic acid, the 20mg/L vitamin B1, the 2mg/L vitamin B6,30g/L glucose cephalo thiophene hip (cefotaxim) and kanamycin or add cephalo thiophene hip and hygromycin (Hggromycin), 0.1mg/L 2,4-D selects to cultivate 6-8 week on the MSB5 medium of 0.1mg/L kinetin;

(5) with the root induction to the root media of the resulting material transfer of step (4), transplant to the field then, or the material grafting that step (4) is obtained is to the cotton seedling with 1-2 sheet true leaf;

(6) material that step (5) is obtained carries out the field evaluation.

3, the breeding method of quick acquisition transgenic cotton plant according to claim 1 and 2, it is characterized in that, with the 10-50mg/L kanamycin, or the 5U/L hygromycin joins on the selection transformant medium, express the Rapid identification transformant by GFP gene or gus gene in the fluorescence microscope transformant.

4, a kind of breeding method that obtains transgenic cotton plant according to claim 1 and 2, it is characterized in that, transgenic seedling transferred to be added with 0.1mg/L IAA, 0.1mg/L, kinetin cultivates on the Hsu root media of 0.1mg/L gibberellic acid (GA3), and the seedling after taking root is transferred in the greenhouse soil of sterilization again and cultivated.

5, a kind of breeding method that obtains transgenic cotton plant according to claim 1 and 2 is characterized in that, with material that tissue culture obtained by grafting to the cotton seedling of 1-2 sheet true leaf and transfer to field cultivation.