CN1110320A

CN1110320A - Method and device for conversion of plant

Info

Publication number: CN1110320A
Application number: CN 94103393
Authority: CN
Inventors: 丁群星; 谢友菊
Original assignee: Individual
Current assignee: Individual
Priority date: 1994-04-07
Filing date: 1994-04-07
Publication date: 1995-10-18
Anticipated expiration: 2014-04-07
Also published as: CN1035304C

Abstract

The present invention relates to a method and device for conversion of plant, belonging to a gene introduction technology of plant genetic engineering. Said method is as follows: a separated object gene is made into an injection buffer containing 20mM/l Mgcl2, 1.5mM/l boric acid, 5% PEG 6000 (W/V) and object group DNA; an acceptor plant is covered by using bag and insulated, and after artificial pollination for a period of time, the above-mentioned injection buffer is injected into ovary; the injection trauma region and its periphery are lightly coated with vaseline ointment; the injected ovary is again insulated until the seed is matured. Said injection device includes microneedle, microneelle fixing button, needle tube and pushing button, and can make sampling and injection by pushing button, and can raise conversion effect and repeatability.

Description

Method and device for conversion of plant

The present invention relates to the gene leading-in technique of genetically engineered plant, especially foreign gene is imported the method and apparatus of recipient plant by injection means.

Present gene leading-in technique at plant has multiple, comprising agrobacterium-mediated transformation, and PEG(polyethylene glycol) mediated method, electric shocking method (electroporation), particle bombardment or the like.Yet these technology are subjected to floristic strong restrictions, and are main only effective to dicotyledons as agrobacterium-mediated transformation; Some technology then is subjected to the restriction of plant tissue culture requirement, as PEG mediated method and electric shocking method, requires sophisticated plant protoplast separation, cultivation and regeneration system; The particle gun method also needs the cooperation of the inducing of plant callus, cultivation and plant regeneration technology.The method for transformation of having broken away from the plant tissue culture technique restriction at present has three classes, i.e. pollen-mediated method, pollen tube passage method and injection.Yet, exist many uncontrollable factors, so be difficult for repeating and checking each defectiveness, explanation successively below because the acceptor of these methods is whole plant.

The pollen-mediated method is foreign gene and plant pollen co-blended, or with microinjection, after means such as particle gun import pollen with foreign gene, award corresponding female fringe the pollen that is subject to processing, in the hope of obtaining containing the seed (Ohta of foreign gene, 1986.Proc.Natl.Acad.Sci.USA, 83: 715-719).Because for some time vigor promptly can descend even death behind the in-vitro pollen, be subjected to handle the back vigor and also can descend, and no matter the foreign gene that carries can only with quite low ratio by style enter ovary and blastular in the surperficial still inside of pollen granule.So it is also few to possess the experimental evidence of abundant cogency so far.

Pollen tube passage method is meant on the plant style column cap after the pollination or on the column cap section, smear exogenous DNA solution, enter the passage that blastular stays by pollen tube by style, spontaneously foreign gene is absorbed and remove to transform zygote in the blastular, thereby obtain containing seed (the Gong bitter edible plant bitter edible plant etc. of foreign gene, 1988, Chinese science (B collects), 6: 611-614).Because the locomotory mechanism of exogenous DNA molecule in said pollen tube channel it be unclear that, in entering the process of blastular, be subjected to the threat of nuclease, the foreign gene quantity and the integrity that can enter zygote all have problems, so it is also very limited to have an example of complete evidence.

Injection is meant with syringe directly injects plant ovary (Liu Bolin etc. with foreign gene, 1989, Chinese science (B collects), 89(7): 699-705) or young fringe chamber (de la Pena etc., 1987, Nature, 325: 274-276), make foreign gene enter gamete or zygote cell, obtain containing the seed of foreign gene after reaching maturity.Because injection device commonly used is general injector for medical purpose, micro-injector or the device of repacking slightly, bigger to the injury of plant ovary in the injection process, injection volume is wayward, operation inconvenience.And the easy abortion of ovary after the injection, the skill that injection opportunity and status requirement are certain is so also be very limited.

The object of the present invention is to provide a kind of method and device, overcome above-mentioned defective of the prior art, improve transformation efficiency and repeatability by injection means conversion plant.

Purpose of the present invention can realize by following measure:

A kind of method that transforms plant may further comprise the steps: a. separation and purification goal gene DNA; B. isolated goal gene is mixed with and contains 20mM/l MgCl ₂1.5mM/l boric acid, 5%PEG 6000(w/v) and the injection damping fluid of goal gene DNA; C. recipient plant is carried out bagging and isolate, after artificial pollination for some time, above-mentioned injection damping fluid is injected ovary with injecting method; D. dab soft petroleum ointment around injection trauma region reaches infects with mould proof; E. isolate the ovary of being injected again,, carry out the molecule checking after the results until seed maturity.The amount of buffer of injecting each ovary is 2 μ l.

The injection device that is used for above-mentioned conversion comprises micropin, micropin fixedly knob, syringe and propelling knob, and this device advances sampling and injection by rotation.

The invention has the advantages that: 1. compare with other transformation technologies, range of application of the present invention is not subjected to floristics and genotypic restriction, can implement any interested crop strain, helps the application of goal gene in agricultural breeding and land for growing field crops production; Present technique does not need tissue culture and plant regeneration process, has shortened the work period.

2. compare with other injection technique, injection buffer formulation special among the present invention is protected goal gene, avoids the degraded of nuclease in the vegetable cell; Injection device makes entry needle reduce greatly the injury of ovary, and can quantitatively control injection volume; Effectively protective devices (soft petroleum ointment is smeared) makes ovary after the injection not be subject to mould to infect, and the seed yield improves greatly, brings up to about 50 percent from percentum, and transformation efficiency also improves thereupon.

3. simple, be easy to repetition and checking.

Fig. 1 is that the present invention transforms the stereographic map with injection device;

Fig. 2 is the sectional view of device shown in Figure 1;

Fig. 3 is the assembling synoptic diagram of device shown in Figure 1;

Fig. 4 is a rotaring gene plant blade DNA Southern results of hybridization;

Fig. 5 is that self progeny's leaf DNA of transformed plant is through the pcr amplification result;

Fig. 6 is No. 4 plant leaf DNA of fourth pcr amplification result; Fig. 7 is that No. 4 plant leaf DNA of fourth multienzyme is cut the Southern results of hybridization;

Fig. 8 is a rotaring gene plant blade DNA Southern results of hybridization;

Fig. 9 changes Bar gene plant leaf DNA Southern results of hybridization;

Figure 10 is the situation of field herbicide spraying Basta, and most of plant is withered, has only the transfer-gen plant survival;

Figure 11 is that responsive plant and transfer-gen plant (green) are in the contrast situation that has sprayed weedicide Basta.

Introduce the present invention in detail below in conjunction with drawings and Examples:

Embodiment 1:

The Bt gene is imported corn inbred line P136

Adopt the method for narrating among the present invention the toxoprotein gene (Bacillus thuringiensis. is called for short Bt) of Bacillus thuringiensis to be injected the blastular of corn inbred line P136, with the planting seed of gathering in the crops, the seedling that grows is carried out pcr amplification and molecular hybridization evaluation, the result shows that in the seed that detects a strain being arranged is transfer-gen plant, and has also detected the Bt gene order in its offspring plant.Illustrate that present method can import plant with foreign gene effectively, and the foreign gene that imports can be genetic to the offspring, referring to Fig. 4 and Fig. 5.

Embodiment 2:

The Bt gene is imported corn seed of single cross agricultural university 60

For further checking effect of the present invention, carry out the injection of corn ovary with the Bt gene as goal gene again, from the seed that obtains, identify through pcr amplification and molecular hybridization, identify a strain transgenic corns again, name and be No. 4, fourth.

Fig. 7 is the results of No. 4 plant leaf DNA of fourth through pcr amplification, Fig. 8 is that No. 4 plant leaf DNA of fourth are through EcoR I, the Southern results of hybridization of three kinds of enzymes of Bgl II and Acc I after enzyme is cut respectively, confirmed that not only the Bt gene has been incorporated into corn and has dyed on the body, and have only a copy, the results are shown in Figure 6 and Fig. 7.

Embodiment 3:

The Bt gene is imported corn inbred line P138

In order to enlarge transfer-gen plant colony, P138 has carried out the ovary injection with Bt gene pairs corn inbred line.Go out three strain transfer-gen plants through Screening and Identification, simultaneously also detected the Bt gene in the progeny plant of No. 4, fourth, the result as shown in Figure 8.

Result shown in the last figure shows, the Bt gene that enters No. 4, fourth through injection has been delivered to the offspring; The Bt gene can enter among the corn inbred line P138 equally.

Embodiment 4:

The Bar gene is imported corn inbred line P138

By above three examples as seen, the Bt gene can be imported corn inbred line P136 and P138 and seed of single cross agricultural university 60 respectively, confirm that present technique is not subjected to the restriction of plant species system with injection means; And the goal gene that injects plant can be delivered to the offspring.In order to verify the effect of other genes, also anti-herbicide gene Bar gene is imported corn inbred line P138, detected the sequence of Bar gene, as shown in Figure 9, and the plant that obtains has tangible resistibility to weedicide, confirm that further the foreign gene that adopts the technology of the present invention to import plant is complete, can work orderly, shown in Figure 10,11.

Referring to Fig. 1,2,3, the injection device that is used to transform, comprise micropin (1), micropin is knob (2) fixedly, syringe (6) and propelling knob (7), advance sampling and injection by rotation, micropin (1) is to draw with the glass capillary heating to form, micropin fixedly knob (2) central authorities is provided with the hole of placing micropin, syringe (6) two ends inwall is provided with screw thread, be used to install micropin fixedly knob (2) and propelling knob (7), also be provided with a flange at the inwall that micropin one end is installed, advance the end portion of knob (7) push rod that one groove is arranged, be used to install sealing-ring (5), the front end of syringe is provided with elastic washer (3) and rigidity packing ring (4).

Claims

1, a kind of method that transforms plant may further comprise the steps:

A. the gene DNA of separation and purification purpose;

B. isolated goal gene is mixed with and contains 20mM/l MgCl ₂1.5mM/l the injection damping fluid of boric acid, 5%PEG 6000 (w/v) and goal gene DNA;

C. recipient plant is carried out bagging and isolate, after artificial pollination for some time, above-mentioned injection damping fluid is injected ovary with injecting method;

D. dab soft petroleum ointment around injection trauma region reaches infects with mould proof;

E. isolate the ovary of being injected again,, carry out the molecule checking after the results until seed maturity.

2, the method for claim 1 is characterized in that used goal gene is the toxoprotein gene Bt gene of Bacillus thuringiensis.

3, the method for claim 1 is characterized in that used goal gene is an anti-herbicide gene Bar gene.

4, the method for claim 1 is characterized in that recipient plant is corn inbred line P136, P138 or corn seed of single cross agricultural university 60.

5, the method for claim 1, the injection damping fluid that it is characterized in that injecting each ovary is 2 μ l.

6, a kind of injection device that is used to transform comprises micropin (1), micropin fixedly knob (2), syringe (6) and propelling knob (7), and this device advances sampling and injection by rotation.

7, device as claimed in claim 6 is characterized in that described micropin (1) is to draw with the glass capillary heating to form, and micropin fixed knob (2) central authorities are provided with the hole of placing micropin.

8, device as claimed in claim 6 is characterized in that syringe (6) two ends inwall is provided with screw thread, is used to install micropin fixed knob (2) and advances knob (7), also is provided with a flange at the inwall that micropin one end is installed.

9, device as claimed in claim 6 is characterized in that advancing the end portion of knob (7) push rod that one groove is arranged, and is used to install sealing-ring (5).

10, device as claimed in claim 6 is characterized in that the front end of syringe is provided with spring washer (3) and rigidity packing ring (4).