JPS63196270A - Production of genetically transformed plant - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to genetic engineering of plants and more particularly to a method for preparing genetically transformed plants, the method comprising:
certain properties, such as specific herbicide resistance, specific resistance to pathogens and microorganisms, modified amino acid composition,
It is used for the selection of new types of plants, including agricultural plants, characterized by enhanced useful biomass content, enhanced stability against adverse environmental factors, and others.

[Conventional technology]

Methods for the transformation of protogellasts of higher plants and the subsequent selection of cell lines and plants with novel genetic properties therefrom are known in the art.

It is known to add to the serum solution and then subject the resulting mixture to the effect of physical and chemical factors, which lead to the entry of the DNA into the protogellasts (Potrykua et al. 1..5hillit.

R@D-I 5aul M, W, + Paszkuws
ki J,. Direct gene transfer, its technological status and future possibilities, '("Dir@ct G@
ne Transfer+5tate of Art a
nd Future Potential+tsu Plan
t117-128].

genetic material contained in lipid bubbles (sfsomes),
Methods of adding Nicotiana tapachie glotopase K in the presence of a flocculant (which leads to fusion of the protogellast with the liposomes and penetration of the genetic material into the protogellast) are also known in the art. known to (Natata)
T, "Liposomes as carriers of Tl-grasmid into protocilasts"("Liposomes a
s a Carrier of Ti-plasmmld
Into Protoplasts
ar Genetics of the Bactert
a-Plant Interacticin'+ Ed
.

Puhler, Springer-Verlag,
Berlin s, a.

1983.268-273].

The above methods have the disadvantage that their application is limited. This is because they can only be applied to a -1 limited number of plant species, namely plants and cell cultures. For this purpose, protoplast production and culture techniques and subsequent regeneration of plants have been developed. Besides, the methods cited above are characterized by low transformation efficiencies (up to 1%) and require considerable consumption of transforming factors.

Furthermore, methods for preparing genetically transformed plants are known in the art, in which nicoteanatapachyame protogellasts are used as receptors. Plasmid pcGN561 is injected into protoplasts by microbial/noexy transfusion (Crosaway A
, etc., @Incorporation of exogenous DNA following microinjection into protoplasts of tobacco mesophiles''
(”rntegration of @ign
DNA following m1croinject
ionof tobacco mesophyl
protoplast'+Mt已.

Gen, Gen5t-+ 1986 + 202 volumes, 1
79-185].

The disadvantage is that by using the above method, neither cell lines nor plants with novel genetic properties were prepared and no expression of the introduced genetic material was demonstrated. The method can be applied independently to protogellasts,
It is achieved only under laboratory conditions and is practically not applied.

The following margin [Problems to be solved by the invention] To enhance transformation efficiency and reduce consumption of transforming factors,
It is then an object of the present invention to provide a method that makes it possible to increase the number of plant species (including higher plants) involved in genetic engineering manipulations.

[Means for solving problems]

Said object is, in accordance with the present invention, a method for preparing genetically transformed plants by introduction of a transforming factor into a receptor and subsequent selection of cell lines and plants with novel genetic properties. (proposed in 1999), a DNA molecule or an autonomously replicating organ is used as the transforming factor, and a grotonorlast, a single cell or a cell in a construct, is used as the receptor, and the A transforming factor is introduced into the receptor by microinjection. The microinjection seal is
This can be achieved with the aid of any suitable method. microinjection under pressure or under the action of an electric field,
It is appropriate to do so.

The method proposed herein increases the transformation efficiency to 40% (
This enhances the transformation efficiency of conventional methods by a factor of 10-20) and significantly reduces the consumption of transforming factors.

The method of the invention uses protogelast as the receptor.

Single cells and cells in structures can be used; DN
A molecules and autonomously replicating organelles are used as transforming factors. This increases the number of species, such as important agricultural plants (eg, cereals), that are involved in genetic engineering operations.

The present invention is achieved by the following method. Protogellasts, single cells or cells in structures (microcallus, embryogenic callus, embryoid bodies) in the corresponding nutrient solution, used as receptors, are placed in a microchamber mounted on a microscope stage. Place and fix by pouring into agar, for example with the help of a sucker or mechanically.

Transforming factor (Grasmid) in the corresponding medium.

A glass micropipette filled with cosmid, chlorocilast) is injected into the receptor with the aid of a micromanipulator, and the injection is carried out under the control of an optical microscope.

Micro-/no-exchange is achieved under pneumatic or hydraulic pressure or under the effect of an electric field. The volume of solution injected is
It differs from 1 to 10 picoliters. All operations are performed under sterile conditions. The injected receptors are transferred to a selective nutrient medium, and the selection is determined by the transforming factor. Selection of transformants and accumulation of their biomass is carried out on the basis of repeated transfer of receptors on selection media. Plants are regenerated from transformed cell lines and cell constructs on selective media.

In order to further understand the invention, specific examples are provided below by way of illustration.

Example 1 A micropipette with a diameter of approximately 1 μm at the tip end is inserted into a protogellast isolated by Nicoci et al. and a buffer solution containing psV2N.0 glasmid (10 μM Trys, pH
7,2r1mM sodium ethylenediaminetetraacetate), 5mMf) kcj 1-2 picoliters are pressurized with the aid of a hydraulic system.

During microinjection, until the first split, the protogelasts were incubated with Cab containing 0.1% by weight agarose.
rche nutrient medium. Microcolonies formed from injected protoplasts were incubated for 60 Q/
Transfer onto Gamburg's medium containing dm' antibiotic, kanamycin sulfate. Eight cell lines were obtained from 53 injected protogellasts, and the cell lines were 120
1f! The transformation efficiency was 15%. From the three resistant lines obtained on Gamburg's medium without the addition of plant hormones, Plants were grown on medium containing kanamycin and regenerated that were able to root.

Example 2 This method is similar to that described in Example 1.

A single cell of Nicotinatapachyame is used as receptor and PLGV23N@07''') sumid is used as transforming agent. Microinjector 1 is carried out with the help of air system. From 20 injected cells: 1
Three cell lines were selected that were able to grow on Gamburg medium containing up to 20 m9/dm' of kanamycin sulfate. - Example 3 This method is similar to the method described in Example 1.

5achin (8-30 cells) containing 0.3% by weight agarose in medium is used as receptor. ! Lasmid pGV0319 is used as a transformation agent. According to the characteristics encoded by the grasmids, selection of transformants is carried out on hormone-deficient 5aehln medium. Insertion of Grasmid into 95 cells yielded 19 cell lines that could be grown in hormone-free medium and the transformation efficiency was 19%.

Example 4 This method is similar to that described in Example 1.

A single cell of Nicotiana tapacheum was used as the receptor and cosmid pGA471 as the transforming agent. 5% by several 0.5% pulses with current strength up to 0.1μ ania.
Performed under the action of an electric field of ~IOV. 1201119/dm from 23 cells transformed with K as above.
We obtained a 6a cell line that could grow on medium containing caramycin sulfate.The transformation efficiency was about 25%. We regenerated plants from two types of cell lines.

Example 5 This method is similar to that described in Example 4.

As a receptor, Triticum aestipam (Tri
t i cuma@stivm Wheat (Miro
novskaya rannyaja) cells are used and the cells are mechanically fixed. 20~
100 cells were injected into each of 8 calluses. The calli were transferred to Murashlge-Skoog medium containing 601n9/dm of calamycin sulfate and 21n9/dm of 2°4-dichlorophenoxyacetic acid. Six cell lines were obtained.

example The method is similar to that described in Example 1.

Using white single cells of a gelastomic mutant of IP type nicotina tapakiame as a receptor,
Chlorogherasts isolated from leaves of Nicotinatapachyame 2 lines were used as autonomously replicating organelles. The chlorogellasts are characterized by antibiotic resistance (streptomycin), and the resistance is encoded by chlorocilasts. micropipes with an end diameter of 8 μm) were filled with a suspension of chlorocilast in Jensan-Basshem medium, and
10 chloroplasts were injected under pressure into the cells. After the first division, the injected cells were transferred onto Gamburg's medium containing 1q/CR'' of stregtomycin sulfate.

From 16 cells injected in this way, green and i
Three cell lines were selected that were characterized by their ability to grow on medium containing mp/cit'' antibiotics.

Claims (1)

[Scope of Claims] 1. A method for producing a genetically transformed plant, the transformation of the plant being carried out by DNA molecules or autonomously replicating into cells of protoplasts, single cells or structures. The method is carried out by microinjection of organelles containing the novel genetic characteristics and subsequent selection of cells or plants with novel genetic characteristics. 2. The method according to claim 1, wherein the microinjection is carried out under pressure or under the effect of an electric field.