JPS62239994A - Method for transformation of cell of gramineous plant using spheroplast of escherichia coli - Google Patents

Abstract

PURPOSE:To carry out transformation of gramineous plant readily, by using a spheroplast of Escherichia coli having a desired gene with a protoplast of a cell derived from gramineous plant and transducing the gene to the cell of gramineous plant. CONSTITUTION:A cell is treated with lysozyme by the use of Escherichia coli in logarithmic growth phase cultivated in a medium such as bouillon medium, glucose M9 medium, etc., capable of multiplying Escherichia coli to give a spheroplast of Escherichia coli having a desired gene. On the other hand, leaves, roots, buds or culture cell of gramineous plant are treated with an enzyme solution obtained by dissolving cellulase and pectoliase in mannitol (0.4mol) to give a protoplast of gramineous plant. The spheroplast of Escherichia coli is fused with the protoplast of gramineous plant and the gene contained in Escherichia coli is transduced to a cell of gramineous plant to carry out transformation.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for transforming the cells of a grass family plant by introducing a desired gene into the cells thereof. The present invention relates to a method of transforming E. coli genes by introducing them into grass plant cells.

Furthermore, the present invention can be utilized for breeding useful varieties such as disease resistance, cold resistance, and chemical resistance by imparting new traits to rice.

[Conventional technology]

Generally speaking, the soil bacterium Agrobacterium tumefaciens (Agrobacterium tumefaciens) is used for transformation by introducing a heterologous gene into plant cells.
iens, hereinafter referred to as A, tumefashens. ) is carried out using a Ti plasmid as a vector. In this case, the most efficient and safe method to introduce genes into plant cells is the coculture method (C
o-culture method) is known, and transformed cells may be obtained at a frequency of 10-' in some cases.

However, A. tumefaciens can infect many dicotyledonous plants and gymnosperms and transform plant cells, but there is a problem in that it cannot infect monocotyledonous plants. That is, as long as the introduction of Ti plasmid into plant cells depends on the infectivity of A. tumefaciens,
The range of plants that can be applied is limited by the host range of A. tumefaciens. Therefore, since many of the major agricultural crops belong to monocotyledonous plants, it is difficult to apply to agricultural products of the Poaceae family such as rice and wheat. For A.

Transformation by gene introduction using the infectivity of C. tumefaciens cannot be performed.

Therefore, transformation methods that do not depend on the infectivity of A. tumefaciens have been developed. There are methods in which plasmids are injected by microinjection ■There are methods in which plasmids are introduced by wrapping them in liposomes.

Among these methods, there are reports that method (1) is successful in transformation, but the transformation rate is low at 10-5 or less. In addition, there are still problems with method (2) when it can be used for plant protoplasts, and the method is still in the development stage. Furthermore, although method (2) is effective for introducing tobacco mosaic virus RNA, there is no success story for Ti plasmid.

[Problem that the invention seeks to solve]

An object of the present invention is to provide a transformation method by gene introduction that can solve the problems of conventional transformation methods by introducing genes into plant cells and is also applicable to grasses. It is.

[Means for solving problems]

In the present invention, when a desired gene is introduced into a grass plant cell to perform transformation, the spheroplast of Escherichia coli containing the desired gene is fused with the protoplast of a cell derived from a grass plant. This is achieved by introducing the gene contained in the Poaceae plant cells into the Poaceae plant cells and performing transformation.

That is, the present invention is a method for transforming a plant by introducing a gene into the cells of a grass family plant, which is generally widely used for genetic manipulation instead of utilizing the infectivity of A. tumefaciens, which has been conventionally used. Transformation is performed by converting Escherichia coli into spheroplasts and introducing genes into plant cells.

The transformation method by gene introduction of the present invention can be widely applied to all gramineous plants such as rice, wheat, barley, corn, turf, and bamboo.

The gene to be introduced is not particularly limited, but it is preferable to connect a promoter region in front of the gene so that it functions properly in plant cells.

Spheroplasts are obtained by treating cells with lysozyme using logarithmically growing E. coli cultured in any medium in which E. coli can grow, such as broth medium or crucose inter-9 medium.

Protoplasts are obtained by processing methods similar to those for other plant cells. In other words, the leaf blades, roots, and
It is obtained by treating sprouts or cultured cells with an enzyme solution in which cellulase and pectolyase are dissolved in mannitol (0.4 mol).

Fusion of rice protoplasts and Escherichia coli spheroplasts obtained in this way is achieved by mixing protoplasts and spheroplasts, and adding an equivalent amount of polyethylene glycol solution or polyvinyl alcohol solution dissolved in MS medium to the mixture. Perform processing. In this case, the mixing ratio of protoplasts and spheroplasts is preferably 100 to 1000 parts of spheroplasts per minute of protoplasts. If the number of spheroplasts is less than 100 parts, the transformation rate will be low, while if it is more than 1000 parts, the transformation effect will be inhibited.

Next, the present invention will be explained in detail with reference to Examples.

〔Example〕

Materials for higher plants for fusion treatment include I+
Cultured rice cells cultured in Murashige and Skoog's liquid medium (MS medium) containing 2.4-D at 1/l were used. 2% Cellulase Onozuka R for late logarithmic growth cells.
-10 (or 1% Cellulase Onozuka R3)
(Yakult) and 0.05% pectolyase Y-23
(Seishin Pharmaceutical Co., Ltd.) was dissolved in 0.4 M mannitol (pH 5.5) and treated with an enzyme solution (pH 5.5) at 30°C for 1 hour to isolate protoplasts.

In addition, the E. coli used for the fusion treatment is a Ti plasmid with a kanamycin resistance gene (neomycin phosphoranferase) of transboson Tn5, which is known in bacteria, as a drug resistance used to select transformed plant cells. A plasmid inserted after the promoter of the tubalin synthesis gene above was constructed, and (H
, W. Bevan.

et al+Nature, Vol. 304, pp. 184-7 (1983), etc.) E. coli strain C-600 obtained by transformation with this plasmid was used. Escherichia coli spheroplasts were prepared by treating cells with logarithmic growth phase that had been cultured overnight in a broth medium with lysozyme. Lysozyme melt: 0.4g Lysozyme (Boehringer Mannheim), 3.72g 2 Na-E
D.T.A.

100 m/0.025M) Squirrel buffer (pH 8,
A solution of 2) in 1) in 0.4M sorbitol was used.

The rice protoplasts and Escherichia coli spheroplasts thus obtained were mixed at a ratio of 1:100, and the mixture was mixed with 40% (wt/wt) dissolved in an equivalent amount of MS medium.
Fusion treatment was performed by adding a PEG (mol, wt, 4000. Wako Pure Chemical Industries) solution.

Observation using an electron microscope showed that 10 minutes after treatment, the cell membrane had already begun to invaginate around the point where the spheroplast adhered to the protoplast surface, and the invagination had already begun to the point where it was incorporated into the cytoplasm. Progressing images and images of vesicles wrapped in cell membranes in the cytoplasm were observed.

In addition, fusion treatment was performed using Escherichia coli spheroplasts that had been previously treated with the fluorescent dye DAPI, which specifically binds to DNA, and when observed using an epifluorescence microscope, approximately 80% of the treated protoplasts showed at least One or more spheroplasts were observed. Furthermore, on average, about 10 spheroplasts were incorporated into each protoplast, and in some cases, about 100 spheroplasts were incorporated.

After 10 minutes, 0.5 M CaC1z and 0.4 M? :
0.05M glycine buffer (pH 10,
5) was slowly added to dilute. Another 20 minutes, 3
After incubation at 0°C, the cells were washed repeatedly with a 0.4 M mannitol solution by centrifugation to remove as much of the remaining Escherichia coli spheroplasts as possible. After washing, treated protoplasts were treated with 2.4-D O as the phytohormone.

The cells were suspended in MS medium containing 5 mg/l, kinetin 0.37N, fosmicin 25-50 Mg/mf and vancosine 100-200 μg/mf as antibiotics, and 0.4 M mannitol, and cultured at 25°C for several weeks. At the stage when the protoplast divides and becomes a dozen cell clusters, M
Wash with S medium, collect 3 x 105 colonies per plate, and add 50 to 20 G-418, a kind of kanamycin.
The cells were cultured by embedding them in MS agar medium containing 0.0%/-.

After about 3 weeks at least 200 drug resistant colonies per plate had developed. On the other hand, no drug-resistant colonies were generated from protoplasts that were not fused with spheroplasts.

As described above, a drug-resistant gene could be introduced into plant cells, and the transformation rate was about 10-3.

〔Effect of the invention〕

In conducting genetic recombination research, the present invention involves amplifying genes in E. coli and then extracting the genes in most cases.
Compared to the conventional method of introducing DNA into plant cells and transforming them using several known appropriate methods, it is possible to directly introduce DNA from E. coli and transform it into plant cells without extracting it. This is a simple method.

This provides a powerful means for breeding innovative and superior varieties by imparting new traits such as disease resistance, cold resistance, and chemical resistance that do not currently exist in plants.

Claims (4)

[Claims] (1) When transforming a desired gene into the cells of a grass family plant, spheroplasts of Escherichia coli containing the desired gene and protoplasts of cells derived from a grass family plant are fused, and 1. A method for transforming gramineous plant cells, characterized by carrying out transformation by introducing a gene contained in the gramineous plant cells into the gramineous plant cells. (2) The method for transforming grass plant cells according to claim 1, characterized in that a promoter region for proper function in grass plant cells is connected in front of the desired gene. (3) The ratio of Escherichia coli spheroplasts to be fused to protoplasts of cells derived from gramineous plants is 100 to 100.
2. The method for transforming gramineous plant cells according to claim 1, wherein the ratio is 1000:1. (4) The method for transforming gramineous plant cells according to claim 1, which comprises sterilizing cells with fosmicin and vancomycin after the fusion treatment.