JPH0782009B2 - Cell fusion method and device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention is a marker for identifying homologous and heterologous cell fusions that are mixed when cell fusion is performed by rapidly labeling the nucleus of plant protoplasts. As to the technology used as.

[Conventional technology]

The method of cell fusion of two types of plant protoplasts to create a new plant is well known.

When cell fusion is carried out using protoplasts, homologous and heterologous fusions occur at the same time, so if the fused plants are regenerated without selecting them, the efficiency is very poor.

Conventionally, as a method of selecting fused cells of plants, fusion of a mutant cell line (yellow) and a normal cell line (green) of tobacco cells (Nagao Teruyoshi:
A method of distinguishing by color, such as the Japan Society of Crop Science, Vol. 47, 491 (1978)) or the fusion of Hanakirin (red) and Ceribaouren (yellow) (Yasuyuki Yamada: Cell Engineering, Vol. 220, 1982). , There is a discrimination method utilizing auxotrophy, a discrimination method of staining one protoplast with a neutral red dye, and the like. However, while sorting methods that utilize color and auxotrophy are effective for certain plants, they cannot be universally applied to all plants.

There are the following problems with the more general method of identification by staining. That is, the cell surface of protoplasts is covered with starch granules, but the neutral red dye is difficult to stain when many starch granules remain on the cell surface, and the smaller the starch granules, the better the stain. Separation of starch on the surface of protoplasts improves the dyeability, but reduces the viability of the stained protoplasts.

On the other hand, when crystal violet, which stains starch granules well, was used, a decrease in cell viability was observed.

[Problems to be Solved by the Invention]

It is an object of the present invention to provide a cell fusion method and device for protoplasts, which allows easy selection of heterotopically fused protoplasts.

[Means for Solving the Problems]

The present inventor has conducted extensive studies in view of the above problems, and as a result, 2
One of protoplasts of different kinds of plants is stained with a fluorescent dye having an acridine group by an electric pulse, the other is stained with a fluorescent dye having a phenylindole group by an electrical pulse, and then cell fusion is performed. It has been found that selection of heterogeneous fusions containing various types of fluorescent dyes can be easily performed. The protoplasts stained with the fluorescent dye used in the present invention have a high cell survival rate and also sufficiently retain the ability to perform cell fusion.

Staining microorganisms and animal cells, especially blood cells, with fluorescent dye,
It is known to perform clinical examinations and diagnoses (see, for example, Japanese Patent Laid-Open No.
60-22661, JP 61-135769, JP 53
-20479, JP-A-50-87395, JP-A-60-21
0997 publication). However, these publications
Simply fluorescently staining the microorganisms or blood cells collected, or cells of the tissue to be diagnosed, and only performing counting, etc., and the cells after fluorescent staining may be dead if they can be stained, Very intense dyeing conditions can also be used. Furthermore, nothing is described about how the fluorescent staining affects cell fusion.

The present inventor also conducted a study with another fluorescent dye, Luciferer CH, but the fluorescent dye was such that the dye slightly adhered to the protoplast surface and the color was not clear.

The present invention provides the following cell fusion method and device.

(1) In the cell fusion method of two different plant protoplasts, the cell nucleus of one protoplast is stained with a fluorescent dye having an acridine group by electric pulse,
A method characterized in that the cell nucleus of the other protoplast is stained with a fluorescent dye having a phenylindole group by electric pulse, and then cell fusion is carried out to select a heterogeneous fusion containing two types of fluorescent dyes.

(2) For use in the production and cell fusion of the two types of plant protoplasts described in (1) above, the first
Connect the protoplast making tank and the first electric pulse applying dyeing tank, and also connect the second protoplast making tank and the second electric pulse applying dyeing tank to make the first electric pulse applying dyeing tank and the second electric pulse applying A device with a cell fusion cell between dyeing tanks.

Protoplasts are obtained by lysing and removing the cell wall, and are in an inconvenient state for plant cells to survive. For this reason, it is preferable to culture the protoplasts for a short time in order to keep the proliferation ability of the fused cells high.

In an attempt, spinach (scientific name: Spinacea oleracea)
When the young leaves on the 30th day after germination are protoplasted and labeled with acridine orange dye, about 50% of the cells are labeled in about 10 minutes, which is faster than the adult leaf-derived protoplasts. The labeling rate of protoplasts varies depending on the species of the plant and which part of the growth process is used, and although it cannot be said unequivocally, protoplasts derived from adult leaves do not receive external stimulation such as electric pulse. When labeled with a fluorescent dye, the labeling rate was about 1 to 2%. Even if the labeling rate is 1 to 2%, labeled protoplasts can be easily selected from unlabeled protoplasts, and therefore there is no problem even if the labeling rate is low for cell fusion.

The apparatus of the present invention has two kinds of protoplast-producing tanks and a staining tank for applying electric pulses, and can perform protoplast formation and fluorescent dye staining by electric pulses simultaneously on two different plant cells in a short time. it can.

The contents of the present invention will be specifically described below with reference to examples.

Example 1 In order to avoid contamination by microorganisms, which is a problem when culturing plant cells, it is desirable to use a device that allows all operations to be performed in a clean bench. Therefore, an instrument as shown in FIG. 1 was manufactured to prepare a sample. Explaining the application of the device of FIG. 1, 1 is a protoplast making cell, 2 is a dye labeling and washing cell, and 3 is an electrofusion cell. No. 4 is a double nylon or stainless net for separating protoplast cells and leaf leaf residue, and the upper part is 100 mesh and the lower part is 30 mesh to prevent clogging. It would be convenient if the mesh size could be changed according to the size of the cells. First, in order to prepare protoplast cells, the cell wall lysing enzyme solution and the debarked leaves are put together in cell 1 and left standing for a certain period of time. In the case of a sample with a solid cell wall, a suction operation is first performed from the 10 stomatal openings to obtain good separation results. However, if the static treatment is sufficient,
Even after the treatment, the cells are not damaged and there are few debris. Two
The reason for tilting the cells is to prevent protoplast cells from breaking as much as possible when they are transferred to the cells below. When the protoplast cells are separated, open the cock of 5 and transfer to the cell of 2. Dye solution is injected from 7 and DC pulse is applied to the cylindrical electrode of 6 using the electric pulse generator of 9 to perform dyeing. After that, the unnecessary enzyme and dye are washed off with a cleaning solution. Two types of labeled protoplast cells can be prepared by inserting another leaf into the other cell of the same type and performing the same operation. This is sent to the fusion cell 3 for cell fusion.

Example-2 Dutch honeywort (scientific name: Apume Graveoleas)
The leaves are sterilized and the back skin is peeled off, and a cell wall lysing enzyme is placed in a 0.6 M mannitol solution adjusted to pH 5.7, and the cell wall is lysed in the cell of FIG. 1 to give protoplast cells. This is transferred to cell 2 and washed with a sterilizing solution. This suspension was mixed with acridine orange (AO) at a concentration of 50 to 250 μM and
Adjust the cell concentration to approximately 10 ³ cells / ml. Apply a pulse width of 0.1 ms and a pulse voltage of 300 to 500 V from both ends of the electrode to this.
When applied three times, labeling is performed within 3 to 5 minutes, and the nucleus or cytoplasm can be observed with a fluorescence microscope. The fluorescence wavelength range of acridine orange is yellow at 520 nm, which makes it easy to see. The pulse voltage can be further reduced by properly adjusting the type and adjustment of the sample.

Example 3 The leaf of a bok choy (Scientific name: Brassica paratinensis) was sterilized, the back skin was peeled off, and the cell wall lysing enzyme was put into 0.7 M mannitol solution adjusted to pH 5.7 to dissolve the cell wall. Use protoplast cells. The following operating method is described in Example 2.
Is almost the same as. This suspension is transferred to the cell 2 in FIG. 1 and adjusted to a concentration of 4-6-diamidino-2-phenylindole (DAPI) 1-2 mM. When a pulse width of 0.1 ms and a pulse voltage of 300 to 500 V are applied 1 to 5 times from both ends of the electrode, the nuclei can be observed within 3 to 5 minutes by labeling with an optical microscope. A fluorescence wavelength range of DAPI shines blue at 360 nm A
It is easily distinguishable from those labeled with O.

In this way, by staining each cell with two types of fluorescent dyes, it is easy to understand the contact state of each other and convenient for selection.

〔The invention's effect〕

According to the present invention, various plant protoplast cells used for cell fusion can be introduced with a fluorescent dye in a short time by applying an electric pulse. Therefore, compared with the case where the dye is simply attached to the cell surface, it does not cause color transfer to the other cell, and since it can be labeled with a small amount of dye, it causes less damage to cells and is advantageous for selection of heterogeneous fusions. To work. Therefore, it is possible to provide a simpler and more effective means than when the identification marker is attached by other methods.

[Brief description of drawings]

FIG. 1 shows a principle configuration diagram of an instrument for labeling a plant protoplast cell with a fluorescent dye. 1 ... Protoplast preparation cell, 2 ... dye labeling and cleaning cell, 3 ... electrofusion cell, 4 ... double wire mesh, 5
…… Cock, 6 …… Cylindrical electrodes, 7 and 8 …… Cleaning solution inlet and outlet, 9 …… Electric pulse generator, 10 …… Pore or suction port.

Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number for FI C12N 15/02 (56) References JP-A-60-22661 (JP, A) JP-A-62-135769 (JP , A) JP 53-20479 (JP, A) JP 50-87395 (JP, A) JP 60-210997 (JP, A) JP 61-70972 (JP, A)

Claims (2)

[Claims] 1. A method for cell fusion of two different plant protoplasts, wherein one protoplast cell nucleus is stained with a fluorescent dye having an acridine group by electric pulse, and the other protoplast cell nucleus has a phenylindole group. A method which comprises staining cells with a fluorescent dye with an electric pulse, and then performing cell fusion to provide for selection of a heterogeneous fusion containing two types of fluorescent dyes. 2. A method for producing two kinds of plant protoplasts according to claim 1 and for cell fusion, the first method comprises:
Connect the protoplast making tank and the first electric pulse applying dyeing tank, and also connect the second protoplast making tank and the second electric pulse applying dyeing tank to make the first electric pulse applying dyeing tank and the second electric pulse applying A device with a cell fusion cell between dyeing tanks.