JPH0427393A - Transduction of gene into plant cell - Google Patents

Abstract

PURPOSE:To transduce a breeding gene into a plant cell without damaging the cell by immersing a plant cell in a buffer solution containing target DNA and introducing the DNA into the cell by electroporation in a plasmolyzed state. CONSTITUTION:Target DNA 4 is mixed into a buffer solution and a plant cell 1 such as a piece of tobacco leaf is immersed in the solution to separate the cell membrane 3 from the cell wall 2 of the cell 1 and cause the plasmolysis. The cell 1 is electroporated by applying 1-10 pulses having a pulse height of 0.5-0.8kV/cm, pulse-width of 6mus to 10ms and pulse spacing of 0.5-10sec to effect the gene transduction of the target DNA 4 into the cell 1. The cell is put into a dilute buffer solution to restore the plasmolyzed cell 1, planted on a medium 7 in a test tube 6, transplanted into a flask 8 after sprouting and cultivated to obtain a plant body 10.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a gene introduction method for improving plant varieties.

(Conventional technology) To introduce genes by electroporation,
Since the cell wall was a hindrance, the cells were transformed into protoplasts and then the genes were introduced by electroporation.

(Problem to be solved by the invention) For this reason, it is necessary to convert cells into protoplasts, and the conditions for regenerating protoplasts into plants, such as the medium and temperature, are very difficult, making it extremely difficult to regenerate them. there were. Another drawback was that cells were damaged when turning them into protoplasts.

(Means for Solving the Problems) In order to solve the above problems, the present invention mixes target DNA into a buffer solution, immerses plant cells in the solution, causes plasma separation, and then electrolyzes the cells. The target DNA was introduced by poration.

(Effect) Since cells undergo plasma separation, target DNA easily enters through the cell wall. In this way, if the gene is then introduced into the cell membrane by electroporation,
Gene introduction can be performed using the same procedure as making protoplasts and then introducing them.

(Example) Embodiments of the present invention will be described with reference to the drawings.

Soak cell 1 of a tobacco leaf in a solution containing 0.4-0.6M mannitol in a normal buffer solution (see Figure 1).
. At this time, target DNA 4 is mixed into the buffer solution.

Then, the cell membrane 3 of the cell separates from the cell wall 2, causing plasma separation. At this time, target DN from cell wall 2
A4 size fits (see Figure 2).

Next, electroporation conditions were set for cell 1 at a voltage of 0. 5-0. 8kV/cm, pulse duration 6
Target DNA 4 is introduced with a LL of 5 to 10 ms, a pulse interval of 0.5 to 10 s, and a pulse application number of 1 to 10 times (see FIG. 3).

Next, the cell 1, which has been subjected to plasma separation in a dilute buffer solution, is returned to its original state (see FIGS. 4 and 5).

The cells thus obtained are planted in a medium 7 in a test tube 6 as shown in FIG. After sprouting, they are transplanted into a medium 9 in a flask 8, cultured, and regenerated into a plant 10 (see FIG. 7).

(Effects of the Invention) Compared to the conventional method using protoplasts in which the cell wall is removed by enzymatic treatment, gene introduction by electroporation can be performed more easily since there is no need to remove the cell wall. Moreover, since there is no danger of cells being destroyed by enzymes, this has the effect of making it easier for plants to regenerate.

[Brief explanation of drawings]

Figure 1 shows a plant cell immersed in a buffer solution, Figure 2 shows a state in which plasma separation has occurred, Figure 3 shows a state in which electroporation is being performed, and Figure 4 shows a gene Figure 5 shows the state in which cells that had undergone plasmodesmation have been restored to their original state, Figure 6 shows the state in which these cells have been planted and cultured in a medium, and Figure 7 shows the state in which the cells have been cultured. It is a diagram of a state in which it has been regenerated into the body. 1 is a plant cell, 2 is a cell wall, 3 is a cell membrane, 4 is a target DNA, 5 is a nucleus, 6 is a test tube, 7 and 9 are culture media, 8 is a flask, and 10 is a plant body. Patent applicant: Benma Noki Co., Ltd. Agent: Tobe Maki (and 3 others)

Claims (1)

[Claims] Gene introduction into plant cells characterized by mixing target DNA in a buffer solution, immersing plant cells in this to cause plasma separation, and then introducing the target DNA into the cells by electroporation. Law.