JPH01179635A - Preparation of plant of somatic cell hybrid of rice plant - Google Patents

Abstract

PURPOSE:To enable easy preparation of somatic cell hybrid of rice plant in high frequency, by using a differentiating marker gene existing in a fused cell. CONSTITUTION:A protoplast produced by a well-known method, for example, is fused by using polyethylene glycol method or electric fusing method under fixed conditions. The prepared fused cell is cultivated to form a colony, which is transplanted on a plant reproducing medium to reproduce a plant. The plant is trained, grown in a greenhouse, protoplasts are examined and a somatic cell hybrid showing only dominant character but not recessive character as a phenotype by development of differentiating marker gene is selected and prepared.

Description

[Detailed description of the invention] <Industrial application field> The present invention relates to the production of somatic cell hybrid plants of rice.

This invention can be used to improve rice varieties by using cell fusion to obtain new rice plants that have not been obtained before.

<Prior Art> Regarding the production of somatic cell hybrids, there have been many reports on plants belonging to the Solanaceae, Apiaceae, Brassicaceae, Rutaceae, and the like. This technology makes it possible to hybridize plant species and varieties that are said to be difficult or impossible to hybridize, depending on the combination.

Furthermore, one of the remarkable features of somatic cell hybrids is that it is possible to mix and cross the genetic elements of the cytoplasms of both parents, which was not possible with conventional crossbreeding methods.

Although this technology is excellent in principle, there are only a few reports of the production of somatic cell hybrid plants in the Poaceae family, to which many of the world's important crops belong. No plants were obtained.

Traditionally, rice protoplast culture was thought to be impossible, but in recent years, an efficient protoplast culture method using suspension cultured cells has been developed, which means that somatic cell hybrid plants can be produced in rice as well. It shows that it is possible.

<Problems to be solved by the invention> Conventionally, in the production of somatic cell hybrid plants, selectable marker genes, such as auxotrophic genes, which are expressed at the cellular level are used, and when the fused cells are obtained, Cells were selected using the method, hybrid plants were efficiently obtained, and hybrid plants were then regenerated.

However, even if it is possible to form somatic cell hybrids in rice, no plant with effective characteristics for selecting such hybrids has yet been developed. On the other hand, it is possible to add a marker gene to select hybrids, but this process requires a long culture period. However, in rice, the redifferentiation ability of cultured cells easily decreases, so it is extremely difficult to maintain a high plant regeneration ability during the period when marker genes are provided.

The object of the present invention is to solve these problems, create a rice somatic cell hybrid, and provide a method for selecting a rice somatic cell hybrid plant.

<Means for Solving the Problems> In order to solve these problems, the present inventors have developed a method that allows cultured rice cells to develop redifferentiation ability by using identification marker genes that originally exist in rice. It is possible to induce cell fusion and redifferentiation of plants before they are lost.
Furthermore, they discovered that the hybrid plants formed can be easily distinguished from the parent plants and can be selected, leading to the completion of the present invention.

That is, in the present invention, rice cell fusion is carried out using, as parent cells, cells having an identification marker gene that is expressed when a plant is formed, and when the plant is regenerated from the resulting fused cells, This is a method for producing rice somatic cell hybrid plants, which is characterized by selecting hybrid plants using these markers.

The term "identification marker gene" as used in the present invention refers to a gene that can clearly identify a hybrid by analyzing the morphology of the plant. Examples include genes that exhibit distinct morphological characteristics.

Furthermore, during cell fusion, protoplasts of one parent are treated with iodoacetamide (hereinafter referred to as ■0^) to lose their ability to divide, or protoplasts with extremely low differentiation ability are used as one parent to generate hybrids. Only the plant body can be efficiently redifferentiated.

The present invention will be explained in more detail below.

How to culture protoplasts and regenerate plants is provided by Fu.
The method of Pujimura et al.
al., 1985, Plant Ti5sue C1
ture Letters+2 (2) Near 4-75)
I followed.

First, when performing cell fusion, a combination of identification marker genes possessed by both parents of the fusion is selected from one of the following.

■(Parent cell with dominant genetic trait) x (Parent cell with other dominant genetic trait) ■(Parent cell with recessive genetic trait) x (Parent cell with other recessive genetic trait) ■(Parent cell with recessive genetic trait and Parent cells with dominant genetic traits)
× (Parent cells that do not have identification marker genes) Two types of rice that meet these conditions are used as materials, and each cup plate,
Immature embryo and non-F! Callus is induced from the panicle and a suspension culture cell line is established.

Next, protoplasts are isolated and purified from those cells,
Submit to fusion. In some cases, selection of hybrids may be further facilitated by treatment with IOA or the like, for example, treatment with IOA at a concentration of 25-30 mM at 25°C for 15 minutes.

The prepared protoplast was mixed with polyethylene glycol (P
They are fused by the EG method or the electrofusion (EF) method under the conditions shown below.

PEG method: Two types of protoplasts each adjusted to a cell density of 2×10' cells/al are mixed at a ratio of 1:1. Add an equal amount of 20 to 40 χP to this protoplast suspension.
An EG (average molecular weight 7,800-9,000) solution is added and the PEG is removed after standing for 15 minutes.

EF method: Two types of protoplasts each adjusted to a density of 2×10′ and 7 ml are mixed at a ratio of 1:1. This protoplast suspension was placed between parallel electric coils for cell fusion, and the following electrical stimulations were applied in the order of ① to ②.

■IMHz, high frequency of 120-200V/cm 3~
10 seconds.

■IMHz, 200-400V/cmO high frequency 0.1
~0.3 seconds.

■ 1 square or attenuated wave of 2000-3500V/cm
Apply 1 to 3 times for 0 to 1000 microseconds.

The above voltage is applied between the electrodes, and the electrodes are left for 15 minutes and then washed.

The fused cells obtained as described above are cultured to form colonies. The formed colonies are transplanted to a plant regeneration medium and the plants are regenerated.

The regenerated plants are acclimatized and grown in a greenhouse, their traits are investigated, and plants that exhibit only dominant traits and no recessive traits are selected based on the expression of the selected identification marker gene.

<Effect> According to the method of the present invention, hybrid plants can be easily selected by using identification marker genes originally present in rice, and alpha hybrids can be efficiently produced.

<Example> Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 A rice variety ``Shidaikoku'' has two traits: a dominant trait of purple coloring and a recessive trait of mellowness, and a wild type (i.e., normal type) for both traits. F
Suspension culture cell lines were established according to the method of Uj Imura et al. (1985), and protoplasts were isolated and purified from these cells. The two types of protoplasts obtained were adjusted to a density of 2XIO' cells/ml and mixed at a ratio of 1:1. This protoplast suspension was placed between the fusion electrodes and the following electrical stimulation was applied to perform fusion. That is; IMHz, 150V/c for 11.5 seconds; IMHz,
250V/cm, 0.1 seconds; 2500V/cn,
A voltage of 50 microseconds was applied between the electrodes in this order. After the fusion treatment, the cells were left at 4'C for 15 minutes, washed once with 0.4 M glucose solution, and cultured.

Colonies formed two weeks after culturing were allowed to proliferate for another two weeks and then transplanted to a differentiation medium to regenerate plants. These plants were further grown in a greenhouse and their traits were investigated. As a result, a plant with a normal height and a purple color was obtained. That is, the genetic trait for purple color is derived from 'Shidaikoku' and the genetic trait for normal plant height is derived from 'Norin 8tan', and the resulting plants are somatic cell hybrids.

Example 2 Seeds of two types of rice varieties "Old" and "D2" with different rice cultivars "Old" and "D2" were used as materials for Fuji imur
We established suspension culture cell lines according to the method of A et al. (1985), isolated and purified protoplasts from these cells, and
D1' protoplasts were treated with a 0.41 glucose solution supplemented with 30mM IOA at 25°C for 115 minutes.

These protoplasts and untreated D2° protoplasts were adjusted to a density of 2×10′ cells/ml and mixed at a ratio of 1:1. This protoplast suspension was placed between the fusion electrodes and the following electrical stimulation was applied to perform fusion. That is; IMHz, 150V/cm.

5 seconds; IMHz, 300V/cm, 0.1 seconds;
A voltage of 2500 V/Cm for 50 microseconds three times was applied between the electrodes in this order. 1 at 4°C after fusion treatment.
The cells were left for 5 minutes, washed once with 0.4-glucose solution, and cultured.

The cell mass formed by the protoplast division was propagated and differentiated by the method described in Example 1 to form a plant body.

When the traits of the plants were investigated, most of the regenerated plants were erect, but 2% had normal plant height. In other words, normal plant height is the result of complementation of the respective correcting genes, and the resulting plants are somatic cell hybrids.

Example 3 Suspension culture cell lines were established using seeds of rice varieties Norin No. 8 and Shidaikoku according to the method of Fuji imura et al. (1985), and protoplasts were isolated and purified from these cells and cultured. did. Among these cell lines, a suspension cultured cell line of Shidaikoku, which has high protoplast division activity and has lost the ability to regenerate plants, was used as one of the parent lines, and protoplasts were isolated and subjected to fusion. As a partner parent strain, protoplasts of Norin 8-Yo which had been treated with 20mM IO-8 for 30 minutes were used.

These two types of protoplasts were adjusted to a density of 2 x 10' cells/silicon and mixed at a ratio of 1:1. An equal volume of 30χPEG (average molecular ff 17,800 to 9,000) solution was added to this protoplast Hg5 solution, and after leaving it for 15 minutes, 0.4M grape #! Wash 3 times with solution and PE
G was removed. This protoplast was subjected to culture.

Colonies formed two weeks after culturing were allowed to proliferate for another two weeks and then transplanted to a regeneration medium to regenerate plants. Furthermore, these regenerated plants were grown in a greenhouse and their traits were investigated. As a result, all the regenerated plants had normal plant heights. Among them, 4% of the individuals exhibited purple color. These two genetic traits are derived from two different parents, and these individuals, which are purple in color and have normal plant height, are somatic hybrids.

<Effects of the Invention> The present invention has made it possible to easily and frequently produce somatic cell hybrids in rice, which had previously been considered impossible.

Patent applicant Mitsui Toatsu Chemical Co., Ltd.

Claims (1)

[Claims] 1) Cell fusion is performed using cells having identification marker genes expressed in plants as parent cells, and when plants are regenerated from the resulting fused cells, hybrid plants are selected using these markers. Method for producing rice somatic cell hybrid plants