JPS6265680A - Breeding of gramineous crop by protoplast cultivation - Google Patents

Abstract

PURPOSE:To enable the conservation of the redifferentiation capability and genetic characteristics of cultured cell, by culturing a culture cell separated from a gramineous crop in a medium having high salt or sugar concentration. CONSTITUTION:It has been found that the presence of salt or sugar having high concentration in addition to auxin in a medium is essential for the induction of callus or the subculture of the induced callus while maintaining the redifferentiation capability and the genetic stability of the culture cell. The salt is sodium, potassium, etc., and the amount is preferably about 3-20g per 1l of the medium in the case of sodium chloride. The sugar to be added to the medium is e.g. mannitol, sorbitol, dextrin, etc., and its content in the medium is preferably about 0.3-0.6M.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for restoring and growing a plant of bacterial cell origin by producing and culturing protoplasts of gramineous grains.

There are the following methods or principles for creating new varieties.

(1) By changing the combination of specific traits through cross-breeding, new genetic traits are introduced and mutations occur.

(2) Variation in traits is caused by an increase or decrease in the number of chromosomes, as seen in polyploids.

(3) Cause mutations by chemical substances, radiation, etc.

(4> Introducing a heterologous gene using recombinant DNA.

(5) New traits are imparted by incorporating heterologous cytoplasm or genes through cell fusion.

Therefore, growing plants from protoplasts obtained by removing the cell walls of plants is an essential technique for producing mutants, selecting them, introducing heterologous genes, and breeding by cell fusion.

Production of protoplasts has been known for a long time, and a production method using enzymes (Takabe et al., 1968) has been extensively studied, and as a result, it has become possible to isolate protoplasts from a wide variety of plants.

For example, in tobacco, it has become possible to culture isolated protoplasts and restore them to the plant (Takabe
et al., 1971). It has also become possible to restore the plant body by fusing protoplasts of two separated plant species and then culturing them (Carlson et al., 1972).

However, in monocots, it has been extremely difficult to isolate and culture protoplasts.

In grasses other than rice, only two cases of restoration of plant bodies from protoplasts have been reported (1981). Although we succeeded in restoring the plant body, any callus originating from pollen or seeds
Restoration of plants from I-weave has not been successful.

In addition, in rice, protoplast culture was not successful for a long time, and only slight callus production and rooting were observed (1'1eka and Sen, 1
976).

In view of the above-mentioned state of the art, the present inventors investigated the influence of culture conditions, medium components, etc. on the regeneration ability and genetic characteristics of cultured cells in mori culture of rice, and investigated the effects of culture conditions, medium components, etc. on the regeneration ability and genetic characteristics of cultured cells in rice mori culture, and As a result of detailed studies on regeneration methods, we succeeded in subculturing callus stably for a long period of time and restoring rice plants from callus originating from pollen or seeds, which may or may not be subcultured. This is the first time for a grass grain that we have been able to restore a plant from callus originating from pollen or seeds, and we were also able to subculture seed callus over a long period of over four years and then restore the plant from this callus. Of course, this is also my first knowledge.

By the method developed by the present inventors and detailed below, various mutagenesis operations, transfer of heterologous genes, protoplast fusion between heterologous plants, and combinations of these operations are performed during the culture period of callus cells. It is now possible to easily select cells based on the selected cells.

According to the method of the present invention, in order to induce callus and subculture the induced callus while maintaining the redifferentiation ability and genetic stability of cultured cells, in addition to auxin in the medium, The presence of high concentrations of salt or sugar is essential. Examples of such salts include chlorides of sodium, potassium, calcium, magnesium, manganese, etc., and sulfates. These salts may be used alone,
Moreover, two or more types may be blended. For example, in the case of salt, the content is 2 to 30 g per 1ρ of the medium, preferably 3 to 30 g.
It is about 20g.

On the other hand, the sugar contained in the medium together with the salt may be, for example, mannitol, sorbitol, dextrin, etc., and these may also be used alone or in combination. The sugar content in the medium is 0.2-1.0M, preferably 0.3-0.0M. That's just what I heard.

The callus cultured in this manner is differentiated into plants by transferring it from the above medium to a regeneration medium. The regeneration medium has a composition normally known for that purpose, e.g. plant hormones such as auxin, cytokinin and sugars, yeast extract,
It contains amino acids such as casamino acids and proline, and in some cases may also contain plant hormones such as abscisic acid and gibberellin.

In the present invention, the plant material used as the material may be any part, such as leaves, seeds, pollen, stems, roots, etc., but pollen and seeds are particularly suitable. The callus obtained by culturing protoplasts separated from the plant body may be immediately redifferentiated, or the callus may be subcultured under the above conditions and removed at any time and subjected to redifferentiation treatment. Good too.

Furthermore, fused protoplasts obtained by fusing protoplasts of rice with protoplasts of other grasses can also be subcultured according to the method of the present invention and differentiated as necessary, and therefore, the present invention is applicable to the present invention. Included.

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

Example 1 Pollen-derived callus was induced by liquid suspension culture of rotten rice (variety: Nipponbare), and 80 single pollen-derived callus derived from one panicle were isolated. After growing each of these calli, their total DNA was extracted and cut with the restriction enzyme Bam.
Ern blotting) was performed to form a hybrid with phosphorylated rice liposome gene DNA.

In 3 out of 50 calli, in addition to the two original 3.8 to 3.9 kh hosomal DNA hunts, a large molecular weight hunt around 7.7 kb was found. Including such analysis of cells? Protoplasts were isolated from one callus with a stable 1' length and cultured.

The enzyme conditions were Mase [2 Team Old 00.5%, Pectolyase Y-230.04%, Cellulase RS 2%, Mannitol 0.45M, and the medium was Chu → Naphthalene Sodium Acetate 10-5M + Kinetin 10-
It was 6M.

From colonies obtained from three independent isolations and cultures, 4
One piece was isolated and subjected to callus growth.

Of the total 41 calluses obtained, 9 calluses had different color tones, and 15 calluses required auxin 3F. There was a large difference in the growth of each colony, with the fresh weight per plate ranging from 1.5-2g to 4.5-2g.
It was distributed up to 5g. A part of the callus was placed in differentiation medium (
Ohno, 1975) and examined the presence or absence of redifferentiation ability. One callus differentiated into buds and roots, and two calluses showed differentiation of roots, confirming redifferentiation and restoration.

The protoplast callus used for differentiation here was
Eight subcultures have been carried out since callus induction, and approximately 15 months have passed including the protoplast isolation and culture period.

This indicates that it is possible to maintain regeneration ability in long-term cultured calli even in rice.

Example 2 Seeds carefully collected from rice (variety: Nipponbare) by single breeding were demixed and used. Seeds in 70% ethanol
The sample was disinfected for 1 minute, then sterilized by immersion in a supernatant solution of a 10% bleached powder aqueous solution for 20 minutes, and then washed with sterile water. Then add the seeds
The cells were placed on a pI 16.0 agar medium prepared by adding 10 −5 M 2,4-dichlorophenoxyacetic acid to the basal medium of Urashige and Skoog (1962) or the basal medium of Miller (1963).

After 40 to 50 days, the induced callus was treated with 5 to 18 g of table salt.
The cells were cultured in a dark blue medium for 4 years in a medium containing /l.

Such callus was treated with 32% Cellulase R, 5% Pectolyase Y-230, 0.05% Mannitol, 0.2M Sorbitol, 3mM Calcilla J chloride, 0.35mM potassium dihydrogen phosphate, and MFS lli÷i.
The cells were treated with 3mM of the solution, and the protoplast 1 released was selected using a stainless steel mesh, washed with a mixture of nitol and sorbitol, and then cultured. The protoplasts separated in this way undergo division after 10 days, and when they become a sufficiently large mass of callus, they are placed in the Murash
ige and Skoog basal medium with 2+4-
The cells were grown in medium containing 10-'M dichlorophenoxyacetic acid.

2,4-dichlorophenoxyacetic acid was removed from the callus thus produced, and then transplanted to a differentiation medium supplemented with naphthaleneacetic acid, hensyl adenine, zeatin, and yeast extract, and after 20 to 40 days, plants were reestablished. I got a body.

Claims (2)

[Claims] (1) A method for maintaining the redifferentiation ability and genetic characteristics of cultured cells, which comprises culturing cells isolated and cultured from grass grains in a medium with a high salt or sugar concentration. (2) Restoration, which is characterized by continuously culturing protoplasts isolated and cultured from grass grains, and culturing the obtained cultured cells in a differentiation medium containing plant hormones.
Cultivation method.