JPH08172952A - Method for improving plant - Google Patents

Abstract

PURPOSE: To provide a method for improving a plant by which extranuclear genetic information about two or more desired cytoplasmic parents can efficiently be transduced without mixing the nuclear genetic information about unnecessary cytoplasmic parents. CONSTITUTION: This method for improving a plant is carried out by the protoplast fusion. The method is to simultaneously or successively bind a protoplast which is a nuclear parent having an inactivated cytoplasm prepared from a cell derived from a plant tissue without any redifferentiating ability to a karryorrhectic protoplast which is a cytoplasmic parent prepared from two or more other cells according to the cell fusion and prepare a plant simultaneously having the nuclear genetic information of the karryorrhectic parents and the extranuclear genetic information about the two or more kinds of cytoplasmic parents. Furthermore, the characteristic of this method comprises the plant tissue that is a young leaf.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a plant improving method by fusion of protoplasts, which comprises producing a plant having nuclear genetic information of a nuclear parent and extranuclear genetic information of two or more types of cytoplasmic parents at the same time. The present invention relates to a method for improving plants.

[0002]

2. Description of the Related Art For many years, most of genetic information of plants has been considered to be controlled by the nucleus, but recently, many important genetic information of cytoplasm, such as male sterility and male fertility,
It has been revealed that genetic information such as disease resistance, high photosynthetic ability, and environmental stress resistance exists. The genetic information of these cytoplasms (hereinafter referred to as extranuclear genetic information) exists in mitochondria and chloroplasts. Therefore, in order to utilize these extranuclear genetic information for plant improvement,
(1) Mating varieties and strains with the characteristics you want to introduce,
A cross breeding method in which the operation of selecting the subsequent generations is repeated, (2)
Methods such as the F1 hybrid breeding method, which creates a hybrid first generation having high productivity and superior properties due to hybrid vigor, are used.

[0003]

However, in the cross breeding method, a big problem is that it requires a long period of time, a large site, and much labor. Furthermore, it was difficult to efficiently introduce the desired extranuclear genetic information and stably express it, which was not a sufficiently satisfactory method. Also, F1
In the hybrid breeding method, in order to cross a maternal line and a paternal line, a method of manual stamen removal and mating, a method of utilizing self-incompatibility, a male sterility of nuclear control and a male sterility of cytoplasm control are used. Although the method of using is used, none of them is still satisfactory in terms of efficiency and labor. In particular, cytoplasmic male sterility, that is, Cytoplasmic Male Steril
rile, hereinafter referred to as CMS. ), A male fertile line having the same nucleus as that of the CMS line, that is, a male sterile line, is required for the maintenance / seed of the mother CMS line. This is a major obstacle because the backcrossing operation that is performed to create is time-consuming and labor-intensive.

[0004]

Under these circumstances, the inventors of the present invention have conducted extensive studies and as a result, as a result, protoplasts and cytoplasms, which are inactive cytoplasm as a nuclear parent and have no redifferentiation ability, have been found. By combining two or more nuclear-disruptive protoplasts as parents at the same time or sequentially by cell fusion, two or more desired cytoplasms can be obtained without mixing unnecessary nuclear cytoplasmic parental nuclear genetic information. The inventors have found that the extranuclear genetic information of the parent can be efficiently introduced, and completed the present invention. That is, the present invention is
A method for improving a plant by fusion of protoplasts, in which a protoplast, which is a nuclear parent with inactivated cytoplasm prepared from cells derived from plant tissue and having no redifferentiation ability, and a cytoplasmic parent prepared from two or more kinds of other cells are used. A certain nuclear destructive protoplast is simultaneously or sequentially combined by cell fusion to prepare a plant having the nuclear genetic information of the nuclear parent and the extranuclear genetic information of the two or more cytoplasmic parents at the same time. A plant improving method (hereinafter, referred to as a method of the present invention) and a plant improving method as described above, wherein the plant tissue is a young leaf, and a plant improving method by protoplast fusion, which is derived from a plant tissue. Prepared from protoplasts of cytoplasmic male sterility, a nuclear parent with inactivated cytoplasm and inability to redifferentiate prepared from cells and two or more other cells. The nuclear disruption cytoplasmic male fertile protoplasts, which are the cytoplasmic parents, are simultaneously or sequentially combined by cell fusion to simultaneously obtain the nuclear genetic information of the nuclear parent and the extranuclear genetic information of the two or more types of cytoplasmic parents. It is intended to provide a plant improving method characterized by producing a male sterile maintenance line and a plant improving method described above characterized in that the plant tissue is young leaves.

The present invention will be described in detail below. The method of the present invention can be applied to plant species capable of plant regeneration from protoplasts. For example, rice, wheat, barley, soybean, potato, corn, sorghum, sugar beet, sugar cane, rapeseed, panicum, clover, alfalfa, citrus, grape, tomato, eggplant, pepper, cabbage, Chinese cabbage, broccoli, asparagus, carrot, lettuce. , Chrysanthemum, tobacco, petunia and the like. Further, the method of the present invention is capable of cell fusion between closely related species and distantly related species in addition to cell fusion of plant species belonging to the same species.

The plant tissue used in the method of the present invention may be a plant tissue in which cells capable of easily leading to protoplasts are present, but preferably a plant having a high callus-inducing ability and a high regeneration ability from protoplasts. It is desirable to use tissue. Further, among the plants produced by the method of the present invention, in plant species that vary widely among individuals, for example, carrot species, etc., plant tissues that can be used in the present invention after investigating the characteristics and properties of the individual Is particularly effective. For example, when only one plant tissue per individual is used in the present invention, it becomes impossible for a plant body that has lost such plant tissue to sustain life,
Since there are problems such as the inability to preserve the plant body and the fact that a plant body having the properties required for breeding cannot be selected after being cultivated once, a plurality of plant tissues exist in one individual. Specifically, young leaves (growing point of side buds) can be mentioned.

The protoplast used in the method of the present invention is prepared, for example, as follows. First, cultured cells are prepared from cells derived from plant tissue via callus or somatic embryo. For example, various plant tissues are sterilized with antiformin or the like having an effective chlorine concentration of about 0.5 to 5%, and then thoroughly washed with sterile water. The washed plant tissue is washed with, for example, Mu
Plant hormones such as auxin and cytokinin were added to a medium such as MS medium described in rashige et al., Physiol. Plant, Volume 15, pages 473 to 497 (1962), and the mixture was solidified with a gelling agent such as agar. Place on a solid medium, 20 ~
Callus and somatic embryos are obtained by culturing at 30 ° C. The obtained callus or adventitious embryo is transplanted to a solid medium prepared by adding plant hormones such as auxin and cytokinin to a medium such as MS medium and hardening with a gelling agent such as agar, and thereafter about every 2 weeks to 2 months. Pass on. The callus or adventitious embryo thus passaged can be used as it is as a material for protoplast isolation, but it is desirable to use it once as a liquid culture cell and then as a material for protoplast isolation.
In that case, for example, transplanting to a medium such as MS liquid medium containing plant hormones such as auxin and cytokinin,
Shake culture is performed at -30 ° C and 60-120 rpm for about 2 weeks to 1 month. Then, once every two weeks, add 1 to medium of the same composition.
Subculture / 4 to 1/20 volume of culture. Liquid culture cells from passage 2 to 10 days are collected by a method such as centrifugation, cellulase, hemicellulase, in a sugar alcohol isotonic solution such as mannitol containing cell wall degrading enzymes such as pectinase,
A crude protoplast solution is obtained by treating at about 25 to 35 ° C. for about 2 to 6 hours. The crude protoplast solution is filtered through a mesh having a pore size of 20 to 100 μm to remove the cell wall residue. After that, for example, the protoplasts obtained by low-speed centrifugation are gently suspended in a sugar alcohol isotonic solution such as mannitol (hereinafter referred to as a washing solution), and then centrifuged again at low speed. The supernatant is removed, the washing solution is added to the collected protoplasts, the mixture is gently stirred, and the protoplasts are suspended again. Further, the same washing operation by centrifugation is repeated about 2 to 3 times to obtain purified protoplasts. When the plant species is carrot, protoplasts can be prepared, for example, as follows. After sterilizing plant tissues such as young leaves and roots of carrot with antiformin having an effective chlorine concentration of about 0.5 to 5% for about 5 to 30 minutes, they are thoroughly washed with sterile water. Washed plant tissue, for example, in MS medium 2.
Callus is obtained by adding about 4-D of about 0.1 to 2.0 mg / L, placing it on a solid medium solidified with agar, and culturing at 20 to 30 ° C. The obtained callus is subdivided, for example, 0.1-2.0 mg / l of 2.4-D is added to MS medium, transplanted to a solid medium solidified with agar, and then subcultured about every one month. The callus thus subcultured is subdivided again, and transplanted to, for example, MS liquid medium supplemented with 0.1-2.0 mg / l of 2.4-D.
Shake culture for 2 weeks to 1 month at -30 ° C and 60-120 rpm. Then, once every two weeks, add 1 / to the medium of the same composition.
Subculture 4 to 1/20 volume of culture. The liquid culture cells on the 2nd to 6th day of passage were collected by centrifugation, and a sugar alcohol solution (pH 4.5 to 6.0M) containing a cell wall-degrading enzyme such as cellulase, hemicellulase, pectinase or the like at a concentration of about 0.3 to 0.6M.
), Desirably in a mannitol solution of about 0.35 to 0.55 M, at about 25 to 35 ° C. and about 50 rpm for about 2 to 5 hours to obtain a crude protoplast solution. The crude protoplast solution is filtered through a mesh with a pore size of 20-40 μm to remove cell wall residue. Then, for example, the protoplasts obtained by centrifugation at 150 xg for 3 minutes are gently suspended in a sugar alcohol solution such as mannitol of about 0.3 to 0.6 M, preferably a mannitol solution (washing solution) of about 0.35 to 0.55 M, and suspended at 150 xg.
Centrifuge for 3 minutes at. The supernatant is removed, the washing solution is added to the collected protoplasts, the mixture is gently stirred, and the protoplasts are suspended again. Further, the same washing operation by centrifugation is repeated about 2 to 3 times to obtain purified protoplasts.

The cell fusion used in the present invention can be performed, for example, as follows.

First, a protoplast, which is a nuclear parent having an inactivated cytoplasm and incapable of redifferentiation obtained by the above-mentioned method (for example, (1) protoplasts are treated with a drug to partially inhibit metabolic pathways). Representative examples thereof include protoplasts that have lost redifferentiation ability, and (2) protoplasts that genetically lack regeneration ability. Among the above, protoplasts other than protoplasts genetically lacking in redifferentiation ability, for example, iodoacetic acid, iodo compounds such as iodoacetamide, preferably in a fusion solution containing about 5-40 mM iodoacetamide, about 2 ~ 30
About 5 to 30 minutes at ℃, preferably about 15 to 25 at about 2 to 3.5 ℃
Process for minutes. Then gently suspend with fusion solution, eg, centrifuge at 150xg for 3 minutes, remove the supernatant,
Add the fusion solution again and gently stir to suspend the protoplasts. Further, the same washing operation is repeated about 2 to 3 times to sufficiently remove the iodine compound to inactivate the cytoplasm, and thus protoplasts artificially losing the redifferentiation ability can be obtained. On the other hand, protoplasts genetically lacking the ability to redifferentiate can be used as they are for the cell fusion described below without performing the above-described treatment for inactivating the cytoplasm.

On the other hand, by irradiating the cytoplasmic protoplasts obtained by the above method with radiation such as soft X-rays, ultraviolet rays and γ rays, nuclear-destructive protoplasts can be obtained. As the type of radiation, soft X-rays are desirable because of their effectiveness depending on the wavelength and ease of handling. The irradiation is, for example, 0.35 containing about 0.1 mM calcium chloride.
A protoplast suspended in a 0.55 M mannitol solution (hereinafter referred to as a fusion solution) is spread on a thin layer having a height of about 0.1 mm, and then the soft X-ray irradiation device is used. The effective dose of soft X-ray is about 50 to 150 krad, but about 85 to 150 krad is particularly effective.
100krad is preferred. In this process, if irradiation is insufficient, the nucleus is not completely destroyed, and if irradiation is excessive, it also damages the cytoplasm, so it is not possible to obtain nuclear-destructive protoplasts. I won't.

The two species thus obtained (nuclear parent / cell
Protoplasts), for example, each with a fusion solution.
1 x 10⁶~ 1 x 10⁸Cells / ml, preferably about 0.5-2.0
× 10 ⁷Adjust to a cell concentration of cells / ml and mix equal amounts of both.
The combined solution is electrically fused. As an electric fusion condition
Is, for example, a frequency of about 0.1 to 2.0 MHz, an electric field strength of 10 to
200Vp-p / cm, pulsed following AC electric field for about 1 to 20 seconds
A DC power supply with a width of about 10 to 200 μs and an electric field strength of about 0.4 to 3 kV / cm.
Give Ruth and make fused cells. Desirably, the frequency is about 0.
4-0.6MHz, electric field strength approx. 80-120Vp-p / cm, time approx. 5-
AC electric field for 15 seconds and pulse width about 30-80 μs, strong electric field
It is a DC pulse of about 0.75 to 1.25 kV / cm. AC electric field
Is adjusted to the condition that several protoplasts are arranged in a row.
However, the DC pulse has the highest probability of fusion of protoplasts.
Also choose to be high. These conditions depend on the plant species, variety and
Since it depends on the condition of the cell, etc.
Increasing the production efficiency of fused cells by selecting
Can be. In addition, compounds such as polyethylene glycol
The cell fusion used is also possible. The above-mentioned fused cell obtained
, Sucrose and sugar alcohol, auxin and
MS medium containing plant hormones such as cytokinin
Suspend in medium. Cell density when suspended is approximately 1 x 10³~ 1
× 10⁷Cells / ml, preferably about 1 x 10^Four~ 1 x 10⁶cell
/ ml, and cultivate at about 20-30 ° C for about 3-6 weeks.
Feed. Nuclear destruction treatment such as soft X-ray treatment and iodine compound treatment
If cytoplasmic inactivation treatment such as
Single non-fused cells or same cells (nuclear parent and nuclear parent,
Fusion cells of cytosolic and cytoplasmic parents) cannot divide and
Selectively obtaining only fusion cells of (nuclear parent and cytoplasmic parent)
As a result, the nuclear genetic information of the nuclear parent and the cytoplasm
Obtaining protoplasts that simultaneously carry the extranuclear genetic information of the parent
be able to. Of course, cytoplasm such as iodine compound treatment
Instead of inactivated protoplasts, genetically
Using protoplasts lacking regeneration potential as nuclear parents
If so, only fusion cells of the nuclear parent and the cytoplasmic parent are selected as described above.
Can be obtained as an alternative.

The obtained protoplasts are further added to about 3 to 8
By culturing for a week, a cell mass that is visible to the naked eye is formed. Then, the obtained fused cell-derived cell mass is transplanted to a medium such as MS liquid medium to which plant hormones such as auxin and cytokinin are added, and the cell mass is about 20 to
Shake culture is performed at 30 ° C. and about 60 to 120 rpm for about 2 weeks to 1 month. Then, about 1/4 to 1/20 of the culture is subcultured to the medium having the same composition about once every two weeks. Protoplasts prepared in the same manner as described above are isolated from the thus obtained cultured cells and used as the nuclear parent. The obtained nuclear parent protoplasts are subjected to cytoplasmic inactivation treatment similar to the above to obtain again protoplasts artificially losing the redifferentiation ability. On the other hand, nuclear destructive protoplasts are obtained from cells other than the cells used above by the same method as described above. And these two [nuclear parent (fused cell origin)
Other cytoplasmic parents] are fused by the same method as described above. The obtained fused cells are suspended in a medium such as MS medium containing sucrose, sugar alcohol, and plant hormones such as auxin and cytokinin. The cell density in suspension is adjusted to about 1 × 10 ³ to 1 × 10 ⁷ cells / ml, preferably about 1 × 10 ⁴ to 1 × 10 ⁶ cells / ml, and the cell density at about 20 to 30 ° C. Incubate for 3-6 weeks. As a result, a protoplast having the nuclear genetic information of the nuclear parent and the extranuclear genetic information of the two types of cytoplasmic parents at the same time can be obtained.

Further, the obtained fused cells, protoplasts, are used as a nuclear parent, and the nuclear-destructive protoplasts prepared by the same method as above from other cells than the cells used above are used as cytoplasmic parents. By repeating cell fusion in the same manner as described above, protoplasts having the nuclear genetic information of the nuclear parent and the extranuclear genetic information of three or more types of cytoplasmic parents at the same time can be obtained.

Protoplasts having the nuclear genetic information of the nuclear parent thus obtained and the extranuclear genetic information of two or more types of cytoplasmic parents at the same time are, for example, about 0.2 to 0.7 M of sugar alcohol such as sorbitol and about 1 Cell density of about 1 × 10 ⁴ to 1 × 10 ⁶ cells / ml, preferably about 1.0 × 10 ^{4 in a} medium such as MS medium containing -10% sucrose, glucose and plant hormones.
Suspend at ~ 1.0 x 106 cells / ml, about 20-30
After culturing at ℃ for about 3 to 8 weeks, protoplasts form macroscopic cell clusters. The cell mass is then transplanted to an adventitious embryo formation medium or a regeneration medium to obtain a regenerated plant.
As described above, it is possible to create a plant having the nuclear genetic information of the nuclear parent and the two or more types of extranuclear genetic information of the cytoplasmic parent at the same time.

When the plant species is carrot, for example, a plant can be prepared from protoplasts having the extranuclear genetic information of two or more cytoplasmic parents at the same time as follows. Protoplasts with about 0.2-0.5 M, preferably about 0.2-0.4 M sorbitol and about 1.5-4%, preferably about 2-3.5%.
Sucrose and about 0-2.0 mg / l, preferably about 0.05-0.5 mg / l
Cell densities of about 1 × 10 ⁴ to 1 × in MS medium containing 2.4-D
When the cells are suspended at 10 ⁷ cells / ml, preferably about 1.0 × 10 ^{5 to} 1.0 × 10 ⁶ cells / ml, and cultured at about 20 to 30 ° C for about 3 to 6 weeks, protoplasts can be obtained in about 3 weeks. , Forming a cell mass that is visible to the naked eye. Cell mass then about 1.5
~ 4%, preferably about 2-3.5% sucrose and about 0-1.0 m
After transplanting to MS medium containing 2.4-D of g / l, preferably about 0.05-0.5 mg / l and growing for about 2 weeks, about 1.5-4
%, Preferably about 2-3.5% sucrose and a lower concentration of 2.4-D than in the preculture medium, and transplanted to MS medium to form somatic embryos. The obtained somatic embryo is transplanted to a solid MS medium solidified with agar or the like to be redifferentiated. This process is carried out at about 20 to 30 ° C., preferably about 22 to 28 ° C. and about 4 to 24 hours under sunshine conditions. Regenerated plants grow to a height of about 10 cm in about 1 to 3 months.
After that, the soil is acclimated to about 15 to 30 ℃, preferably the daytime temperature.
After cultivating under conditions of 18 to 25 ° C and night temperature of about 18 to 22 ° C for about 3 months, low temperature treatment is performed at about 2 to 10 ° C, preferably about 3 to 6 ° C for about 2 months, and the lunch temperature is about 18 again. By cultivating at -25 ° C and a night temperature of about 18-22 ° C, it is possible to bolt and flower.

In the method of the present invention, protoplasts which are inactivated cytoplasm prepared from cells derived from plant tissue and have no redifferentiation ability, and nuclei which are cytoplasmic parents prepared from two or more kinds of other cells. As a method for linking destructive protoplasts by cell fusion, it is possible to introduce extranuclear genetic information one by one by sequentially linking them as described above, but it also has an inactivated cytoplasm and redifferentiation. Introducing two or more types of extranuclear genetic information at one time by simultaneously mixing inactive nuclear parent protoplasts and cytoplasmic parent nuclear-destructive protoplasts prepared from two or more types of other cells and fusing them You can also In this case, a mixture of protoplasts that are the nuclear parent and one type of cytoplasmic parent, protoplasts, and a mixture of the protoplasts that are the nuclear parent and two or more types of protoplasts, which are the cytoplasmic parents, are mixed. Therefore, it is necessary to select a cell fusion of the protoplast, which is the nuclear parent, and the protoplasts, which are two or more types of cytoplasmic parents, by investigating the properties of the regenerated plant.

Utilizing the method of the present invention, for example, cell fusion of protoplasts prepared from cells derived from tissues of cytoplasmic male sterile plants and protoplasts of nuclear-disrupting cytoplasmic male sterile plants prepared from two or more kinds of other cells. The male sterility maintaining line can be created by combining them with each other. CMS can be easily obtained by using the male sterile line.
Will be able to maintain / seed. Further, the obtained male sterile maintenance line can be used as a parent of F1 hybrid. In addition, extranuclear genetic information, for example, resistance to various diseases caused by infection with filamentous fungi, bacteria, viruses, high photosynthetic bright reaction ability under normal conditions, high CO ₂ fixing ability in dark reaction, strong light. high photosynthetic capacity under, or high photosynthetic capacity under low CO ₂ concentration, lack of water, excess water, low temperatures, high temperature, salt, the properties such as resistance to various environmental stresses such as metal is introduced efficiently into plant Is also possible. For example, in the case of making disease resistant plants,
Each protoplast is prepared from the plant cultivar A susceptible to disease and the plant cultivars B and C having different disease resistance controlled by cytoplasm by the method according to the above description or Example 1 described later. Protoplasts prepared from cells of plant variety A (ie, protoplasts that are the nuclear parent) and protoplasts prepared from cells of plant variety B (ie, protoplasts that are the cytoplasmic parent) were according to the above description or Example 2 below. The cells are combined by a cell fusion method to prepare a fused cell having the nucleus of plant variety A (ie, nuclear genetic information of nuclear parent) and the cytoplasm of plant variety B (ie, extranuclear genetic information of cytoplasmic parent) at the same time. Next, the protoplasts prepared from the fused cells (that is, the protoplasts which are the nuclear parent) and the protoplasts prepared from the cells of the plant variety C (that is, the protoplasts which are the cytoplasmic parent) were subjected to the above description or Example 2 described later. Having the nucleus of plant variety A (ie, nuclear genetic information of nuclear parent) and the cytoplasm of plant breeds B and C (ie, extranuclear genetic information of two or more cytoplasmic parents) at the same time by combining by cell fusion method Create fused cells. By culturing, redifferentiating, acclimatizing, and growing the fused cells, it is possible to grow disease-resistant plants having two different types of disease resistance controlled by the cytoplasm. That is, it is possible to exemplify that two types of disease resistance possessed by the plant variety B and the plant variety C can be imparted to the plant variety A at the same time without changing the nuclear genetic information of the plant variety A. Further, in the case of producing a plant having a high photosynthetic ability, the plant cultivar D having a low photosynthetic ability and the plant varieties E and F having different high photosynthetic abilities controlled by the cytoplasm were subjected to the above description or Example 1 described later. Each protoplast is prepared by the method. Protoplasts prepared from cells of plant variety D (ie, protoplasts that are the nuclear parent) and protoplasts prepared from cells of plant variety E (ie, protoplasts that are the cytoplasmic parent) were as described above or in Example 2 below. The cells are combined by a cell fusion method to prepare a fused cell having the nucleus of the plant variety D (that is, the nuclear genetic information of the nuclear parent) and the cytoplasm of the plant variety E (that is, the extranuclear genetic information of the cytoplasmic parent) at the same time. Next, the protoplasts prepared from the fused cells (that is, the protoplasts that are the nuclear parent) and the protoplasts prepared from the cells of the plant variety F (that is, the protoplasts that are the cytoplasmic parent) were subjected to the above description or Example 2 described later. Having the nucleus of plant variety D (ie, nuclear genetic information of the nuclear parent) and the cytoplasm of plant varieties E and F (ie, extranuclear genetic information of two or more cytoplasmic parents) at the same time by combining by the method of cell fusion Create fused cells. By culturing, redifferentiating, acclimatizing and growing the fused cells, it is possible to grow high photosynthetic plants having two different photosynthetic abilities controlled by the cytoplasm. That is, it is possible to exemplify that two types of high photosynthetic ability of the plant variety B and the plant variety C can be simultaneously given to the plant variety C without changing the nuclear genetic information of the plant variety A. Further, in the case of producing plants having environmental stress resistance, plant varieties G vulnerable to a certain environmental stress and plant varieties H and I having different environmental stress resistance controlled by cytoplasm are used in the above description or Example 1 below. Prepare each protoplast by the method according to. Protoplasts prepared from cells of plant variety G (ie, protoplasts that are the nuclear parent) and protoplasts prepared from cells of plant variety H (ie, protoplasts that are the cytoplasmic parent) were according to the above description or Example 2 below. The cells are combined by a cell fusion method to prepare a fused cell having the nucleus of the plant variety G (that is, the nuclear genetic information of the nuclear parent) and the cytoplasm of the plant variety H (that is, the extranuclear genetic information of the cytoplasmic parent) at the same time. Next, the protoplasts prepared from the fused cells (that is, the protoplasts that are the nuclear parent) and the protoplasts that are prepared from the cells of the plant variety I (that is, the protoplasts that are the cytoplasmic parent) were subjected to the above description or Example 2 described later. Having the nucleus of plant variety G (that is, the nuclear genetic information of the nuclear parent) and the cytoplasm of plant varieties H and I (that is, the extranuclear genetic information of two or more cytoplasmic parents) at the same time by combining by the method of cell fusion Create fused cells. By culturing, redifferentiating, acclimatizing, and growing the fused cells, it is possible to grow an environmental stress-resistant plant having two different types of environmental stress resistance controlled by the cytoplasm.
That is, it is possible to exemplify that the two environmental stress resistances of the plant variety H and the plant variety I can be simultaneously imparted to the plant variety G without changing the nuclear genetic information of the plant variety G. Hereinafter, examples will be described in more detail, but the present invention is not limited to these examples.

【Example】

Example 1 (Preparation of Protoplasts) CMS carrot individuals were obtained by cultivating and flowering the commercial and varieties of the present generation and progeny, and selecting individuals in which all flowers were male sterile. MS-1 which is one of the CMS carrot individuals thus obtained was used as a nuclear parent. M
Young leaves of S-1 were treated with antiformin with an effective chlorine concentration of 1%.
After sterilizing for 20 minutes, it was washed with sterile water 3 to 4 times. The washed young leaves were treated with 1 mg / l of 2.4-D (2,4-dichlorophenoxyaceti
acid), 0.8% agar and 3% sucrose were placed on an MS solid medium and cultured at 25 ° C. in the dark for 1 to 2 months to form callus. Carrot varieties as cytoplasmic parents, "Kokubun Kokucho Daicho" and "Koizumi Ideal Five Sun"
Was used. Callus was formed from both young leaves in the same manner as above. Each of the obtained callus was subdivided, and each was transplanted to MS solid medium containing 0.5 mg / l of 2.4-D, 0.8% agar and 2% sucrose, and subcultured about every 1 month thereafter. Cultured. The callus subcultured in this way is subdivided,
The cells were transferred to an MS liquid medium containing 0.5 mg / l 2.4-D and 2% sucrose, and cultured with shaking at 25 ° C. and 100 rpm for 3 weeks. Then, once every two weeks, about 1/20 of the shaking culture was subcultured in a medium having the same composition to prepare liquid cultured cells. Approximately 1 to 2 months after the start of liquid culture, the liquid culture cells on the 3rd day of subculture were collected by centrifugation at 3000 rpm for 10 minutes, and the collected liquid culture cells were supplemented with 1% of dolicerase (Kyowa Hakko Kogyo Co., Ltd.). 0.5% Cellulase Onozuka RS (Yakult Honsha), 0.01% Pectolyase Y
-23 (manufactured by Seishin Pharmaceutical Co., Ltd.), 0.1% MES [2- (N-morphol
ino) ethanesulfonic acid] and 0.5M mannitol in an enzyme solution (pH 5.7) at 30 ° C and 50 rpm in the dark.
A crude protoplast solution was obtained by performing time treatment. The crude protoplast solution was filtered through a stainless mesh having a pore size of 32 μm to remove the cell wall residue. 150xg,
The protoplasts were precipitated by centrifugation for 3 minutes and the supernatant was removed with a pipette. The obtained protoplasts
The suspension was gently suspended in a wash solution of 0.5 M mannitol (pH 5.7), centrifuged at 150 xg for 3 minutes, and the supernatant was removed with a pipette. Next, a 0.5 M mannitol (pH 5.7) washing solution was added and gently stirred to suspend the protoplasts. Further, the same purification operation by centrifugation was repeated twice to obtain each purified protoplast.

Example 2 (Preparation of fused cells and improved plants) The protoplasts of "Kokubu Benin Daicho" obtained in Example 1 were suspended in a fusion solution consisting of 0.1 mM calcium chloride and 0.5 M mannitol. After being spread in a thin layer having a height of 0.1 mm, it was irradiated with soft X-ray of 85 krad. On the other hand, the MS-1 protoplasts obtained in Example 1 were suspended in the above fusion solution containing 15 mM iodoacetamide, treated at 3 ° C. for 20 minutes, then centrifuged at 150 × g for 3 minutes, and pipetted. The supernatant was removed with. The above fusion solution was added again and the mixture was gently stirred to suspend the protoplasts. Further, the same purification operation by centrifugation was repeated twice. The obtained two protoplasts were each treated with the above fusion solution at 1 × 10 ⁷ cells / m 2.
After adjusting the cell concentration to l, both were mixed at 0.4 ml. This mixed solution was put into a fusion chamber (FTC-03) of an electric cell fusion device (Shimadzu Corporation, Shimazu cell fusion device SSH-1), frequency 1.0 MHz, electric field strength 10
Fused cells were prepared under the conditions of an AC electric field of 0 Vp-p / cm for 10 seconds, followed by a DC pulse having a pulse width of 50 μs and an electric field strength of 1.0 kV / cm. The fused cells thus obtained were suspended in ⁵ ml of MS medium (pH 5.7) containing 0.3 M sorbitol, 0.1 mg / l 2.4-D and 2% sucrose at 2 × 10 ⁵ cells / ml. The cells were cultured at 25 ° C in the dark at 50 rpm. About 1 month after culturing, a cell mass having a diameter of about 0.5 mm was formed. The obtained cell mass was subdivided, transferred to an MS liquid medium containing 0.5 mg / l 2.4-D and 2% sucrose, and cultured with shaking at 25 ° C. and 100 rpm for 3 weeks. Then, once every two weeks, about 1/20 of the shaking culture was subcultured in a medium having the same composition to prepare liquid cultured cells. Approximately 1 to 2 months after the start of liquid culture, the liquid culture cells on the 3rd day after passage were collected by centrifugation at 3000 rpm for 10 minutes, and the collected liquid culture cells were collected at 1%.
Doriserase (manufactured by Kyowa Hakko Kogyo Co., Ltd.), 0.5% cellulase Onozuka RS (manufactured by Yakult Headquarters), 0.01% pectriase Y-23 (manufactured by Seishin Pharmaceutical Co., Ltd.), 0.1% MES
In an enzyme solution (pH 5.7) containing [2- (N-morpholino) ethanesulfonic acid] and 0.5M mannitol, 30 ℃, 50rp
A crude protoplast solution was obtained by performing the treatment under a dark condition of m 3 for 3 hours. This crude protoplast solution has a pore size of 32 μm.
The residue was filtered through a stainless steel mesh to remove the cell wall residue. The protoplasts were pelleted by centrifugation at 150 xg for 3 minutes and the supernatant removed with a pipette. The obtained protoplasts were gently suspended in a wash solution of 0.5 M mannitol (pH 5.7), centrifuged at 150 xg for 3 minutes, and the supernatant was removed with a pipette. The above washing solution was again added and gently stirred to suspend the protoplasts. Further, the same purification operation by centrifugation was repeated twice to obtain a purified protoplast of the fused cells. The purified protoplasts obtained were suspended in the above fusion solution containing 15 mM iodoacetamide and treated at 3 ° C. for 20 minutes, then 150 ×
g, centrifuged for 3 minutes, and the supernatant removed with a pipette. The above fusion solution was added again and the mixture was gently stirred to suspend the protoplasts. Further, the same purification operation by centrifugation was repeated twice. On the other hand, the "Koizumi ideal five dimensions" protoplast obtained in Example 1 was suspended in a fusion solution consisting of 0.1 mM calcium chloride and 0.5 M mannitol, and spread on a thin layer having a height of 0.1 mm. , 85 krad of soft X-ray. The two protoplasts thus obtained were each adjusted to a cell concentration of 1 × 10 ⁷ cells / ml with the above fusion solution.
Both were mixed by 0.4 ml. This mixed solution was put into a fusion chamber (FTC-03) of an electric cell fusion device (Shimadzu Corporation, Shimazu cell fusion device SSH-1), and the frequency was set.
Fused cells were prepared under the conditions of an alternating electric field of 1.0 MHz, electric field strength of 100 Vp-p / cm, and 10 seconds, followed by a DC pulse having a pulse width of 50 μs and an electric field strength of 1.0 kV / cm. The fused cells obtained were treated with 0.3 M sorbitol, 0.1 mg / l 2.4-D and 2%.
2 × 10 ⁵ cells in ⁵ ml of MS medium (pH 5.7) containing sucrose
The cells were suspended at a concentration of / ml and cultured at 25 ° C. in the dark at 50 rpm. About 1 month after culturing, a cell mass having a diameter of about 0.5 mm was formed. After subdividing the obtained cell mass, 0.1 mg / l of 2.4
It was diluted to 1/100 with an MS liquid medium (pH 5.7) containing -D and 2% sucrose, and this was cultivated with shaking at 25 ° C and 100 rpm for 2 weeks. Further, this culture was transplanted to an MS liquid medium (pH 5.7) containing 2% sucrose and shake-cultured at 25 ° C. and 100 rpm for 1 month to obtain an adventitious embryo (torpedo-shaped embryo). Obtained somatic embryos, 0.8% agar and 3%
The cells were transferred to an MS medium (pH 5.7) containing sucrose and the plant cells were redifferentiated. The regenerated plants were grown to a plant height of about 10 cm and acclimated to pots filled with soil. Acclimated plants,
After cultivating for about 3 months under the conditions of day temperature 20 ℃, night temperature 18 ℃ (14 hours during daytime, 10 hours at night), low temperature treatment is performed for 4 ℃ for 2 months, 25 ℃ for daytime, 20 ℃ for nighttime It was cultivated and flowered for about 2 months under the conditions of (14 hours in the daytime and 10 hours in the night). As a result, all 16 evaluated individuals were male fertile individuals having normal pollen. As a result of investigating the properties of the male fertile individuals obtained, two types of extranuclear inheritance, nuclear genetic information of the nuclear parent (MS-1) and cytoplasmic parent ("Kokubun-Kurei Taicho" and "Koizumi ideal five dimensions") Information, that is, the property that nuclear DNA components have the same agronomic traits as MS-1 and MS-1,
And the components of mitochondrial DNA have both "Kokubun Sengoku Daicho" and "Koizumi Ideal Five Sun" components, and have the same male fertility as "Kokubun Fresh Bentai" and "Koizumi Ideal Five Sun". It was confirmed to have both at the same time.

Example 3 (Maintenance of MS-1) MS-1 and the male fertile individual obtained in Example 2 were crossed with bees or flies or manually to give MS-1.
Seeds can be obtained. Since the nuclear genetic information of the male fertile individual obtained in Example 2 and the nuclear genetic information of MS-1 are exactly the same, it is possible to maintain MS-1 by crossing. From the above, the produced plant can be used as a male sterility maintenance line of MS-1.

[0021]

EFFECTS OF THE INVENTION According to the present invention, desired extranuclear genetic information of two or more types of cytoplasmic parents can be efficiently introduced without mixing unnecessary nuclear genetic information of cytoplasmic parents.

Claims (4)

[Claims] 1. A method for improving a plant by fusion of protoplasts, which comprises a protoplast, which is a nuclear parent having an inactivated cytoplasm and incapable of redifferentiation prepared from cells derived from plant tissue, and two or more other cells. A plant having the nuclear genetic information of the nuclear parent and the extranuclear genetic information of the two or more types of cytoplasmic parents at the same time by combining the prepared cytoplasmic nuclear destructive protoplasts simultaneously or sequentially by cell fusion. A method for improving a plant, which comprises: 2. The method for improving plants according to claim 1, wherein the plant tissue is young leaves. 3. A method for improving plants by fusion of protoplasts, which comprises two or more types of protoplasts of cytoplasmic male sterility, which are nuclear parents having inactivated cytoplasm prepared from cells derived from plant tissues and having no redifferentiation ability. The cytogenetic cytoplasmic male-sterile protoplasts, which are cytoplasmic parents prepared from other cells, are combined simultaneously or sequentially by cell fusion, and the nuclear genetic information of the nuclear parent and the two or more types of cytoplasmic parents. A method for improving plants, which is characterized in that a male sterility maintenance line having at the same time extra-nuclear genetic information as described above is created. 4. The method for improving plants according to claim 3, wherein the plant tissue is young leaves.