JPH08163936A - Zoysia plant transformant and preparation thereof - Google Patents

Abstract

PURPOSE: To efficiently create an antibiotic-resistant plant by introducing an antibiotic-resistant gene into a Zoysia plant. CONSTITUTION: The protoplast of a Zoysia plant to be transformed and a plasmid containing an antibiotic-resistant gene are suspended and the palsmid is introduced into the protoplast by the polyethylene glycol treatment or electroporation. Then, cell aggregate is formed, adventitious bud or embryo is formed from the aggregate and the transformant of the Zoysia plant is regenerated from the adventitious bud or embryo. In the introduction according to the particle gun technique, the antibiotic-resistant gene is shot together with metallic particles to introduce the gene into the cell aggregate and the transformant of the Zoysia plant is regenerated.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a Zoisia genus plant transformant and a method for producing the same, which efficiently introduces an antibiotic resistance gene into a Zoisia plant, and frequently produces an antibiotic resistance Zoisia plant and the same. It produces protoplasts, colonies, calli, somatic embryos, and somatic buds. INDUSTRIAL APPLICABILITY The present invention is extremely effective in creating a new variety of Zoisia plants.

[0002]

2. Description of the Prior Art Plants of the genus Zoisia are widely used in parks, playgrounds, golf courses and the like, and attempts have been made to utilize gene recombination for imparting disease resistance or insect resistance. The technology of gene recombination has emerged as a method for modifying the genetic traits of plants. Although only a few years have passed since its introduction, the rate of progress and spread has been remarkable, and many transformants have already been obtained in plants such as rice, tobacco and tomato.

As a method of gene recombination and introduction of a foreign gene into plants, there is a method of utilizing Agrobacterium, which is one of soil bacteria used for tobacco, tomato and the like.
However, since this genus plant is less susceptible to Agrobacterium infection, this method cannot be used.

Therefore, it is necessary to introduce a foreign gene directly into cells, not by such indirect means. In particular, the introduction method (polyethylene glycol method, electroporation method, etc.) in which a foreign gene is introduced into a protoplast prepared from a cell aggregate having redifferentiation ability and redifferentiated into a plant body treats a large amount of cells at one time. In addition, since it is possible to introduce into a single cell, the regenerated plant does not become a so-called chimera (a phenomenon in which the gene-introduced cell is localized) that can occur when it is introduced into a multi-cell, and This is an effective means because it becomes a transformant.

[0005]

However, this method of introduction has problems in the efficiency of gene introduction, the efficiency of regeneration from the introduced cells, and the reliability of the method of introduction. Further, this introduction method has a problem that it is indispensable to establish a culture method for protoplasts or a method for regenerating a plant from protoplasts. Recently, an introduction method has been developed in which metal particles having a gene to be introduced adsorbed into a cell clump are implanted. Also in this method called the particle gun method, induction of cell clumps that are frequently redifferentiated is also essential, and there are many conditions to be solved including the above-mentioned problem of chimera.

That is, although the plant of the genus belongs to the family Gramineae, its characteristics are quite different from those of other plants belonging to the family Gramineae, such as rice.
It was almost impossible to obtain a transformant by applying a known introduction method. This is because the number of cells into which the gene had been introduced was small, and because these cells could not be efficiently selected and regenerated into plants, the introduction conditions were extremely difficult.

[0007] That is, in the case of introduction into protoplasts, if the intensity of the gene introduction treatment is too strong, the changes produced in the membrane of protoplasts are destroyed without being repaired, and conversely if it is too weak. However, it is impossible to obtain a cell into which a gene has been introduced unless truly appropriate conditions such as the case where the gene is not introduced. Also, regarding selection by antibiotics, not all cells into which the antibiotic resistance gene has been introduced will obtain sufficient resistance as compared to cells into which they have not been introduced, so we will also examine methods for efficient selection. It is necessary. further,
The fact that the resistance of the plants of this genus to the antibiotics originally possessed was remarkably higher than that of other gramineous plants also contributed to the difficulty in selection. Furthermore, when the introduction treatment is performed, the redifferentiation ability may decrease.

Therefore, the present inventors have examined many conditions such as the concentration of the polyethylene glycol solution to be treated and the treatment time as a method for efficiently introducing the antibiotic resistance gene into cells. Furthermore, prior to the introduction, we attempted to use techniques such as a method for heating protoplasts. Further, the present invention has been completed by studying a method for treating an antibiotic that efficiently selects introduced cells, and a method for regenerating a plant from a cell into which a gene has been introduced.

That is, the present invention aims at efficiently producing an antibiotic resistant plant in a Zoisia genus plant which has been made difficult or impossible by the methods that have been tried so far. .

[0010]

To achieve the above object, the present invention provides a Zoisia plant transformant and a method for producing the same. That is, it is characterized in that a plant of the genus Zoisia is transformed by the introduction of an antibiotic resistance gene to be a plant resistant to antibiotics. At this time, it is considered that the cell cluster is transformed by the introduction of the antibiotic resistance gene and becomes a cell aggregate having resistance to the antibiotic. It is also considered that the cells transformed by the introduction of an antibiotic resistance gene are transformed into cells having resistance to antibiotics. Further, it is considered that the seeds are obtained from the Zoisia plant transformants and propagate the Zoisia plant transformants. It is also considered that the Zoisia plant transformant is propagated by seed propagation. Further, it is considered that the Zoisia plant transformant is propagated by vegetative propagation. The present invention also provides a method for producing a Zoisia plant transformant, which comprises introducing an antibiotic resistance gene into a Zoisia plant. At this time, it is considered that the target to which the antibiotic resistance gene is introduced is a cell clump derived from a Zoisia plant.
It is also considered to be protoplasts isolated from cell aggregates derived from Zoisia plants. It is also possible to introduce the antibiotic resistance gene into the cell clumps or the protoplasts and then make the plants. It is also conceivable to introduce an antibiotic resistance gene into the cell clumps or the protoplasts and culture the cells in an antibiotic-containing medium to form antibiotic-resistant cell clumps. Also, culturing the antibiotic resistant cell clumps in a medium containing antibiotics,
It is possible to form somatic embryos resistant to antibiotics.
It is also considered that the antibiotic-resistant cell clumps are cultured in a medium containing antibiotics to form antibiotic-resistant adventitious buds. It is also conceivable to culture the antibiotic-resistant somatic embryo or antibiotic-resistant somatic bud in a medium containing an antibiotic to form an antibiotic-resistant plant body. It is also possible to use a polyethylene glycol solution for the introduction of the antibiotic resistance gene. It is also possible that the polyethylene glycol solution has a concentration of about 20 to about 60%. It is also possible to apply an electric pulse when introducing the antibiotic resistance gene. The electric pulse has an electric field strength of about 300 to about 600 V / cm,
It is considered that the time constant is about 10 to about 50 ms.
Further, when introducing the antibiotic resistance gene, it is considered that the metal particles having the antibiotic resistance gene adsorbed therein are driven into the cell clump under high pressure. It is also contemplated that the pressure is about 800 to about 1500 PSI. It is also possible to heat the protoplasts prior to the introduction of the antibiotic resistance gene. Further, it is considered that the heating is performed at about 30 ° C. to about 55 ° C. for about 1 to about 15 minutes. When the antibiotic resistance gene is the hygromycin phosphotransferase gene, it is possible to use about 200 to about 400 μg / mL hygromycin as the antibiotic. When the antibiotic resistance gene is the neomycin phosphotransferase gene, the antibiotic resistance is about 100 to about 500 μg /
It is conceivable to use mL of Geneticin.

[0011]

In the present invention, a protoplast of a Zoisia plant for transforming and a plasmid containing an antibiotic resistance gene are suspended together, and the plasmid is treated with polyethylene glycol, electroporation or the like. After the introduction, the cell agglomerates are formed, somatic embryos or adventitious buds are formed from the resulting cell agglomerates, and the transformants of the Zoisia plant are regenerated from the adventitious embryos or adventitious buds thus obtained. To do. In addition, in the introduction by the particle gun method, the gene is introduced into the cell clumps by driving the antibiotic resistance gene guide together with the metal particles, and the cell clumps are formed. From the cell clumps thus obtained, somatic embryos or An adventitious bud is formed, and a somatic embryo or a transformant of a Zoisia genus plant is regenerated from the adventitious bud thus obtained.

[0012] In the present invention, a method for introducing an antibiotic resistance gene into the protoplasts and cell clumps of the genus Zoisia at a high frequency was established by conducting a detailed examination of the conditions for introduction, and the genes originally belonging to the genus Zoisia The resistance to the existing antibiotics has been investigated in detail, and only the cell clumps and tissues into which the established antibiotic resistance gene has been introduced are selected. This has been established, and this has made it possible to introduce a gene of the genus Zoisia, which has been impossible so far. Furthermore, in the particle gun method, it became possible to prevent the appearance of chimeras by adding antibiotics during the entire period until the plants were regenerated. As a result, it is possible to efficiently produce an antibiotic-resistant plant in a plant of the genus Zoisia which has been difficult or impossible in the past.

[0013]

DETAILED DESCRIPTION OF THE INVENTION Hereinafter, the present invention will be described specifically. The method for preparing the material used in the present invention from the plant of the genus Zoisia is the 1992 patent application publication No. 30 proposed by the present inventor.
It may be performed according to the method described in Japanese Patent No. 4825. Hereinafter, the antibiotic resistance gene used in the present invention, three types of gene transfer methods, a method for culturing an antibiotic resistance cell clump,
A method for selecting an antibiotic-resistant cell clump, a method for regenerating an antibiotic-resistant plant of the genus Zoisia, and the like will be described in detail.

<Antibiotic resistance gene used in the present invention> Antibiotic resistance genes used in the present invention include promoters that function in Zoisia plants, antibiotic resistance genes, and terminators that function in Zoisia plants. It is possible to use constructed plasmids. For example,
As the promoter, the promoter of the 35S RNA gene (CaMV35S) derived from cauliflower mosaic virus can be used. Hygromycin phosphotransferase gene (hph), neomycin phosphotransferase II gene (nptII) and the like can be used as the antibiotic resistance gene. Further, as the terminator, a terminator derived from NOS gene and the like can be used.

[0015] <gene transfer methods (polyethylene glycol treatment)> For example, the above plasmid 1 to 100 [mu] g / mL, of 10 ⁵ to 010 ⁷ cells / mL and Zoishia genus plant-derived protoplasts, as a tonicity modifier 0.
Suspend in a buffer containing 4-0.6 M mannitol or the like. A polyethylene glycol solution is added to this so that the final concentration is 10 to 30%, and the mixture is left at room temperature for 5 to 30 minutes. Thereafter, the gene is introduced into protoplasts by diluting polyethylene glycol.
If the protoplast is heated at 30 to 55 ° C. for 1 to 15 minutes prior to this introduction, the introduction efficiency is dramatically improved. The protoplasts derived from plants of the genus Zoisia are made from callus.

<Gene Transfer Method (Electroporation Method)> For example, 1 to 100 μg / mL of the above plasmid and 10 ^{5 to} 10 ⁷ cells / mL of Zoisia plant-derived protoplast are used as osmotic pressure regulators. Suspend in a buffer containing 4 to 0.6 M mannitol or the like. An electric pulse having an electric field strength of 300 to 600 V / cm and a time constant of 10 to 50 ms is applied to this, and electrical stimulation is applied to introduce the gene into protoplasts. Prior to this introduction, the protoplasts were incubated at 30-55 ° C for 3 hours.
The heating efficiency is drastically improved by heating for 15 minutes.
The protoplasts derived from plants of the genus Zoisia are made from callus.

<Gene Transfer Method (Particle Gun Method)> For example, a cell aggregate derived from the genus Zoisia is filtered with a filter paper (Whatman No. 4, φ7c) of about 0.3 to 0.5 mL.
m) Evenly spread and adsorb onto the surface. Metal particles having a diameter of 0.1 to 5 μm, which are obtained by adsorbing the above-mentioned plasmid using calcium chloride and spermidine, are introduced by using a particle gun at an air pressure of 800 to 1500 PSI or a pressure at the time of explosive explosion. Tungsten or gold can be used for the metal particles.

<Cultivation of protoplasts after introduction of antibiotic resistance gene by polyethylene glycol method or electroporation method> Protoplasts derived from the genus Zoisia obtained by the above paragraph No. "0015" or paragraph No. "0016" are gene-transfected. After the treatment, the plant tissue culture medium is cultured in a liquid or solid medium containing a plant growth regulator. Examples of plant tissue culture media include N6 medium, MS medium, R2 medium, K8P medium, and AA.
There is a medium etc. Examples of plant growth regulators include auxins such as 2.4-dichlorophenoxyacetic acid and naphthaleneacetic acid, and cytokinins such as kinetin. Culturing is performed in the dark, and the temperature is preferably 20 to 30 ° C. By such culture, cell aggregates are formed from protoplasts.

<Culture culture of cell mass after introduction of antibiotic resistance gene by particle gun method> Paragraph No. "00"
The cell aggregate derived from the genus Zoisia obtained in 17) is subjected to gene introduction treatment, and then cultured in a liquid or solid medium in which a plant growth regulator is added to a plant tissue culture medium. Examples of the plant tissue culture medium include N6 medium, MS medium, R2 medium, K8P medium, AA medium and the like. Examples of plant growth regulators include auxins such as 2.4-dichlorophenoxyacetic acid and naphthaleneacetic acid, and cytokinins such as kinetin. Culturing is performed in the dark, and the temperature is preferably 20 to 30 ° C. By such culture, cell aggregates are formed from the cells into which the gene has been introduced.

<Selection Method of Antibiotic Resistant Cell Aggregates>
After 7 to 28 days of culture in the above paragraph number “0018” or paragraph number “0019”, for example, by treating the cell clumps with an antibiotic such as hygromycin, kanamycin, geneticin, etc., the desired antibiotic resistance can be obtained. It is possible to select only the cell clumps of. As a method for treating the cell clumps with an antibiotic, a method of adding a liquid medium containing an antibiotic, a method of developing on an agar medium containing an antibiotic, and the like are possible.

<Method of Producing Antibiotic-Resistant Adventitious Embryo or Adventitious Bud> A medium (eg, N6 medium, R2 medium, MS) for regenerating a plant body of antibiotic-resistant cell clumps.
A plant growth regulator (eg, medium, LS medium, etc.)
2. Auxins such as 4-dichlorophenoxyacetic acid and naphthaleneacetic acid, and cytokinins such as kinetin)
Are further added, and the cells are further cultured on an agar medium to which antibiotics such as hygromycin, kanamycin and geneticin are added. Subculture to fresh medium every 14 to 40 days,
Continue the culture. The culture is preferably performed in the light. Antibiotic-resistant somatic embryos or somatic buds are obtained 24 to 48 days after the start of culture.

<Method of Regenerating Antibiotic-Resistant Zoisia Plant> A medium for regenerating a plant from the antibiotic-resistant adventitious embryo or adventitious bud obtained above (eg, N6 medium, R2 medium, MS medium, LS medium, etc.)
Plant growth regulators (eg, auxins such as 2.4-dichlorophenoxyacetic acid and naphthaleneacetic acid, and cytokinins such as kinetin) are added, and further cultured on an agar medium to which antibiotics such as hygromycin and geneticin are added. . Subculture to fresh medium at intervals of 14 to 40 days to continue culturing. The culture is preferably performed in the light. Antibiotic-resistant Zoisia plants can be obtained 24 to 48 days after the start of culture.

<Vegetative Propagation of Antibiotic-Resistant Zoisia Plants> In the present invention, the term “plant” refers to an individual plant composed of roots, stems, leaves, flowers and the like according to the usual concept. The plant of the genus Zoisia used in the present invention can be propagated vegetatively by a stalk (runner) and a rhizome. This growth is self-replicating and all inherited traits are maintained. That is, the Zoisia genus plant transformant having resistance to the antibiotic produced by the present invention can grow without the antibiotic resistance being lost by the growth by the runner and the rhizome.

<Seed Propagation of Antibiotic-Resistant Zoisia Plants> The antibiotic-resistant gene introduced into a plant according to the present invention is transferred from the plant to the plant to the progeny in accordance with Mendelian inheritance rule through seeds as a vector. Be inherited. Therefore, the introduced gene is present in the seeds formed from pollen or ovary of the plant of the present invention, and the inherited trait is inherited to the offspring. That is, the Zoicia plant transformant having resistance to the antibiotic produced by the present invention can grow without the antibiotic resistance being lost by the seed growth.

[0025]

The present invention will be described in more detail with reference to the following two examples. The present invention is not limited to these examples.

<Embodiment 1> Zoysia jap
onica ) was used as a test material, protoplasts isolated from a liquid culture system having a redifferentiation ability derived from mature seeds or growing points. P35Sh for the antibiotic resistance gene
ph (CaMV35S promoter + hygromycin phosphotransferase gene + NOS terminator) was used.

<Introduction of Gene into Protoplasts> Protoplasts were heated at 45 ° C. for 5 minutes to give a density of 2 × 10 ^6.
/ ML, the antibiotic resistance gene was suspended in an introduction buffer of 0.4 M mannitol, 15 mM magnesium chloride, 0.1% MES so as to be 50 μg / mL, and treated with polyethylene glycol. That is, the final concentration is 2
A polyethylene glycol solution was added to 0%, the mixture was allowed to stand for a while, a diluent containing sodium chloride, calcium chloride and potassium chloride was added, and centrifugal washing was performed.

<Cultivation of introduced protoplasts and selection of antibiotic-resistant cell clumps and regeneration of antibiotic-resistant plants> Protoplasts were cultured in a liquid medium based on K8p medium. Fourteen days after the introduction, hygromycin 200 to 400 μg / mL was added to the culture medium to start selection of antibiotic-resistant cell clumps. After 28 days of selective culture, the selected antibiotic-resistant cell clumps were transplanted to an agar medium containing 200 to 400 μg / mL of hygromycin. After culturing and proliferating for 1 month, callus was transplanted to a regeneration medium to regenerate somatic embryos, thereby regenerating antibiotic-resistant plants (Fig. 1).

<Confirmation of Antibiotic Resistance Gene in Plant> DNA was isolated from the obtained plant by a conventional method, and the presence of the introduced hygromycin phosphotransferase gene was confirmed by Southern hybridization ( (Fig. 2).

In FIG. 2, column 1 shows the introduced antibiotic resistance gene. Column 2 shows the DNA of the zoysiagrass plant that has not been introduced. Rows 3 and 4 show the DNA of antibiotic-resistant zoysiagrass plants.

<Example 2> Zoysia ( Zoysia)
matrella ) mature seeds or liquid cultured cells derived from the growing point were used as test materials. As the antibiotic resistance gene, p35Shph (CaMV35S promoter + hygromycin phosphotransferase gene + NOS terminator) was used.

<Introduction of Gene into Cell Aggregate> About 0.3 to 0.5 mL of the cell aggregate was filtered (Whatman No. 4,
(7 cm in diameter) is uniformly spread and adsorbed to form a sample, and gold particles (diameter 1 μm) on which an antibiotic resistance gene is adsorbed
m) was driven in using a particle gun. As the particle gun, Bio-Rad PDS1000 / He was used.

<Cultivation of introduced cell clumps and selection of antibiotic-resistant cell clumps and regeneration of antibiotic-resistant plants> Cell clumps were cultured on an agar medium based on MS medium. . Seven days after the introduction, the cell clumps together with the filter paper were transferred to a selection medium containing 200 to 400 μg / mL hygromycin, and selection of antibiotic-resistant cell clumps was started. After 28 days of the selective culture, the selected antibiotic-resistant cell clumps were transplanted to an agar medium containing 200 to 400 μg / mL hygromycin. After culturing and proliferating for 1 month, cell clumps were transplanted to a redifferentiation medium to regenerate somatic embryos, thereby regenerating antibiotic-resistant plants (Fig. 3).

<Confirmation of Antibiotic Resistance Gene in Plant> DNA was isolated from the obtained plant by a conventional method and the presence of the introduced hygromycin phosphotransferase gene was confirmed by Southern hybridization ( (Fig. 4).

In FIG. 4, column 1 shows the introduced antibiotic resistance gene. Column 2 shows the DNA of the L. serrata plant without the introduction treatment. Columns 3 and 4 show the DNA of antibiotic-resistant plants of C. perfringens.

[0036]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a product and a method which are extremely effective in the creation of new varieties of Zoisia plants to which useful traits will be developed in the future.

[Brief description of drawings]

FIG. 1 is a photograph showing the morphology of a plant obtained by the present invention.

FIG. 2 is a view prepared based on a photograph of the plant DNA shown in FIG. 1 analyzed by Southern hybridization.

FIG. 3 is a photograph showing the morphology of another plant obtained by the present invention.

FIG. 4 is a view prepared based on a photograph of the plant DNA shown in FIG. 3 analyzed by Southern hybridization.

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Claims (24)

[Claims] 1. A Zoisia genus plant transformant, which is obtained by transforming a Zoisia genus plant by introduction of an antibiotic resistance gene and has resistance to antibiotics. 2. A Zoysia genus plant transformant, which is obtained by transforming a Zoisia genus plant by introducing an antibiotic resistance gene into a cell clump having resistance to an antibiotic. 3. A Zoisia genus plant transformant, which is obtained by transforming a Zoisia genus plant by introducing an antibiotic resistance gene into cells having antibiotic resistance. 4. The Zoisia genus plant transformant according to claim 1, which is a seed obtained from the Zoisia genus plant transformant for propagating the Zoisia genus plant transformant. A transformant of the genus Zoisia, which is characterized by: 5. The Zoisia plant transformant according to any one of claims 1 to 3, wherein the Zoisia plant transformant is propagated by seed propagation. Method of breeding. 6. The Zoisia plant transformant according to any one of claims 1 to 3, wherein the Zoisia plant transformant is propagated by vegetative propagation. Method of breeding. 7. A method for producing a Zoisia plant transformant, which comprises introducing an antibiotic resistance gene into a Zoisia plant. 8. The method for producing a Zoisia plant transformant according to claim 7, wherein the target to which the antibiotic resistance gene is introduced is a cell aggregate derived from a Zoisia plant. A method for producing a plant transformant. 9. The method for producing a Zoisia plant transformant according to claim 7, wherein the Zoisia plant transformant is a protoplast isolated from a cell mass derived from a Zoisia plant. Method. 10. The method for producing a Zoisia plant transformant according to claim 8 or 9, wherein the plant is obtained by introducing an antibiotic resistance gene into the cell clumps or the protoplasts. A method for producing a transformant of a genus Zoisia. 11. The method for producing a Zoisia genus plant transformant according to claim 10, wherein an antibiotic resistance gene is introduced into the cell clumps or the protoplasts, followed by culturing in a medium containing an antibiotic to obtain antibiotic resistance. A method for producing a Zoicia plant transformant, which comprises forming a sexual cell clump. 12. The method for producing a Zoisia plant transformant according to claim 11, wherein the antibiotic-resistant cell clump is cultured in a medium containing an antibiotic to form an antibiotic-resistant somatic embryo. A method for producing a transformant of the genus Zoisia, which comprises: 13. The method for producing a Zoicia plant transformant according to claim 11, wherein the antibiotic-resistant cell clump is cultured in a medium containing an antibiotic to form an antibiotic-resistant adventitious bud. A method for producing a transformant of the genus Zoisia, which comprises: 14. The method for producing a Zocia genus plant transformant according to claim 12 or 13, wherein the antibiotic-resistant somatic embryo or antibiotic-resistant somatic bud is cultured in a medium containing an antibiotic to obtain an antibiotic. A method for producing a transformant of the genus Zoisia, which comprises forming a substance-resistant plant. 15. The method for producing a Zoisia plant transformant according to claim 9, wherein a polyethylene glycol solution is used for introducing the antibiotic resistance gene. 16. The method for producing a Zoisia plant transformant according to claim 15, wherein the concentration of the polyethylene glycol solution is about 20 to about 60%. . 17. The method for producing a transformant of the genus Zoisia according to claim 9, wherein an electric pulse is applied when introducing the antibiotic resistance gene. 18. The method for producing a Zoicia plant transformant according to claim 17, wherein the electric pulse has an electric field intensity of about 300 to about 600 V / cm and a time constant of about 10 to about 5.
A method for producing a transformant of the genus Zoisia, which is 0 ms. 19. The method for producing a transformant of the genus Zoisia according to claim 8, wherein metal particles having an antibiotic resistance gene adsorbed therein are implanted into the cell clump under high pressure when the antibiotic resistance gene is introduced. A method for producing a transformant of the genus Zoisia, which comprises: 20. The method for producing a Zoisia plant transformant according to claim 19, wherein the pressure is about 800 to about 1500 PSI. 21. The method for producing a Zoisia plant transformant according to claim 9, wherein the protoplast is heated prior to the introduction of the antibiotic resistance gene, and the Zoisia plant transformant is produced. Method. 22. The method for producing a Zoisia plant transformant according to claim 21, wherein the heating is performed at about 30 ° C. to about 55 ° C. for about 1 to about 15 minutes. Method for producing transformant. 23. The method for producing a Zoisia genus plant transformant according to any one of claims 7 to 22,
When the antibiotic resistance gene is the hygromycin phosphotransferase gene, about 2 as an antibiotic
A method for producing a Zoisia plant transformant, which comprises using from 00 to about 400 μg / mL of hygromycin. 24. The method for producing a Zoisia genus plant transformant according to any one of claims 7 to 22,
About 1 as an antibiotic when the antibiotic resistance gene is the neomycin phosphotransferase II gene
A method for producing a Zoisia plant transformant, which comprises using from 00 to about 500 μg / mL of geneticin.